AABB Technical Manual 15th Ed. 2005

AABB%20Technical%20Manual%2015TH

User Manual:

Open the PDF directly: View PDF .
Page Count: 931

Download
Open PDF In Browser	View PDF

Technical Manual
15th Edition

Other related publications available from the AABB:
Technical Manual and Standards for Blood Banks and
Transfusion Services on CD-ROM
Transfusion Therapy: Clinical Principles and Practice, 2nd Edition
Edited by Paul D. Mintz, MD
Transfusion Medicine Self-Assessment and Review
By Pam S. Helekar, MD; Douglas P. Blackall, MD; Jeffrey L. Winters, MD;
and Darrell J. Triulzi, MD
Blood Transfusion Therapy: A Physician’s Handbook, 8th Edition
Edited by Jerry Gottschall, MD
Practical Guide to Transfusion Medicine
By Marian Petrides, MD, and Gary Stack, MD, PhD
Transfusion Medicine Interactive: A Case Study Approach CD-ROM
By Marian Petrides, MD; Roby Rogers, MD; and Nora Ratcliffe, MD

To purchase books, please call our sales department at (866)222-2498 (within the United
States) or (301)215-6499 (outside the United States); fax orders to (301)907-6895 or email
orders to sales@aabb.org. View the AABB Publications Catalog and order books on the
AABB Web site at www.aabb.org. For other book services, including chapter reprints and
large quantity sales, ask for the Senior Sales Associate.

Mention of specific products or equipment by contributors to this AABB publication
does not represent an endorsement of such products by the AABB nor does it necessarily indicate a preference for those products over other similar competitive products. Any
forms and/or procedures in this book are examples. AABB does not imply or guarantee
that the materials meet federal, state, or other applicable requirements. It is incumbent
on the reader who intends to use any information, forms, policies, or procedures contained in this publication to evaluate such materials for use in light of particular circumstances associated with his or her institution.
Efforts are made to have publications of the AABB consistent in regard to acceptable
practices. However, for several reasons, they may not be. First, as new developments in
the practice of blood banking occur, changes may be recommended to the Standards for
Blood Banks and Transfusion Services. It is not possible, however, to revise each publication at the time such a change is adopted. Thus, it is essential that the most recent edition of the Standards be consulted as a reference in regard to current acceptable practices. Second, the views expressed in this publication represent the opinions of authors.
The publication of this book does not constitute an endorsement by the AABB of any
view expressed herein, and the AABB expressly disclaims any liability arising from any
inaccuracy or misstatement.

Copyright © 2005 by AABB. All rights reserved. No part of this book may be reproduced
or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or by any information storage and retrieval system, without permission in writing from the Publisher.

AABB
8101 Glenbrook Road
Bethesda, Maryland 20814-2749

ISBN No. 1-56395-196-7
Printed in the United States

Cataloging-in-Publication Data
Technical manual / editor, Mark E. Brecher. —15th ed.
p. ; cm.
Including bibliographic references and index.
ISBN 1-56395-196-7
1. Blood Banks—Handbooks, manuals, etc. I. Brecher, Mark E. II. AABB.
[DNLM: 1. Blood Banks—laboratory manuals. 2. Blood Transfusion—
laboratory manuals. WH 25 T2548 2005]
RM172.T43 2005
615’.39—dc23
DNLM/DLC

Technical Manual
Program Unit
Chair and Editor
Mark E. Brecher, MD

Associate Editors
Regina M. Leger, MSQA, MT(ASCP)SBB, CQMgr(ASQ)
Jeanne V. Linden, MD, MPH
Susan D. Roseff, MD

Members/Authors
Martha Rae Combs, MT(ASCP)SBB
Gregory Denomme, PhD, FCSMLS(D)
Brenda J. Grossman, MD, MPH
N. Rebecca Haley, MD, MT(ASCP)SBB
Teresa Harris, MT(ASCP)SBB, CQIA(ASQ)
Betsy W. Jett, MT(ASCP), CQA(ASQ)CQMgr
Regina M. Leger, MSQA, MT(ASCP)SBB, CQMgr(ASQ)
Jeanne V. Linden, MD, MPH
Janice G. McFarland, MD
James T. Perkins, MD
Susan D. Roseff, MD
Joseph Sweeney, MD
Darrell J. Triulzi, MD
Liaisons
Gilliam B. Conley, MA, MT(ASCP)SBB
Michael C. Libby, MSc, MT(ASCP)SBB

Acknowledgments
The Technical Manual Program Unit extends special thanks to those volunteers who
provided peer review and made other contributions:
James P. AuBuchon, MD
Lucia M. Berte, MA,
MT(ASCP)SBB, DLM,
CQA(ASQ)CQMgr
Arthur Bracey, MD
Linda Braddy,
MT(ASCP)SBB
Donald R. Branch,
MT(ASCP)SBB, PhD
Ritchard Cable, MD
Sally Caglioti,
MT(ASCP)SBB
Loni Calhoun,
MT(ASCP)SBB
Tony S. Casina,
MT(ASCP)SBB
Geoff Daniels, PhD,
MRcPath
Robertson Davenport, MD
Richard J. Davey, MD
Walter Dzik, MD
Ted Eastlund, MD
Anne F. Eder, MD, PhD
Ronald O. Gilcher, MD,
FACP
Lawrence T. Goodnough,
MD
Linda Hahn,
MT(ASCP)SBB, MPM
Heather Hume, MD
Mark A. Janzen, PhD
Susan T. Johnson, MSTM,
MT(ASCP)SBB

W. John Judd, FIBMS,
MIBiol
Michael H. Kanter, MD
Louis M. Katz, MD
Debra Kessler, RN, MS
Thomas Kickler, MD
Karen E. King, MD
Joanne Kosanke,
MT(ASCP)SBB
Thomas A. Lane, MD
Alan H. Lazarus, PhD
German F. Leparc, MD
Douglas M. Lublin, MD,
PhD
Dawn Michelle,
MT(ASCP)SBB
Kenneth Moise, Jr., MD
S. Breanndan Moore, MD
Tania Motschman, MS,
MT(ASCP)SBB,
CQA(ASQ)
Marilyn K. Moulds,
MT(ASCP)SBB
Nancy C. Mullis,
MT(ASCP)SBB
Scott Murphy, MD
Patricia Pisciotto, MD
Mark A. Popovsky, MD
Marion E. Reid, PhD,
FIBMS
Jennifer F. Rhamy, MBA,
MA, MT(ASCP), SBB, HP
Scott D. Rowley, MD

Arell S. Shapiro, MD
R. Sue Shirey, MS,
MT(ASCP)SBB
Bruce Spiess, MD, FAHA
Jerry E. Squires, MD, PhD
Marilyn J. Telen, MD
Susan Veneman,
MT(ASCP)SBB
Phyllis S. Walker, MS,
MT(ASCP)SBB
Dan A. Waxman, MD
Robert Weinstein, MD
Connie M. Westhoff, PhD,
MT(ASCP)SBB
Members of AABB committees who reviewed
manuscripts as part of
committee resource
charges
The staff of the Armed
Services Blood Program
Office
The staff of the US Food
and Drug Administration, Center for
Biologics Evaluation
and Research
The staff of the Transplantation and Transfusion
Service, McClendon
Clinical Laboratories,
UNC Hospitals

Special thanks are due to Laurie Munk, Janet McGrath, Nina Hutchinson, Jay Pennington, Frank McNeirney, Kay Gregory, MT(ASCP)SBB, and Allene Carr-Greer,
MT(ASCP)SBB of the AABB National Office for providing support to the Program Unit
during preparation of this edition.

Introduction

he 15th edition of the AABB Technical Manual is the first in the
second half century of this publication. The original Technical Manual (then
called Technical Methods and Procedures)
was published in 1953 and the 14th edition marked the 50th anniversary of this
publication.
Over the years, this text has grown and
matured, until today it is a major textbook
used by students (medical technology and
residents) and practicing health-care professionals (technologists, nurses, and physicians) around the world. Selected editions
or excerpts have been translated into
French, Hungarian, Italian, Japanese, Spanish, Polish, and Russian. It is one of only
two AABB publications that are referenced
by name in the AABB Standards for Blood
Banks and Transfusion Services (the other
being the Circular of Information for the
Use of Human Blood Components). All
branches of the US Armed Services have
adopted the AABB Technical Manual as
their respective official manuals for blood
banking and transfusion medicine activities.
The Technical Manual serves a diverse
readership and is used as a technical refer-

ence, a source for developing policies and
procedures, and an educational tool. The
Technical Manual is often the first reference
consulted in many laboratories; thus, it is
intended to provide the background information to allow both students and experienced individuals to rapidly familiarize
themselves with the rationale and scientific
basis of the AABB standards and current
standards of practice. As in previous editions, the authors and editors have tried to
provide both breadth and depth, including
substantial theoretical and clinical material
as well as technical details. Due to space
limitations, the Technical Manual cannot
provide all of the advanced information on
any specific topic. However, it is hoped that
sufficient information is provided to answer
the majority of queries for which individuals consult the text, or at a minimum, to direct someone toward additional pertinent
references.
Readers should be aware that, unlike
most textbooks in the field, this book is
subjected to extensive peer review (by experts in specific subject areas, AABB committees, and regulatory bodies such as the
Food and Drug Administration). As such,
this text is relatively unique, and represents
ix

AABB Technical Manual

a major effort on the part of the AABB to
provide an authoritative and balanced
reference source.
As in previous recent editions, the content is necessarily limited in order to retain
the size of the Technical Manual to that of a
textbook that can be easily handled. Nevertheless, readers will find extensive new and
updated information, including expanded
coverage of quality approaches, apheresis
indications, cellular nomenclature, molecular diagnostics, hematopoietic progenitor
cell processing, and transfusion-transmitted
diseases.
Techniques and policies outlined in the
Technical Manual are, to the best of the
Technical Manual Program Unit's ability, in
conformance with AABB Standards. They
are not to be considered the only permissible way in which requirements of Standards can be met. Other methods, not included, may give equally acceptable results.
If discrepancy occurs between techniques
or suggestions in the Technical Manual and
the requirements of Standards, authority
resides in Standards. Despite the best efforts of both the Program Unit and the extensive number of outside reviewers, errors
may remain in the text. As with previous
editions, the Program Unit welcomes suggestions, criticisms, or questions about the
current edition.

I would like to thank the members of the
Technical Manual Program Unit for their
dedication and long hours of work that
went into updating this edition. I would
also like to thank all the AABB committees,
the expert reviewers, and the readers who
have offered numerous helpful suggestions
that helped to make this edition possible. I
would particularly like to thank my three
associate editors—Gina Leger, Jeanne Linden, and Sue Roseff—who have provided
countless invaluable hours in the preparation of this edition. Finally I would like to
thank Laurie Munk, AABB Publications Director, whose tireless efforts on behalf of
the Technical Manual never cease to amaze
me, and who has made the publication of
this book a pleasure.
This edition is my third and final Technical Manual. I served as associate editor for
the 13th edition and chief editor for the
14th and 15th editions. It has been an
honor to help shepherd these editions to
fruition and it is my hope that the AABB
Technical Manual will continue to be one
of the AABB's premier publications for decades to come.

Mark E. Brecher, MD
Chief Editor
Chapel Hill, NC

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

Quality Issues
1.

Quality Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Quality Control, Quality Assurance, and Quality Management . . . . . . . . . . . . . 2
Quality Concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Practical Application of Quality Principles . . . . . . . . . . . . . . . . . . . . . . . . . 6
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Appendix 1-1. Glossary of Commonly Used Quality Terms . . . . . . . . . . . . . . . 30
Appendix 1-2. Code of Federal Regulations Quality-Related References . . . . . . . 32
Appendix 1-3. Statistical Tables for Binomial Distribution Used to
Determine Adequate Sample Size and Level of Confidence for
Validation of Pass/Fail Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Appendix 1-4. Assessment Examples: Blood Utilization . . . . . . . . . . . . . . . . . 36

Facilities and Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Facilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Safety Program. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fire Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Electrical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biosafety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chemical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Radiation Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Shipping Hazardous Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Waste Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Disaster Planning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix 2-1. Safety Regulations and Recommendations Applicable to
Health-Care Settings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix 2-2. General Guidelines for Safe Work Practices, Personal Protective
Equipment, and Engineering Controls . . . . . . . . . . . . . . . . . . . . . . .
Appendix 2-3. Biosafety Level 2 Precautions . . . . . . . . . . . . . . . . . . . . .
Appendix 2-4. Sample Hazardous Chemical Data Sheet. . . . . . . . . . . . . . .
Appendix 2-5. Sample List of Hazardous Chemicals in the Blood Bank . . . . . .
Appendix 2-6. Specific Chemical Categories and How to Work Safely with
These Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix 2-7. Incidental Spill Response . . . . . . . . . . . . . . . . . . . . . . . .
Appendix 2-8. Managing Hazardous Chemical Spills . . . . . . . . . . . . . . . .

.
.
.
.
.
.
.
.
.
.
.
.
.

39
40
42
47
48
49
57
63
66
67
67
68
70

. . 71
.
.
.
.

.
.
.
.

73
77
78
80

. . 82
. . 84
. . 87

xi
Copyright © 2005 by the AABB. All rights reserved.

xii

AABB Technical Manual

Blood Utilization Management. . . . . . . . . . . . . . . . . . . . . .
Minimum and Ideal Inventory Levels . . . . . . . . . . . . . . . . . .
Determining Inventory Levels. . . . . . . . . . . . . . . . . . . . . . .
Factors that Affect Outdating . . . . . . . . . . . . . . . . . . . . . . .
Improving Transfusion Service Blood Ordering Practices . . . . . . .
Special Product Concerns . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.
.
.
.

89
89
89
90
91
94
95

Blood Donation and Collection
4.

Allogeneic Donor Selection and Blood Collection . . . . . . . . . . . . . . . . . . . 97
Blood Donation Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Collection of Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Appendix 4-1. Full-Length Donor History Questionnaire . . . . . . . . . . . . . . . 110
Appendix 4-2. Medication Deferral List. . . . . . . . . . . . . . . . . . . . . . . . . . 113
Appendix 4-3. Blood Donor Education Materials . . . . . . . . . . . . . . . . . . . . 114
Appendix 4-4. Some Drugs Commonly Accepted in Blood Donors . . . . . . . . . 115

Autologous Blood Donation and Transfusion . . . . . . . . . .
Preoperative Autologous Blood Collection . . . . . . . . . . . . .
Acute Normovolemic Hemodilution . . . . . . . . . . . . . . . .
Intraoperative Blood Collection . . . . . . . . . . . . . . . . . . .
Postoperative Blood Collection . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.
.
.

117
118
126
130
133
135

Apheresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Separation Techniques . . . . . . . . . . . . . . . . . . . . .
Component Collection . . . . . . . . . . . . . . . . . . . . .
Therapeutic Apheresis . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.
.

139
139
140
144
158

Blood Component Testing and Labeling . . . . . . . . . . . . . .
Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Labeling, Records, and Quarantine . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.
.

163
163
170
174
174

Collection, Preparation, Storage, and Distribution of Components from
Whole Blood Donations . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Blood Component Descriptions . . . . . . . . . . . . . . . . . . . . . . . . . . .
Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prestorage Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Storage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.
.

175
175
178
179
184

.
.
.
.
.

Contents

Inspection, Shipping, Disposition, and Issue
Blood Component Quality Control . . . . . .
References . . . . . . . . . . . . . . . . . . . .
Appendix 8-1. Component Quality Control .

.
.
.
.

xiii

.
.
.
.

194
197
199
202

Immunologic and Genetic Principles
9.

Molecular Biology in Transfusion Medicine . . . . . . . . . . .
From DNA to mRNA to Protein . . . . . . . . . . . . . . . . . . .
Genetic Mechanisms that Create Polymorphism . . . . . . . . .
Genetic Variability . . . . . . . . . . . . . . . . . . . . . . . . . . .
Molecular Techniques. . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix 9-1. Molecular Techniques in Transfusion Medicine .

.
.
.
.
.
.
.
.

203
203
207
208
209
220
221
222

10.

Blood Group Genetics. . . . . . . . . . . . . . . . . . . . . . .
Basic Principles. . . . . . . . . . . . . . . . . . . . . . . . . . .
Genetics and Heredity. . . . . . . . . . . . . . . . . . . . . . .
Patterns of Inheritance . . . . . . . . . . . . . . . . . . . . . .
Population Genetics . . . . . . . . . . . . . . . . . . . . . . . .
Blood Group Nomenclature . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix 10-1. Glossary of Terms in Blood Group Genetics

.
.
.
.
.
.
.
.

223
223
225
232
236
238
239
241

11.

Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Immune Response. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Organs of the Immune System . . . . . . . . . . . . . . . . . . . . . . . .
Cells of the Immune System . . . . . . . . . . . . . . . . . . . . . . . . .
Soluble Components of the Immune Response . . . . . . . . . . . . . .
Immunology Relating to Transfusion Medicine . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix 11-1. Definitions of Some Essential Terms in Immunology .

.
.
.
.
.
.
.
.
.

243
243
249
249
256
263
267
268
269

12.

Red Cell Antigen-Antibody Reactions and Their Detection .
Factors Affecting Red Cell Agglutination . . . . . . . . . . . . . .
Enhancement of Antibody Detection . . . . . . . . . . . . . . . .
The Antiglobulin Test . . . . . . . . . . . . . . . . . . . . . . . . .
Other Methods to Detect Antigen-Antibody Reactions. . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.
.
.

271
272
276
277
283
286

.
.
.
.
.
.
.
.

.
.
.
.
.
.

xiv

AABB Technical Manual

Blood Groups
13.

ABO, H, and Lewis Blood Groups and Structurally Related Antigens
The ABO System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The H System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Lewis System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The I/i Antigens and Antibodies . . . . . . . . . . . . . . . . . . . . . . . .
The P Blood Group and Related Antigens . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.
.
.
.
.

289
289
303
304
306
308
311
312

14.

The Rh System . . . . . . . . . . . . . . . . . . . . . . . . . . .
The D Antigen and Its Historical Context. . . . . . . . . . . .
Genetic and Biochemical Considerations . . . . . . . . . . .
Rh Terminology . . . . . . . . . . . . . . . . . . . . . . . . . .
Serologic Testing for Rh Antigen Expression . . . . . . . . . .
Weak D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Other Rh Antigens . . . . . . . . . . . . . . . . . . . . . . . . .
Rhnull Syndrome and Other Deletion Types . . . . . . . . . . .
Rh Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . .
Rh Typing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.
.
.
.
.
.
.
.
.

315
315
316
318
319
322
324
325
327
328
332
333

15.

Other Blood Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Distribution of Antigens . . . . . . . . . . . . . . . . . . . . . . . . .
MNS System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Kell System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Duffy System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Kidd System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Other Blood Group Systems . . . . . . . . . . . . . . . . . . . . . . .
Blood Group Collections . . . . . . . . . . . . . . . . . . . . . . . . .
High-Incidence Red Cell Antigens Not Assigned to a Blood Group
System or Collection . . . . . . . . . . . . . . . . . . . . . . . . . .
Low-Incidence Red Cell Antigens Not Assigned to a Blood Group
System or Collection . . . . . . . . . . . . . . . . . . . . . . . . . .
Antibodies to Low-Incidence Antigens . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.
.
.
.
.

335
335
337
340
343
345
346
355

.
.
.
.

357
358
358
360

Platelet and Granulocyte Antigens and Antibodies
Platelet Antigens . . . . . . . . . . . . . . . . . . . . . . .
Granulocyte Antigens . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.

361
361
377
380

16.

. .
. .
. .
. .

.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.

. . . . . . . . . 356

Contents

17.

The HLA System . . . . . . . . . . . . . . . . . . . . . . . . .
Genetics of the Major Histocompatibility Complex . . . .
Biochemistry, Tissue Distribution, and Structure . . . . . .
Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . .
Biologic Function . . . . . . . . . . . . . . . . . . . . . . . .
Detection of HLA Antigens and Alleles . . . . . . . . . . . .
The HLA System and Transfusion . . . . . . . . . . . . . . .
HLA Testing and Transplantation . . . . . . . . . . . . . . .
Parentage and Other Forensic Testing . . . . . . . . . . . .
HLA and Disease . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.
.
.
.
.
.
.
.
.

385
386
390
391
393
394
397
400
402
402
404
405

Serologic Principles and Transfusion Medicine
18.

Pretransfusion Testing . . . . . . . . . . . . . . . . . . . . . . . . . .
Transfusion Requests . . . . . . . . . . . . . . . . . . . . . . . . . . .
Blood Sample. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Serologic Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Crossmatching Tests. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Interpretation of Antibody Screening and Crossmatch Results . . .
Labeling and Release of Crossmatched Blood at the Time of Issue .
Selection of Units . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.
.
.
.
.
.
.

407
407
409
410
413
415
416
418
420
420

19.

Initial Detection and Identification of Alloantibodies to Red Cell Antigens .
Significance of Alloantibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . .
General Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Basic Antibody Identification Techniques . . . . . . . . . . . . . . . . . . . . .
Complex Antibody Problems. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Selecting Blood for Transfusion . . . . . . . . . . . . . . . . . . . . . . . . . . .
Selected Serologic Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.
.
.
.
.

423
423
424
427
431
439
443
450

20.

The Positive Direct Antiglobulin Test and Immune-Mediated Red
Cell Destruction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Direct Antiglobulin Test . . . . . . . . . . . . . . . . . . . . . . . .
Immune-Mediated Hemolysis . . . . . . . . . . . . . . . . . . . . . . .
Serologic Problems with Autoantibodies . . . . . . . . . . . . . . . . .
Drug-Induced Immune Hemolytic Anemia . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.
.
.
.

453
454
458
469
472
477
479

.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.

xvi

AABB Technical Manual

Appendix 20-1. An Example of an Algorithm for Investigating a Positive DAT
(Excluding Investigation of HDFN) . . . . . . . . . . . . . . . . . . . . . . . . . . 480
Appendix 20-2. Some Drugs Associated with Immune Hemolysis and/or
Positive DATs Due to Drug-Induced Antibodies . . . . . . . . . . . . . . . . . . . 481

Clinical Considerations in Transfusion Practice
21.

Blood Transfusion Practice . . . . . . . . . . . . . . . . . . .
Red Blood Cell Transfusion . . . . . . . . . . . . . . . . . . . .
Platelet Transfusion . . . . . . . . . . . . . . . . . . . . . . . .
Granulocyte Transfusion . . . . . . . . . . . . . . . . . . . . .
Special Cellular Blood Components. . . . . . . . . . . . . . .
Replacement of Coagulation Factors . . . . . . . . . . . . . .
Cryoprecipitated AHF Transfusion . . . . . . . . . . . . . . .
Special Transfusion Situations . . . . . . . . . . . . . . . . . .
Pharmacologic Alternatives to Transfusion . . . . . . . . . .
Oversight of Transfusion Practice . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.
.
.
.
.
.
.
.

483
483
488
492
492
493
500
508
512
514
515

22.

Administration of Blood and Components
Pre-Issue Events . . . . . . . . . . . . . . . . .
Blood Issue and Transportation . . . . . . . .
Pre-Administration Events . . . . . . . . . . .
Administration . . . . . . . . . . . . . . . . . .
Post-Administration Events . . . . . . . . . .
Quality Assurance . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . .

.
.
.
.
.
.
.
.

521
521
524
525
527
531
532
532

23.

Perinatal Issues in Transfusion Practice . . . . . . . . .
Hemolytic Disease of the Fetus and Newborn . . . . . .
Neonatal Immune Thrombocytopenia . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.

535
535
551
554

24.

Neonatal and Pediatric Transfusion Practice . . . . . . . .
Fetal and Neonatal Erythropoiesis . . . . . . . . . . . . . . .
Unique Aspects of Neonatal Physiology . . . . . . . . . . . .
Cytomegalovirus Infection . . . . . . . . . . . . . . . . . . . .
Red Cell Transfusions in Infants Less than 4 Months of Age .
Transfusion of Other Components . . . . . . . . . . . . . . .
Neonatal Polycythemia . . . . . . . . . . . . . . . . . . . . . .
Extracorporeal Membrane Oxygenation . . . . . . . . . . . .
Leukocyte Reduction . . . . . . . . . . . . . . . . . . . . . . .
Transfusion Practices in Older Infants and Children . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.
.
.
.
.
.
.
.

557
557
558
562
562
568
572
572
573
574
577

. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .

Contents

xvii

25.

Cell Therapy and Cellular Product Transplantation . . . .
Diseases Treated with Hematopoietic Cell Transplantation . .
Sources of Hematopoietic Progenitor Cells . . . . . . . . . . .
Donor Eligibility . . . . . . . . . . . . . . . . . . . . . . . . . . .
Collection of Products. . . . . . . . . . . . . . . . . . . . . . . .
Processing of Hematopoietic Progenitor Cells. . . . . . . . . .
Freezing and Storage . . . . . . . . . . . . . . . . . . . . . . . .
Transportation and Shipping. . . . . . . . . . . . . . . . . . . .
Thawing and Infusion . . . . . . . . . . . . . . . . . . . . . . . .
Evaluation and Quality Control of Hematopoietic Products. .
Regulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.
.
.
.
.
.
.
.
.
.

581
583
583
589
591
596
604
606
607
607
608
609
609

26.

Tissue and Organ Transplantation . . . . . . . . . . . . . .
Transplant-Transmitted Diseases and Preventive Measures.
Bone Banking. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Skin Banking . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Heart Valves. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Records of Stored Tissue Allografts . . . . . . . . . . . . . . .
FDA Regulation of Tissue . . . . . . . . . . . . . . . . . . . . .
The Importance of ABO Compatibility . . . . . . . . . . . . .
The Role of Transfusion in Kidney Transplants . . . . . . . .
Liver Transplants. . . . . . . . . . . . . . . . . . . . . . . . . .
Other Organ Transplants . . . . . . . . . . . . . . . . . . . . .
Transfusion Service Support for Organ Transplantation . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.
.
.
.
.
.
.
.
.
.

617
617
623
625
625
626
626
627
627
627
629
629
630

27.

Noninfectious Complications of Blood Transfusion
Manifestations . . . . . . . . . . . . . . . . . . . . . . . .
Acute Transfusion Reactions . . . . . . . . . . . . . . . .
Evaluation of a Suspected Acute Transfusion Reaction.
Delayed Consequences of Transfusion . . . . . . . . . .
Records of Transfusion Complications . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.
.
.
.

633
633
639
652
656
660
661

28.

Transfusion-Transmitted Diseases . . . . . . . . . . . . . .
Hepatitis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Human Immunodeficiency Viruses . . . . . . . . . . . . . . .
Human T-Cell Lymphotropic Viruses . . . . . . . . . . . . . .
West Nile Virus . . . . . . . . . . . . . . . . . . . . . . . . . . .
Herpesviruses and Parvovirus . . . . . . . . . . . . . . . . . .
Transmissible Spongiform Encephalopathies . . . . . . . . .
Bacterial Contamination . . . . . . . . . . . . . . . . . . . . .
Syphilis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tick-Borne Infections . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.
.
.
.
.
.
.

667
667
675
682
683
686
689
690
695
695

.
.
.
.
.
.
.

xviii

AABB Technical Manual

Other Nonviral Infectious Complications of Blood Transfusion
Reducing the Risk of Infectious Disease Transmission . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . .

.
.
.
.

697
699
703
711

Methods
Methods Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713
1.

General Laboratory Methods . . . . . . . . . . . . . . . . . . . . .
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 1.1. Transportation and Shipment of Dangerous Goods .
Method 1.2. Treatment of Incompletely Clotted Specimens . . . . .
Method 1.3. Solution Preparation—Instructions . . . . . . . . . .
Method 1.4. Serum Dilution . . . . . . . . . . . . . . . . . . . . . .
Method 1.5. Dilution of % Solutions. . . . . . . . . . . . . . . . . .
Method 1.6. Preparation of a 3% Red Cell Suspension . . . . . . .
Method 1.7. Preparation and Use of Phosphate Buffer . . . . . . .
Method 1.8. Reading and Grading Tube Agglutination . . . . . . .

.
.
.
.
.
.
.
.
.
.

715
715
716
722
723
725
726
727
728
728

Red Cell Typing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 2.1. Slide Test for Determination of ABO Type of Red Cells . . . . . . . . .
Method 2.2. Tube Tests for Determination of ABO Group of Red Cells and
Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 2.3. Microplate Test for Determination of ABO Group of Red Cells
and Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 2.4. Confirmation of Weak A or B Subgroup by Adsorption and Elution. .
Method 2.5. Saliva Testing for A, B, H, Lea, and Leb . . . . . . . . . . . . . . . . . . .
Method 2.6. Slide Test for Determination of Rh Type . . . . . . . . . . . . . . . . .
Method 2.7. Tube Test for Determination of Rh Type . . . . . . . . . . . . . . . . .
Method 2.8. Microplate Test for Determination of Rh Type . . . . . . . . . . . . . .
Method 2.9. Test for Weak D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 2.10. Preparation and Use of Lectins . . . . . . . . . . . . . . . . . . . . . .
Method 2.11. Use of Sulfhydryl Reagents to Disperse Autoagglutination . . . . . .
Method 2.12. Gentle Heat Elution for Testing Red Cells with a Positive DAT . . . .
Method 2.13. Dissociation of IgG by Chloroquine for Red Cell Antigen
Testing of Red Cells with a Positive DAT . . . . . . . . . . . . . . . . . . . . . . . .
Method 2.14. Acid Glycine/EDTA Method to Remove Antibodies from
Red Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 2.15. Separation of Transfused from Autologous Red Cells by
Simple Centrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 2.16. Separation of Transfused Red Cells from Autologous
Red Cells in Patients with Hemoglobin S Disease . . . . . . . . . . . . . . . . . .

731
731

.
.
.
.
.
.
.
.
.
.

732
733
735
736
739
740
741
741
743
744
745
746
747
748
749

Contents

Antibody Detection, Antibody Identification, and Serologic Compatibility
Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 3.1. Immediate-Spin Compatibility Testing to Demonstrate
ABO Incompatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 3.2. Indirect Antiglobulin Test (IAT) for the Detection of Antibodies
to Red Cell Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 3.3. Prewarming Technique . . . . . . . . . . . . . . . . . . . . . . . .
Method 3.4. Saline Replacement to Demonstrate Alloantibody in the
Presence of Rouleaux . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 3.5. Enzyme Techniques . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 3.6. Direct Antiglobulin Test (DAT) . . . . . . . . . . . . . . . . . . . .
Method 3.7. Antibody Titration . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 3.8. Use of Sulfhydryl Reagents to Distinguish IgM from IgG
Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 3.9. Plasma Inhibition to Distinguish Anti-Ch and -Rg from
Other Antibodies with HTLA Characteristics . . . . . . . . . . . . . . . . . .
Method 3.10. Dithiothreitol (DTT) Treatment of Red Cells . . . . . . . . . . .
Method 3.11. Urine Neutralization of Anti-Sda . . . . . . . . . . . . . . . . . . .
Method 3.12. Adsorption Procedure . . . . . . . . . . . . . . . . . . . . . . . .
Method 3.13. Using the American Rare Donor Program . . . . . . . . . . . . .

. . . 751
. . . 751
. . . 752
. . . 754
.
.
.
.

.
.
.
.

755
756
760
761

. . . 764
.
.
.
.
.

.
.
.
.
.

Investigation of a Positive Direct Antiglobulin Test . . . . . . . . . . . . . . . .
Elution Techniques
Method 4.1. Cold-Acid Elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 4.2. Glycine-HCl/EDTA Elution . . . . . . . . . . . . . . . . . . . . . . . .
Method 4.3. Heat Elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 4.4. Lui Freeze-Thaw Elution. . . . . . . . . . . . . . . . . . . . . . . . . .
Method 4.5. Methylene Chloride Elution. . . . . . . . . . . . . . . . . . . . . . . .
Immune Hemolytic Anemia Serum/Plasma Methods
Method 4.6. Cold Autoadsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 4.7. Determining the Specificity of Cold-Reactive Autoagglutinins. . . .
Method 4.8. Cold Agglutinin Titer . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 4.9. Autologous Adsorption of Warm-Reactive Autoantibodies . . . . . .
Method 4.10. Differential Warm Adsorption Using Enzyme- or ZZAP-Treated
Allogeneic Red Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 4.11. One-Cell Sample Enzyme or ZZAP Allogeneic Adsorption . . . . .
Method 4.12. Polyethylene Glycol Adsorption . . . . . . . . . . . . . . . . . . . . .
Method 4.13. The Donath-Landsteiner Test . . . . . . . . . . . . . . . . . . . . . .
Method 4.14. Detection of Antibodies to Penicillin or Cephalosporins by
Testing Drug-Treated Red Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 4.15. Demonstration of Immune-Complex Formation Involving Drugs.
Method 4.16. Ex-Vivo Demonstration of Drug/Anti-Drug Complexes . . . . . . .

xix

.
.
.
.
.

765
766
767
768
769

. 771
.
.
.
.
.

772
772
773
774
775

.
.
.
.

775
776
778
779

.
.
.
.

781
782
783
784

. 786
. 788
. 789

AABB Technical Manual

Hemolytic Disease of the Fetus and Newborn . . . . . . . . . . . . .
Method 5.1. Indicator Cell Rosette Test for Fetomaternal Hemorrhage
Method 5.2. Acid-Elution Stain (Modified Kleihauer-Betke). . . . . . .
Method 5.3. Antibody Titration Studies to Assist in Early Detection of
Hemolytic Disease of the Fetus and Newborn . . . . . . . . . . . . .

. . . . . . . 793
. . . . . . . 793
. . . . . . . 794
. . . . . . . 796

Blood Collection, Storage, and Component Preparation. . . . . . . . . . .
Method 6.1. Copper Sulfate Method for Screening Donors for Anemia . . . .
Method 6.2. Arm Preparation for Blood Collection . . . . . . . . . . . . . . . .
Method 6.3. Phlebotomy and Collection of Samples for Processing and
Compatibility Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 6.4. Preparation of Red Blood Cells . . . . . . . . . . . . . . . . . . . .
Method 6.5. Preparation of Prestorage Red Blood Cells Leukocytes
Reduced . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 6.6. Rejuvenation of Red Blood Cells . . . . . . . . . . . . . . . . . . .
Method 6.7. Red Cell Cryopreservation Using High-Concentration
Glycerol—Meryman Method . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 6.8. Red Cell Cryopreservation Using High-Concentration
Glycerol—Valeri Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 6.9. Checking the Adequacy of Deglycerolization of Red Blood Cells
Method 6.10. Preparation of Fresh Frozen Plasma from Whole Blood . . . . .
Method 6.11. Preparation of Cryoprecipitated AHF from Whole Blood . . . .
Method 6.12. Thawing and Pooling Cryoprecipitated AHF . . . . . . . . . . .
Method 6.13. Preparation of Platelets from Whole Blood . . . . . . . . . . . .
Method 6.14. Preparation of Prestorage Platelets Leukocytes Reduced
from Whole Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 6.15. Removing Plasma from Platelet Concentrates (Volume
Reduction) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method 7.1. Quality Control for Copper Sulfate Solution . . . . . . . . . .
Method 7.2. Standardization and Calibration of Thermometers . . . . . .
Method 7.3. Testing Blood Storage Equipment Alarms . . . . . . . . . . .
Method 7.4. Functional Calibration of Centrifuges for Platelet Separation
Method 7.5. Functional Calibration of a Serologic Centrifuge . . . . . . .
Method 7.6. Performance Testing of Automatic Cell Washers . . . . . . .
Method 7.7. Monitoring Cell Counts of Apheresis Components . . . . . .
Method 7.8. Manual Method for Counting Residual White Cells in
Leukocyte-Reduced Blood and Components . . . . . . . . . . . . . . .

.
.
.
.
.
.
.
.

. . . 799
. . . 799
. . . 800
. . . 801
. . . 804
. . . 805
. . . 806
. . . 807
.
.
.
.
.
.

.
.
.
.
.
.

810
812
813
814
815
815

. . . 817
. . . 817
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

819
819
821
823
826
828
830
832

. . . . . 832

Appendices
Appendix 1. Normal Values in Adults . . . . . . . . . . . . . . . . . . . . . . . . . . . 835
Appendix 2. Selected Normal Values in Children . . . . . . . . . . . . . . . . . . . . 836
Appendix 3. Typical Normal Values in Tests of Hemostasis and Coagulation
(Adults). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 837

Contents

Appendix 4. Coagulation Factor Values in Platelet Concentrates . . . .
Appendix 5. Approximate Normal Values for Red Cell, Plasma, and
Blood Volumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix 6. Blood Group Antigens Assigned to Systems. . . . . . . . .
Appendix 7. Examples of Gene, Antigen, and Phenotype Terms . . . .
Appendix 8. Examples of Correct and Incorrect Terminology . . . . . .
Appendix 9. Distribution of ABO/Rh Phenotypes by Race or Ethnicity
Appendix 10. Suggested Quality Control Performance Intervals . . . .
Appendix 11. Directory of Organizations . . . . . . . . . . . . . . . . . .
Appendix 12. Resources for Safety Information . . . . . . . . . . . . . .

xxi

. . . . . . . 838
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

839
840
844
844
845
846
848
850

Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 853

Chapter 1: Quality Systems

Chapter 1
1

Quality Systems

PRIMARY GOAL OF blood centers
and transfusion services is to promote high standards of quality in
all aspects of production, patient care, and
service. This commitment to quality is reflected in standards of practice set forth by
the AABB.1(p1) A quality system includes the
organizational structure, responsibilities,
policies, processes, procedures, and resources established by the executive management to achieve quality.1(p1) A glossary of
quality terms used in this chapter is included in Appendix 1-1.
The establishment of a formal quality assurance program is required by regulation
under the Centers for Medicare and Medi2
caid Services (CMS) Clinical Laboratory
Improvement Amendments (CLIA) and the
Food and Drug Administration (FDA)3-5 current good manufacturing practice (cGMP).
The FDA regulations in 21 CFR 211.22 require an independent quality control or
quality assurance unit that has responsibil-

ity for the overall quality of the finished
product and authority to control the processes that may affect this product.4 (See
Code of Federal Regulations quality-related
citations in Appendix 1-2.) Professional and
accrediting organizations, such as the
AABB,1 Joint Commission on Accreditation
of Healthcare Organizations (JCAHO),6 College of American Pathologists (CAP),7 and
the Clinical and Laboratory Standards Institute (formerly NCCLS),8 have also established requirements and guidelines to address quality issues. The International
Organization for Standardization (ISO)
quality management standards (ISO 9001)
are generic to any industry and describe
9
the key elements of a quality system. In
addition, the Health Care Criteria for Performance Excellence10 published by the
Baldrige National Quality Program provide
an excellent framework for implementing
quality on an organizational level. The
AABB defines the minimum elements that
1

AABB Technical Manual

must be addressed in a blood bank or
transfusion service quality system in its
Quality System Essentials (QSEs).11 The
AABB QSEs were developed to be compatible with ISO 9001 standards and the FDA
Guideline for Quality Assurance in Blood
Establishments.5 Table 1-1 shows a comparison of the AABB QSEs and ISO 9001:2000
requirements.

Quality Control, Quality
Assurance, and Quality
Management
The purpose of quality control (QC) is to
provide feedback to operational staff
about the state of a process that is in progress. It tells staff whether to continue
(everything is acceptable), or whether to
stop until a problem has been resolved
(something is found to be out of control).
Product QC is performed to determine
whether the product or service meets
specifications. Historically, blood banks
and transfusion services have employed
many QC measures as standard practice
in their operations. Examples include reagent QC, clerical checks, visual inspections, and measurements such as temperature readings on refrigerators and volume
or cell counts performed on finished
blood components.
Quality assurance activities are not tied
to the actual performance of a process.
They include retrospective review and analysis of operational performance data to determine if the overall process is in a state of
control and to detect shifts or trends that
require attention. Quality assurance provides information to process managers regarding levels of performance that can be
used in setting priorities for process improvement. Examples in blood banking in-

clude record reviews, monitoring of quality
indicators, and internal assessments.
Quality management considers interrelated processes in the context of the organization and its relations with customers and
suppliers. It addresses the leadership role of
executive management in creating a commitment to quality throughout the organization, the understanding of suppliers and
customers as partners in quality, the management of human and other resources,
and quality planning. The quality systems
approach described in this chapter encompasses all of these activities. It ensures the
application of quality principles throughout the organization and reflects the changing focus of quality efforts from detection
to prevention.

Quality Concepts
Juran’s Quality Trilogy
Juran’s Quality Trilogy is one example of a
quality management approach. This model
centers around three fundamental processes for the management of quality in
any organization: planning, control, and
improvement.12(p2.5)
The planning process for a new product
or service includes activities to identify requirements, to develop product and process specifications to meet those requirements, and to design the process. During
the planning phase, the facility must perform the following steps:
1.
Establish quality goals for the project.
2.
Identify the customers.
3.
Determine customer needs and expectations.
4.
Develop product and service specifications to meet customer, operational, regulatory, and accreditation
requirements.

Chapter 1: Quality Systems

Table 1-1. Comparison of the AABB Quality System Essentials and the ISO 9001
Categories*
AABB Quality System Essentials

ISO 9001:2000

Organization

4.1
5.1
5.2
5.3
5.4
5.5
5.6

Resources

6.1 Provision of resources
6.2 Human resources

Equipment

6.3 Infrastructure
7.6 Control of monitoring and measuring devices

Supplier and Customer Issues

7.2 Customer-related processes
7.4 Purchasing

Process Control

7.1 Planning of product realization
7.3 Design and development
7.5 Production and service provision

Documents and Records

4.2 Documentation requirements

Deviations, Nonconformances, and
Complications

8.3 Control of nonconforming product

Assessments: Internal and External

8.2 Monitoring and measuring
8.4 Analysis of data

Process Improvement

8.1 General
8.4 Analysis of data
8.5 Improvement

Facilities and Safety

6.3 Infrastructure
6.4 Work environment

General requirements
Management commitment
Customer focus
Quality policy
Planning
Responsibility, authority, and communication
Management review

*This table represents only one way of comparing the two systems.

AABB Technical Manual

Develop operational processes for
production and delivery, including
written procedures and resources requirements.
6.
Develop process controls and validate the process in the operational
setting.
The results of the planning process are
9
referred to as design output.
Once implemented, the control process
provides a feedback loop for operations
that includes the following:
1.
Evaluation of actual performance.
2.
Comparison of performance to goals.
3.
Action to correct any discrepancy
between the two.
It addresses control of inputs, production, and delivery of products and services
to meet specifications. Process controls
should put operational staff in a state of
self-control such that they can recognize
when things are going wrong, and either
make appropriate adjustments to ensure
the quality of the product or stop the process. An important goal in quality management is to establish a set of controls that
ensure process and product quality but that
are not excessive. Controls that do not add
value should be eliminated in order to conserve limited resources and to allow staff to
focus attention on those controls that are
critical to the operation. Statistical tools,
such as process capability measurement
and control charts, allow the facility to evaluate process performance during the planning stage and in operations. These tools
help determine whether a process is stable
(ie, in statistical control) and whether it is
capable of meeting product and service
specifications.12(p22.19)
Quality improvement is intended to attain higher levels of performance, either by
creating new or better features that add
value, or by removing existing deficiencies
in the process, product, or service. Opportunities to improve may be related to defi-

ciencies in the initial planning process; unforeseen factors that are discovered upon
implementation; shifts in customer needs;
or changes in starting materials, environmental factors, and other variables that affect the process. Improvements must be
based on data-driven analysis; an ongoing
program of measurement and assessment
is fundamental to this process.

Process Approach
In its most generic form, a process includes all of the resources and activities
that transform an input into an output.
An understanding of how to manage and
control processes in the blood bank or
transfusion service is based on the simple
equation:
INPUT

à PROCESS à OUTPUT

For example, a key process for donor centers is donor selection. The “input” includes 1) the individual who presents for
donation and 2) all of the resources required for the donor health screening.
Through a series of activities including
verification of eligibility (based on results
of prior donations, mini-physical, and
health history questionnaire), an individual is deemed an “eligible donor.” The
“output” is either an eligible donor who
can continue to the next process (blood
collection) or an ineligible donor who is
deferred. When the selection process results in a deferred donor, the resources
(inputs) associated with that process are
wasted and contribute to the cost of quality. One way that donor centers attempt to
minimize this cost is to educate potential
donors before the health screening so that
those who are not eligible do not enter the
selection process.

Chapter 1: Quality Systems

Strategies for managing a process should
consider all of its components, including its
interrelated activities, inputs, outputs, and
resources. Supplier qualification, formal
agreements, supply verification, and inventory control are strategies for ensuring that
the inputs to a process meet specifications.
Personnel training and competency assessment, equipment maintenance and control, management of documents and records, and implementation of appropriate
in-process controls provide assurance that
the process will operate as intended. Endproduct testing and inspection, customer
feedback, and outcome measurement provide information to help evaluate the quality of the product and to improve the process
as a whole. These output measurements and
quality indicators are used to evaluate the
effectiveness of the process and process
controls.
In order to manage a system of processes effectively, the facility must understand how its processes interact and any
cause-and-effect relationships between
them. In the donor selection example, the
consequences of accepting a donor who is
not eligible reach into almost every other
process in the facility. One example would
be a donor with a history of high-risk behavior that is not identified during the selection process. The donated product may
test positive for one of the viral marker assays, triggering follow-up testing, look-back
investigations, and donor deferral and notification procedures. Components must be
quarantined and their discard documented.
Staff involved in collecting and processing
the product are at risk of exposure to infectious agents. Part of quality planning is to
identify those relationships so that quick
and appropriate corrective action can be
taken if process controls fail. It is important
to remember that operational processes include not only product manufacture or service creation, but also the delivery of a

product or service. Delivery generally involves interaction with the customer. The
quality of this transaction is critical to
customer satisfaction and should not be
overlooked in the design and ongoing
assessment of the quality system.

Service vs Production
Quality principles apply equally to a
broad spectrum of activities, from those
involved in processing and production, to
those involving the interactions between
individuals in the delivery of a service.
However, different strategies may be appropriate when there are differing expectations related to customer satisfaction.
Although the emphasis in a production
process is to minimize variation in order
to create a product that consistently meets
specifications, service processes require a
certain degree of flexibility to address customer needs and circumstances at the
time of the transaction. In production,
personnel need to know how to maintain
uniformity in the day-to-day operation. In
service, personnel need to be able to
adapt the service in a way that meets customer expectations but does not compromise quality. To do this, personnel must
have sufficient knowledge and understanding of interrelated processes to use
independent judgment appropriately, or
they must have ready access to higher
level decision-makers. When designing
quality systems for production processes,
it is useful to think of the process as the
driver, with people providing the oversight and support needed to keep it running smoothly and effectively. In service,
people are the focus; the underlying process provides a foundation that enables
staff to deliver safe and effective services
that meet the needs of the customers in
almost any situation.

AABB Technical Manual

Quality Management as an Evolving
Science
It is important to remember that quality
management is an evolving science. The
principles and tools in use today will
change as research provides new knowledge of organizational behavior, as technology provides new solutions, and as the
field of transfusion medicine presents
new challenges. Periodic assessments of the
quality management systems will help
identify practices that are no longer effective or that could be improved through
the use of new technology or new tools.

Practical Application of
Quality Principles
The remainder of this chapter discusses
the elements of a quality system and practical application of quality principles to the
blood bank and transfusion service environment. These basic elements include:
■
Organizational management
■
Human resources
■
Customer and supplier relations
■
Equipment management
■
Process management
■
Documents and records
■
Deviations and nonconforming products and services
■
Monitoring and assessment
■
Process improvement
■
Work environment

Organizational Management
The facility should be organized in a manner that promotes effective implementation and management of its quality system.
The structure of the organization must be
documented and the responsibilities for
the provision of blood, components, products, and services must be clearly defined. These should include a description

of the relationships and avenues of communication between organizational units
and those responsible for key quality
functions. Each facility may define its
structure in any format that suits its operations. Organizational trees or charts that
show the structure and relationships are
helpful.
The facility must define in writing the
authority and responsibilities of management to establish and maintain the quality
system. These include oversight of operations and regulatory and accreditation
compliance as well as periodic review and
assessment of quality system effectiveness.
Executive management support for quality
system goals, objectives, and policies is
critical to the success of the program. Management must participate in the review and
approval of quality and technical policies,
processes, and procedures.
The individual designated to oversee the
facility’s quality functions must report directly to management. This person has the
responsibility to coordinate, monitor, and
facilitate quality system activities and has
the authority to recommend and initiate
5
corrective action when appropriate. The
designated individual need not perform all
of the quality functions personally. Ideally,
this person should be independent of the
operational functions of the donor center
or transfusion service. In small facilities,
however, this may not always be possible.
Depending on the size and scope of the organization, the designated oversight person
may work in a department (eg, transfusion
service), may have responsibilities covering
several areas (eg, laboratory-wide), may
have a staff of workers (eg, quality unit), or
may be part of an organization-wide unit
(eg, hospital quality management). Individuals with dual quality and operational responsibilities should not provide quality
oversight for operational work they have
performed (21 CFR 211.194).

Chapter 1: Quality Systems

Quality oversight functions may include
the following5:
Review and approval of standard op■
erating procedures (SOPs) and training plans.
Review and approval of validation
■
plans and results.
Review and approval of document
■
control and record-keeping systems.
Audit of operational functions.
■
Development of criteria for evaluat■
ing systems.
Review and approval of suppliers.
■
Review and approval of product
■
specifications, ie, requirements to be
met by the products used in the manufacturing, distribution, or transfusion
of blood and components.
Review of reports of adverse reactions,
■
deviations in the manufacturing process, nonconforming products and services, and customer complaints.
■
Participation in decisions to determine blood and component suitability for use, distribution, or recall.
■
Review and approval of corrective
action plans.
■
Surveillance of problems (eg, error
reports, inspection deficiencies, customer complaints) and the effectiveness of corrective actions implemented
to solve these problems.
■
Use of information resources to
identify trends and potential problems before a situation worsens and
products or patients are affected.
■
Preparation of periodic (as specified
by the organization) reports of quality issues, trends, findings, and corrective and preventive actions.
Quality oversight functions may be
shared among existing staff, departments,
and facilities, or, in some instances, may be
contracted to an outside firm. The goal is to
provide as much of an independent evaluation of the facility’s quality activities as pos-

sible. Policies, processes, and procedures
must exist to define the roles and responsibilities of all individuals in the development
and maintenance of these quality goals.
Quality system policies and processes
should be applicable across the entire facility. A blood bank or transfusion service
need not develop its own quality policies if
it is part of a larger entity whose quality
management system addresses all of the
minimum requirements. The quality system must address all matters related to
compliance with federal, state, and local
regulations and accreditation standards applicable to the organization.

Human Resources
This element of the quality system is aimed at management of personnel, including selection, orientation, training, competency assessment, and staffing.

Selection
Each blood bank, transfusion service, or
donor center must have a process to provide adequate numbers of qualified personnel to perform, verify, and manage all
activities within the facility.1(p3),3 Qualification requirements are determined based
on job responsibilities. The selection process should consider the applicant’s qualifications for a particular position as
determined by education, training, experience, certifications, and/or licensure. For
laboratory testing staff, the standards for
personnel qualifications must be compatible with the regulatory requirements es2
tablished under CLIA. Job descriptions are
required for all personnel involved in processes and procedures that affect the
quality of blood, components, tissues,
and services. Effective job descriptions
clearly define the qualifications, responsibilities, and reporting relationships of the
position.

AABB Technical Manual

Orientation, Training, and Competency
Assessment
Once hired, employees must be oriented
to their position and to the organization’s
policies and procedures. The orientation
program should include facility-specific
requirements and an introduction to policies that address issues such as safety,
quality, computers, security, and confidentiality. The job-related portion of the
orientation program covers the operational issues specific to the work area.
Training must be provided for each procedure for which employees have responsibility. The ultimate result of the orientation and training program is to deem new
employees competent to work independently in performing the duties and responsibilities defined in their job descriptions. Time frames should be established
to accomplish this goal. Before the introduction of a new test or service, existing
personnel must be trained to perform
their newly assigned duties and must be
deemed competent. During orientation
and training, the employee should be
given the opportunity to ask questions
and seek additional help or clarification.
All aspects of the training must be documented and the facility trainer or designated facility management representative
and the employee should mutually agree
upon the determination of competence.
FDA cGMP training is required for staff
involved in the manufacture of blood and
blood components.4 It should provide staff
with an understanding of the regulatory basis for the facility’s policies and procedures
as well as train them in facility-specific application of the cGMP requirements as described in their own written operating procedures. This training must be provided at
periodic intervals to ensure that staff remain familiar with regulatory requirements.

To ensure that skills are maintained, the
facility must have regularly scheduled competence evaluations of all staff whose activities affect the quality of blood, components, tissues, or services.2,6 Depending
upon the nature of the job duties, such assessments may include: written evaluations; direct observation of activities; review
of work records or reports, computer records, and QC records; testing of unknown
samples; and evaluation of the employee’s
problem-solving skills.5
A formal competency plan that includes
a schedule of assessments, defined minimum
acceptable performance, and remedial
measures is one way to ensure appropriate
and consistent competence assessments.
Assessments need not be targeted at each
individual test or procedure performed by
the employee; instead, they can be grouped
together to assess like techniques or methods. Written tests can be used effectively to
evaluate problem-solving skills and to rapidly cover many topics by asking one or
more questions for each area to be assessed. For testing personnel, CMS requires
that employees who perform testing be assessed semiannually during the first year
and annually thereafter.2
The quality oversight personnel should
assist in the development, review, and approval of training programs, including the
criteria for retraining.5 Quality oversight
personnel also monitor the effectiveness of
the training program and competence evaluations and make recommendations for
changes as needed. In addition, JCAHO requires the analysis of aggregate competency assessment data for the purpose of
identifying staff learning needs.6

Staffing
Management should have a staffing plan
that describes the number and qualifications of personnel needed to perform the

Chapter 1: Quality Systems

functions of the facility safely and effectively. JCAHO requires that hospitals evaluate staffing effectiveness by looking at
human resource indicators (eg, overtime,
staff injuries, staff satisfaction) in conjunction with operational performance
indicators (eg, adverse events, patient’s
6
complaints). The results of this evaluation should feed into the facility’s human
resource planning process along with
projections based on new or changing
operational needs.

Customer and Supplier Relations
Materials, supplies, and services used as
inputs to a process are considered “critical” if they affect the quality of products
and services being produced. Examples of
critical supplies are blood components,
blood bags, test kits, and reagents. Examples of critical services are infectious
disease testing, blood component irradiation, transportation, equipment calibration,
and preventive maintenance services. The
suppliers of these materials and services
may be internal (eg, other departments
within the same organization) or external
(outside vendors). Supplies and services used
in the collection, testing, processing, preservation, storage, distribution, transport,
and administration of blood, components,
and tissue that have the potential to affect
quality should be qualified before use and
obtained from suppliers who can meet the
facility’s requirements.1(pp8,9) The quality
system must include a process to evaluate
the suppliers’ abilities to meet these requirements. Three important elements
are supplier qualification; agreements;
and receipt, inspection, and testing of incoming supplies.

Supplier Qualification
Critical supplies and services must be
qualified on the basis of defined require-

ments. Similarly, the supplier should be
qualified to ensure a reliable source of
materials. The facility should clearly define requirements or expectations for the
suppliers and share this information with
staff and the supplier. The ability of suppliers to consistently meet specifications
for a supply or service should be evaluated along with performance relative to
availability, delivery, and support. Examples of factors that could be considered to
qualify suppliers are:
Licensure, certification, or accredita■
tion.
Supply or product requirements.
■
Review of supplier-relevant quality
■
documents.
Results of audits or inspections.
■
Review of quality summary reports.
■
Review of customer complaints.
■
Review of experience with supplier.
■
■
Cost of materials or services.
■
Delivery arrangements.
■
Financial security, market position,
and customer satisfaction.
■
Support after the sale.
A list of approved suppliers should be
maintained, including both primary suppliers and suitable alternatives for contingency
planning. Critical supplies and services
should be purchased only from those suppliers who have been qualified. Once qualified, periodic evaluation of the supplier’s
performance helps to ensure its continued
ability to meet requirements. Tracking the
supplier’s ability to meet expectations gives
the facility valuable information about the
stability of the supplier’s processes and its
commitment to quality. Documented failures of supplies or suppliers to meet defined requirements should result in immediate action by the facility. These actions
include notifying the supplier, quality oversight personnel, and management with
contracting authority, if applicable. Supplies may need to be replaced or quaran-

AABB Technical Manual

tined until all quality issues have been resolved.

Agreements
Contracts and agreements define expectations and reflect concurrence of the parties involved.1(p8) Periodic review of agreements ensures that expectations of all
parties continue to be met. Changes must
be mutually agreed upon and incorporated as needed.
Blood banks and transfusion services
should maintain written contracts or agreements with outside suppliers of critical materials and services such as blood components, irradiation, compatibility testing, or
infectious disease marker testing. The outside supplier may be another department
within the same facility that is managed independently, or it may be another facility
(eg, contract manufacturer). The contracting facility assumes responsibility for the
manufacture of the product; ensuring the
safety, purity, and potency of the product;
and ensuring that the contract manufacturer complies with all applicable product
standards and regulations. Both the contracting facility and the contractor are
legally responsible for the work performed
by the contractor.
It is important for the blood bank or
transfusion service to participate in the
evaluation and selection of suppliers. They
should review contracts and agreements to
ensure that all aspects of critical materials
and services are addressed. Examples of issues that could be addressed in an agreement or a contract include: responsibility
for a product or blood sample during shipment; the responsibility of the supplier to
promptly notify the facility when changes
that could affect the safety of blood, components, or patients have been made to the
materials or services; and the responsibility
of the supplier to notify the facility when

information that a product may not be
considered safe is discovered, such as
during look-back procedures.

Receipt, Inspection, and Testing of
Incoming Supplies
Before acceptance and use, critical materials, such as reagents and blood components, must be inspected and tested (if
necessary) to ensure that they meet specifications for their intended use.1(pp8,9),4 It is
essential that supplies used in the collection, processing, preservation, testing,
storage, distribution, transport, and administration of blood and components
also meet FDA requirements.
The facility must define acceptance criteria for critical supplies (21 CFR 210.3) and
develop procedures to control materials
that do not meet specifications to prevent
their inadvertent use. Corrective action
may include returning the material to the
vendor or destroying it. Receipt and inspection records provide the facility with a
means to trace materials that have been
used in a particular process and also provide information for ongoing supplier
qualification.

Equipment Management
Equipment that must operate within defined specifications to ensure the quality
of blood, components, tissues, and services is referred to as “critical” equipment
1(p4)
Critical equipin the quality system.
ment may include instruments, measuring devices, and computer systems (hardware and software). Activities designed to
ensure that equipment performs as intended include qualification, calibration,
maintenance, and monitoring. Calibration,
functional and safety checks, and preventive maintenance must be scheduled and
performed according to the manufacturer’s recommendations and regulatory

Chapter 1: Quality Systems

requirements of the FDA and CMS. Written procedures for the use and control of
equipment must comply with the manufacturer’s recommendations unless an alternative method has been validated by
the facility and approved by the appropriate regulatory and accrediting agencies.
When selecting new equipment, it is important to consider not only the performance of equipment as it will be used in
the facility, but also any supplier issues regarding ongoing service and support. There
should be a written plan for installation,
operational, and performance qualification. After installation, there must be documentation of any problems and the follow-up actions taken. Recalibration and
requalification may be necessary if repairs
are made that affect the critical operating
functions of the equipment. Recalibration
and requalification should also be considered when existing equipment is relocated.
The facility must develop a mechanism
to uniquely identify and track all critical
equipment, including equipment software
versions, if applicable. The unique identifier may be the manufacturer’s serial number or a facility’s unique identification
number. Maintaining a list of all critical
equipment helps in the control function of
scheduling and performing functional and
safety checks, calibrations, preventive
maintenance, and repair. The equipment
listing can be used to ensure that all appropriate actions have been performed and recorded. Evaluation and analysis of equipment calibration, maintenance, and repair
data will assist the facility in assessing the
suitability of the equipment. They will also
allow for better control in managing defective equipment and in identifying equipment that may need replacement. When
equipment is found to be operating outside
acceptable parameters, the potential effects
on the quality of products or test results
must be evaluated and documented.

Process Management
Written, approved policies, processes, and
procedures must exist for all critical functions performed in the facility and must
be carried out under controlled conditions. Each facility should have a systematic approach for identifying, planning, and
implementing policies, processes, and procedures that affect the quality of blood,
components, tissues, and services. These
documents must be reviewed by management personnel with direct authority over
the process and by quality oversight personnel before implementation. Changes
must be documented, validated, reviewed,
and approved. Additional information on
policies, processes, and procedures can
be found in the Documents and Records
section.
Once a process has been implemented,
the facility must have a mechanism to ensure that procedures are performed as
defined and that critical equipment, reagents, and supplies are used in conformance with manufacturers’ written instructions and facility requirements. Table 1-2
lists elements that constitute sound process
control. A facility using reagents, supplies,
or critical equipment in a manner that is
different from the manufacturer’s directions must have validated such use and
may be required to request FDA approval to
operate at variance to 21 CFR 606.65(e) if
the activity is covered under regulations for
blood and blood components (21 CFR
640.120). If a facility believes that changes
to the manufacturer’s directions would be
appropriate, it should encourage the manufacturer to make such changes in the labeling (ie, package insert or user manual).

Process Validation
Validation is used to demonstrate that a
process is capable of achieving planned
results.9 It is critical to validate processes

AABB Technical Manual

Table 1-2. Elements of a Sound Process Control System
■

Systematic approach to developing policies, processes, or procedures and controlling changes.

■

Validation of policies, processes, and procedures.

■

Development and use of standard operating procedures.

■

Equipment qualification processes.

■

Staff training and competence assessment.

■

Acceptance testing for new or revised computer software involved in blood bank procedures.

■

Establishment of quality control, calibration, and preventive maintenance schedules.

■

Monitoring of quality control, calibration, preventive maintenance, and repairs.

■

Monitoring and control of production processes.

■

Processes to determine that supplier qualifications and product specifications are maintained.

■

Participation in proficiency testing appropriate for each testing system in place.

■

Processes to control nonconforming materials, blood, components, and products.

in situations where it is not feasible to
measure or inspect each finished product
or service in order to fully verify conformance with specifications. However, even
when effective end-product testing can be
achieved, it is advisable to validate important processes to generate information that
can be used to optimize performance.
Prospective validation is used for new or
revised processes. Retrospective validation
may be used for processes that are already
in operation but were not adequately validated before implementation. Concurrent
validation is used when required data
cannot be obtained without performance
of a “live” process. If concurrent validation is used, data are reviewed at predefined intervals before final approval for full
implementation occurs. Modifications to
a validated process may warrant revalidation, depending on the nature and extent
of the change. It is up to the facility to determine the need for revalidation based
on its understanding of how the proposed
changes may affect the process.

Validation Plan
Validation must be planned if it is to be
effective. Development of a validation
plan is best accomplished after obtaining
an adequate understanding of the system,
or framework, within which the process
will occur. Many facilities develop a template for the written validation plan to
ensure that all aspects are adequately addressed. Although no single format for a
validation plan is required, the following
elements are common to most:
■
System description
■
Purpose/objectives
■
Risk assessment
■
Responsibilities
■
Validation procedures
■
Acceptance criteria
■
Approval signatures
■
Supporting documentation
The validation plan must be reviewed
and approved by quality oversight personnel. Staff responsible for carrying out the
validation activities must be trained in the
process before the plan is implemented. The

Chapter 1: Quality Systems

results and conclusions of these activities
may be appended to the approved validation plan or recorded in a separate document. This documentation typically contains
the following elements:
Expected and observed results
■
Interpretation of results as accept■
able or unacceptable
Corrective action and resolution of
■
unexpected results
Conclusions and limitations
■
Approval signatures
■
Supporting documentation
■
Implementation time line
■
When a validation process does not produce the expected outcome, its data and
corrective actions must be documented as
well. The responsible quality oversight personnel should have final review and approval of the validation plan, results, and
corrective actions and determine whether
new or modified processes and equipment
may be implemented, or implemented with
specified limitations.

Equipment Validation
Validation of new equipment used in a
process should include installation qualification, operational qualification, and
performance qualification.13
■
Installation qualification demonstrates
that the instrument is properly installed in environmental conditions
that meet the manufacturer’s specifications.
■
Operational qualification demonstrates that the installed equipment
operates as intended. It focuses on
the capability of the equipment to
operate within the established limits
and specifications supplied by the
manufacturer.
■
Performance qualification demonstrates that the equipment performs
as expected for its intended use in

the processes established by the
facility and that the output meets
specifications. It evaluates the adequacy of equipment for use in a specific process that employs the
facility’s own personnel, procedures,
and supplies in a normal working
environment.

Computer System Validation
The FDA considers computerized systems
to include: “hardware, software, peripheral devices, personnel, and documentation.”14 End-user validation of computer
systems and the interfaces between
systems should be conducted in the environment where it will be used. Testing
performed by the vendor or supplier of
computer software is not a substitute for
computer validation at the facility. Enduser acceptance testing may repeat some
of the validation performed by the developer, such as load or stress testing and
verification of security, safety, and control
features, in order to evaluate performance
under actual operating conditions. In addition, the end user must evaluate the
ability of personnel to use the computer
system as intended within the context of
actual work processes. Staff must be able
to successfully navigate the hardware and
software interface and respond appropriately to messages, warnings, and other
functions. Depending upon the nature of
the computer functionality, changes to the
computer system may result in changes to
how a process is performed. If this occurs,
process revalidation must also be performed. As with process validation, quality oversight personnel should review and
approve validation plans, results, and corrective actions and determine whether
implementation may proceed with or
without limitations. Facilities that develop their own software should refer to

AABB Technical Manual

FDA guidance regarding general principles of software validation for additional
information.15

Quality Control
QC testing is performed to ensure the
proper functioning of materials, equipment, and methods during operations.
QC performance expectations and acceptable ranges must be defined and
readily available to staff so that they will
recognize unacceptable results and trends
and respond appropriately. The frequency
for QC testing is determined by the facility in accordance with the applicable
CMS, FDA, AABB, state, and manufacturer’s requirements. QC results must be
documented concurrently with performance.3 Records of QC testing must include
identification of personnel, identification
of reagent (including lot number, expiration dates, etc), identification of equipment, testing date and time (when applicable), results, interpretation, and reviews.
Unacceptable QC results must be investigated and corrective action implemented,
if indicated, before repeating the QC procedure or continuing the operational process. Specific examples of suggested quality control intervals for blood banks and
transfusion services are included in Appendix 10 at the end of the book, and information regarding methods of quality
control are found in the methods section
devoted to QC.

Documents and Records
Documentation provides a framework for
understanding and communication throughout the organization. Documents describe
the way that processes are intended to
work, how they interact, where they must
be controlled, what their requirements
are, and how to implement them. Records
provide evidence that the process was

performed as intended and information
needed to assess the quality of products
and services. Together, documents and
records are used by quality oversight personnel to evaluate the effectiveness of a facility’s policies, processes, and procedures.
An example of quality system documentation is provided in ISO 9001 and includes
the following items9:
1.
The quality policy and objectives.
2.
A description of the interactions between processes.
3.
Documented procedures for the control
of documents, control of records,
control of a nonconforming product,
corrective action, preventive action,
and internal quality audits.
4.
Records related to the quality system, operational performance, and
product/service conformance.
5.
All other documents needed by the
organization to ensure the effective
planning, operation, and control of
its processes.
Written policies, process descriptions, procedures, work instructions, labels, forms,
and records are all part of the facility’s documentation system. They may be paper-based or electronic. Documents provide a description or instructions of what is
supposed to happen; records provide evidence of what did happen. A document
management system provides assurance
that documents are comprehensive, current, and available, and that records are
accurate and complete. A well-structured
document management system links policies, process descriptions, procedures, forms,
and records together in an organized and
workable system.

Documents
Documents should be developed in a format that conveys information clearly and
provides staff with instructions and forms.

Chapter 1: Quality Systems

The Clinical and Laboratory Standards Institute offers guidance regarding general
levels of documentation8 as well as detailed instructions on how to write proce16
dures. General types of documentation
are described below.
Policies. Policies communicate the highest level goals, objectives, and intent of the
organization. The rest of the organization’s
documentation will interpret and provide
instruction regarding implementation of
these policies.
Processes. Process documents describe
a sequence of actions and identify responsibilities, decision points, requirements,
and acceptance criteria. Table 1-3 lists examples of process documents that might be
in place to support a quality system. Process diagrams or flowcharts are often used
for this level of documentation. It is helpful
to show process control points on the diagram as well as flow of information and handoffs between departments or work groups.
Procedures and Work Instructions.
These documents provide step-by-step directions on how to perform job tasks and
procedures. Procedures and work instructions should include enough detail to perform the task correctly, but not so much as
to make them difficult to read. The use of
standardized formats will help staff know
where to find specific elements and facilitates implementation and control.1(p66) Procedures may also be incorporated by reference, such as those from a manufacturer’s
manual. Relevant procedures must be
available to staff in each area where the cor1(p66),3
responding job tasks are performed.
Forms. Forms provide a template for
capturing data either on paper or electronically. These documents specify the data requirements called for in SOPs and processes. Forms should be carefully designed
for ease of use, to minimize the likelihood
of errors, and to facilitate retrieval of information. They should include instructions

for use when it is not immediately evident
what information should be recorded or
how to record it. For quantitative data, the
form should indicate units of measure.
Computer data entry and review screens
are a type of form. Forms must be designed
to effectively capture outcomes and support process traceability.
Labels. Blood component labels are a
critical material subject to the requirements
of a document management system. Many
facilities maintain a master set of labels that
can be used as reference to verify that only
current approved stock is in use. New label
stock must be verified as accurate before it
is put into inventory; comparison against a
master label provides a mechanism for accomplishing this. Change control procedures
must be established for the use of on-demand
label printers to prevent nonconforming
modification of label format or content.
Each facility must have a defined process
for developing and maintaining documents. It should include: basic elements required for document formats; procedures
for review and approval of new or revised
documents; a method for keeping documents current; control of document distribution; and a process for archiving, protecting, and retrieving obsolete documents.
Training must be provided to staff responsible for the content of new or revised documents. Document management systems include established processes to:
1.
Verify the adequacy of the document
before approval and issue.
2.
Periodically review, modify, and reapprove as needed to keep documents
current.
3.
Identify changes and revision status.
4.
Ensure that documents are legible,
identifiable, and readily available.
5.
Prevent unintended use of outdated
or obsolete documents.
6.
Protect documents from unintended
damage or destruction.

AABB Technical Manual

Table 1-3. Examples of Quality System Process Documents
Organization

■

Management review process

Resources

■

Personnel hiring process
Training process
Competence assessment process

■
■

■

Equipment

■

Supplier and Customer Issues

■
■
■
■
■

■
■
■

Process Control

■
■

Equipment management process
Installation qualification process
Supplier qualification process
Contract review process
Process for qualification of critical materials
Ordering and inventory control of critical materials
Receipt, inspection, and testing of incoming critical
materials
Change control process
Validation process
Process for acceptance testing of computer software
Process for handling proficiency testing
Process for handling, storage, distribution, and transport
of blood components

Documents and Records

■
■
■

Process for creation and approval of documents
Document management process
Records management process

Deviations, Nonconformances, and
Complications

■

Event management process
Process for handling customer complaints
Process for notification of external sources

Assessments

■

■
■

Internal audit process
Process for quality monitoring
Process for handling external assessments

Process Improvement

■

Corrective and preventive action processes

Facilities and Safety

■
■

Process for handling disasters
Employee safety management process

Chapter 1: Quality Systems

External documents that are incorporated by reference become part of the document management system and must be
identified and controlled. The facility must
have a mechanism to detect changes to external documents in its system, such as a
manufacturer’s package inserts or user
manuals, so that corresponding changes to
procedures and forms can be made. When
new or revised policies, process descriptions, procedures, or forms are added to or
replaced in the facility’s manual, the documents must be marked with the effective
date. One copy of retired documents must
be retained as defined by existing and applicable standards and regulations.
A master list of all current policies, process descriptions, procedures, forms, and
labels is useful for maintaining document
control. It should include the document title, the individual or work group responsible
for maintaining it, the revision date and
number (if one is assigned), and the area
where it is used. It should also identify the
number and location of controlled copies in
circulation. Copies of documents that will be
used in the workplace should be identified
and controlled to ensure that none are
overlooked when changes are implemented.

Records
Records provide evidence that critical
steps in a procedure have been performed
appropriately and that products and services conform to specified requirements.
Review of records is an important tool to
help evaluate the effectiveness of the quality system. Records must be created concurrently with the performance of each
significant step and clearly indicate the
identity of individuals who performed
3
each step and when it occurred. The
quality system must include a process for
managing records that addresses the following items:

■

Creation and identification of records
Protection from accidental or unauthorized modification or destruction
Verification of completeness, accu■
racy, and legibility
Storage and retrieval
■
Creation of copies or backups
■
Retention periods
■
Confidentiality
■
Record-keeping systems must allow for
ready retrieval within time frames established by the facility and must permit traceability of blood components as required by
federal regulations.3 Specific requirements
for records to be maintained by blood
banks and transfusion services are included
in the AABB Standards for Blood Banks and
Transfusion Services1(pp69-80) and in 21 CFR
606.160.
When forms are used for capturing data
or recording steps or test results, the forms
become records. Data must be recorded in
a format that is clear and consistent. The
facility must define a process and time
frames for the record review to ensure accuracy, completeness, and appropriate follow-up. It must determine how reports and
records are to be archived and define their
retention period. When copies of records
are retained, the facility must verify that the
copy contains complete, legible, and accessible content of the original record before
the original is destroyed.
If records are maintained electronically,
adequate backup must exist in case of system failure. Electronic records must be
readable for the entire length of their retention period. Obsolete computer software,
necessary to reconstruct or trace records,
must be archived appropriately. If the
equipment or software used to access archived data cannot be maintained, the records should be converted to another format or copied to another medium to
permit continued access. Converted data
must be verified against the original to en■

AABB Technical Manual

sure completeness and accuracy. Electronic
media such as magnetic tapes, optical
disks, and online computer data storage are
widely used for archiving documents. Records kept in this manner must meet FDA
requirements for electronic record-keeping.17 Microfilm or microfiche may be used
to archive written records. The medium selected should be appropriate for the retention requirements.
Privacy of patient and donor information must be addressed in the quality
system with established policies and procedures to maintain the security and confidentiality of records. Computer systems must
be designed with security features to prevent unauthorized access and use. This system may include levels of security defined
by job responsibility and administered by
the use of security codes and passwords.
Each facility should have a policy for altering or correcting records. A common
practice is to indicate the date, the change,
the identity of the person making the
change, and evidence of review by a responsible person. The original recording
must not be obliterated in written records;
it may be crossed out with a single line, but
it should remain legible. Electronic records
must permit tracking of both original and
corrected data and include the date and
user identification of the person making
the change. There should be a process for
controlling changes.1(p10) A method for referencing changes to records, linked to the
original records, and a system for reviewing
changes for completeness and accuracy are
essential. Audit trails for changed data in
computerized systems are required by the
17
FDA.
The following are issues that might be
considered when planning record storage:
■
Storage of records in a manner that
protects them from damage and from
accidental or unauthorized destruction or modification.

■

Degree of accessibility of records in
proportion to frequency of their use.
Method and location of record stor■
age related to the volume of records
and the amount of available storage
space.
Availability of properly functioning
■
equipment, computer hardware, and
software to view archived records.
Documentation that microfiched re■
cords legitimately replace original
documents that may be stored elsewhere or destroyed.
Retention of original color-coded re■
cords when only black-and-white reproductions are available.
Considerations for electronic records include:
A method of verifying the accuracy
■
of data entry.
Prevention of unintended deletion of
■
data or access by unauthorized persons.
■
Adequate protection against inadvertent data loss (eg, when a storage
device is full).
■
Validated safeguards to ensure that a
record can be edited by only one
person at a time.
■
Security and access of confidential
data.
A backup disk or tape should be maintained in the event of unexpected loss of
information from the storage medium.
Backup or archived computer records and
databases must be stored off-site.1(p68) The
storage facility should be secure and maintain appropriate conditions, in accordance
with the manufacturer’s recommendations
and instructions. An archival copy of the
computer operating system and applications software should be stored in the same
manner.
The facility should develop and maintain
alternative systems to ensure information
access if computerized data are not avail-

Chapter 1: Quality Systems

able. The backup and recovery procedures
for computer downtime must be defined,
with validation documentation to show
that the backup system works properly. The
associated processes must be checked periodically to ensure that the backup system
remains effective. Special consideration
should be given to staff competence and
readiness to use the backup system.
To link relevant personnel to recorded
data, the facility must maintain a record of
names, inclusive dates of employment, signatures, and identifying initials or identification codes of personnel authorized to
create, sign, initial, or review reports and
records. Magnetically coded employee
badges and other computer-related identifying methods are generally accepted in lieu
of written signatures provided they meet electronic record-keeping requirements.

Deviations and Nonconforming Products
or Services
The quality system must include a process
for detecting, investigating, and responding
to events that result in deviations from accepted policies, processes, and procedures
or in failures to meet requirements, as defined by the donor center or transfusion
service, AABB standards, or applicable regulations.1(p81),3 This includes the discovery
of nonconforming products and services
as well as adverse reactions to blood donation and transfusion.1(pp81-85),2 The facility
should define how to:
■
Document and classify occurrences.
■
Determine the effect, if any, on the
quality of products or services.
■
Evaluate the impact on interrelated
activities.
■
Implement corrective action, including notification and recall, as appropriate.

■

Analyze the event to understand root
causes.
Implement preventive actions as ap■
propriate on the basis of root-cause
analysis.
Report to external agencies, when
■
required.
Facility personnel should be trained to
recognize and report such occurrences. Depending upon the severity of the event and
risk to patients, donors, and products, as
well as the likelihood of recurrence, investigation into contributing factors and underlying cause(s) may be warranted. The
cGMP regulations require an investigation
and documentation of the results if a specific event could adversely affect patient
safety or the safety, purity, potency, or efficacy of blood or components.2,3 Tools and
approaches for performing root-cause
analysis and implementing corrective action are discussed in the section addressing
process improvement. A summary of the
event, investigation, and any follow-up
must be documented. Table 1-4 outlines
suggested components of an internal event
report.
Fatalities related to blood collection or
transfusion must be reported as soon as
possible to the FDA Center for Biologics
Evaluation and Research (CBER) [21 CFR
606.170(b)]. Instructions for reporting to
CBER are available in published guidance20
and at http://www.fda.gov/cber/transfusion.htm. A written follow-up report is submitted within 7 days of the fatality and
should include a description of any new procedures implemented to avoid recurrence.
AABB Association Bulletin #04-06 provides
additional information, including a form to
21
be used for reporting donor fatalities.
Regardless of their licensure and registration status with the FDA, all donor centers, blood banks, and transfusion services
must promptly report biologic product deviations (previously known as errors and

AABB Technical Manual

Table 1-4. Components of an Internal Event Report
WHO

■
■
■
■

WHAT

■
■

18,19

Identity of reporting individual(s)
Identity of individuals involved (by job title) in committing, compounding,
discovering, investigating, and initiating any immediate action
Patient or donor identification
Reviewer(s) of report

■

Brief description of event
Effects on and outcome to patient, donor, or blood component
Name of component and unit identification number
Manufacturer, lot number, and expiration date of applicable reagents and
supplies
Immediate action taken

WHEN

■
■
■
■

Date of report
Date and time of event occurrence
Date and time of discovery
Collection and shipping dates of blood component(s)

WHERE

■
■
■

Physical location of event
Where in process detected
Where in process initiated

WHY/HOW

■
■

Explanation of how event occurred
Contributing factors
Root cause(s)

■
■

■

FOLLOW-UP

■
■
■
■

External reports or notifications (eg, FDA*, manufacturer, or patient’s
physician)
Corrective actions
Implementation dates
Effectiveness of actions taken

*The following are some examples identified by the FDA as reportable events if components or products are released for
distribution:
– Arm preparation not performed or done incorrectly
– Units from donors who are (or should have been) either temporarily or permanently deferred because of their medical
history or a history of repeatedly reactive viral marker tests
– Shipment of unit with repeatedly reactive viral markers
– ABO/Rh or infectious disease testing not done in accordance with the manufacturer’s package insert
– Units from donors for whom test results were improperly interpreted because of testing errors related to the improper
use of equipment
– Units released before completion of all tests (except as emergency release)
– Sample used for compatibility testing that contains the incorrect identification
– Testing error that results in the release of an incorrect unit
– Incorrectly labeled blood components (eg, ABO, expiration date)
– Incorrect crossmatch label or tag
– Storage of biological products at the incorrect temperature
– Microbial contamination of blood components when the contamination is attributed to an error in manufacturing

Chapter 1: Quality Systems

accidents) and information relevant to
these events to the FDA 3,22 using Form
FDA-3486 when the event:
Is associated with manufacturing (ie,
■
testing, processing, packing, labeling,
storing, holding, or distributing).
Represents a deviation from current
■
good manufacturing practice, applicable regulations or standards, or
established specifications, or is unexpected or unforeseen.
May affect the safety, purity, or po■
tency of the product.
Occurs while the facility had control
■
of or was responsible for the product.
Involves a product that has left the
■
control of the facility (ie, distributed).
There must also be a mechanism to report medical device adverse events to the
FDA.23 The JCAHO encourages reporting of
sentinel events, including hemolytic transfusion reactions involving the administration of blood or components having major
blood group incompatibilities.6
Each facility should track reported events
and look for trends. The use of classification schemes may facilitate trend analysis
and typically involves one or more of the
following categories: the nature of the
event, the process (or procedure) in which
the event occurred, event severity, and
causes. If several events within a relatively

short period involve a particular process or
procedure, that process or procedure should
be further investigated. The most useful
schemes involve use of multiple categories
for each event, which allow data to be
sorted in a variety of ways so that patterns
can emerge (see example in Table 1-5).
Such sorting can result in identification of
situations that require closer monitoring or
of problems needing corrective action. The
extent of monitoring and length of time to
monitor processes will depend on the frequency of the occurrence and the critical
aspects of the occurrences. Reporting and
monitoring of events are essential problem
identification methods for process improvement activities in a quality management system.
Occasionally, the blood bank or transfusion service may need to deviate from approved procedures in order to meet the
unique medical needs of a particular patient. When this situation arises, a medically indicated exception is planned and
approved in advance by the facility’s medical director. The rationale and nature of the
planned exception must be documented.
Careful consideration should be given to
maintaining a controlled process and to
verifying the safety and quality of the resulting product or service. Any additional
risk to the patient must be disclosed.

Table 1-5. Example of Event Classification
Event: A unit of Red Blood Cells from a directed donor was issued to an incorrect patient.
■

■

Classification of event
Type of event – patient
Procedure involved – issuing products
Process involved – blood administration
Product involved – Red Blood Cells
Other factors – directed donor
Investigation revealed
Proximate cause – two patients with similar names had crossmatched blood available
Root cause – inadequate procedure for verification of patient identification during issue

AABB Technical Manual

Monitoring and Assessment
The quality system should describe how
the facility monitors and evaluates its
processes. The AABB Standards1(p93) defines assessment as a systematic, independent examination that is performed at
defined intervals and at sufficient frequency to determine whether actual activities comply with planned activities,
are implemented effectively, and achieve
objectives. Evaluations typically include
comparison of actual results to expected
results. Depending on the focus, this can
include evaluation of process outputs (eg,
results), the activities that make up a process as well as its outputs, or a group of
related processes and outputs (ie, the
system). Types of assessments include external assessments, internal assessments,
quality assessments, peer review, and selfassessments.

oversight personnel for any deficiencies
noted in the assessment. Quality oversight
personnel should track progress toward implementation of corrective and preventive
actions and monitor them for effectiveness.
In order to make the best use of these assessments, there must be a process to track,
trend, and analyze the problems identified
so that opportunities for improvement can
be recognized.1(pp86,87) Early detection of trends
makes it possible to develop preventive actions before patient safety or blood components are adversely affected. Evaluation
summaries provide information useful in
correcting individual or group performance
problems and ensuring adequacy of test
methods and equipment. In addition to review of assessment results, executive management must review any associated corrective or preventive action.

Internal Assessments

Quality Indicators

Internal assessments may include evaluation of quality indicator data, targeted audits of a single process, or system audits
that are broader in scope and cover a set
of interrelated processes. These assessments should be planned and scheduled.
The details of who performs the assessments and how they are performed should
be addressed. Assessments should cover
the quality system and major operating
systems found in the blood bank, transfusion service, or donor center.
In addition, there must be a process for
responding to the issues raised as a result of
the assessment, including review processes
and time frames. The results should be
documented and submitted to management personnel with authority over the
process assessed as well as to executive
management. Management should develop
corrective and preventive action plans with
input from operational staff and quality

Quality indicators are specific performance measurements designed to monitor one or more processes during a defined
time and are useful for evaluating service
demands, production, adequacy of personnel, inventory control, and process
stability. These indicators can be process-based or outcome-based. Processbased indicators measure the degree to
which a process can be consistently performed. An example of a process-based
indicator is measurement of turnaround
time from blood component ordering until transfusion. Outcome-based indicators
are often used to measure what does or
does not happen after a process is or is
not performed. Counting incorrect test
result reports is an example of such an indicator. For each indicator, thresholds are
set that represent warning limits and/or
action limits. These thresholds can be determined from regulatory or accreditation

Chapter 1: Quality Systems

requirements, benchmarking, or internally derived data.
Tools frequently used for displaying
quality indicator data are run charts and
control charts. In a run chart, time is plotted on the x-axis and values on the y-axis.
In control charts, the mean of the data and
upper and lower control limits, which have
been calculated from the data, are added to
the chart. Single points outside the upper
and lower control limits result from special
causes. Statistical rules for interpreting consecutive points outside 1 standard deviation (SD), 2 SD, and 3 SD should be used to
recognize a process that is out of control;
the root cause should be determined and
corrective action should be initiated if indicated.

Blood Utilization Assessment
The activities of blood usage review committees in the transfusion setting are an
example of internal assessment. Guidelines are available from the AABB for both
adult and pediatric utilization review.24-26
Peer review of transfusion practices, required by the AABB, is also required by
the JCAHO6 for hospital accreditation, by
the CMS 2 for hospitals to qualify for
Medicare reimbursement, and by some
states for Medicaid reimbursement.
Transfusion audits provide a review of
policies and practices to ensure safe and
appropriate transfusions and are based on
measurable, predetermined performance
criteria. Transfusion services should investigate an adequate sampling of cases (eg,
5% of the number of cases occurring within
a defined time frame or 30 cases, whichever
is larger). Audits assess the facility’s performance and effectiveness in:
■
Blood ordering practices for all categories of blood and components.
■
Minimizing wastage of blood components.

■

Distribution, handling, use, and administration of blood components.
Evaluating all confirmed transfusion
■
reactions.
Meeting patients’ transfusion needs.
■
Informing patients and physicians
■
in a timely and confidential manner
of possible infectious disease transmission.
One method of assessing the blood administration process is to observe a predetermined number of transfusions by following the unit of blood as it is issued for
transfusion and as it is transfused.25
Assessments of transfusion safety policy
and practice may include a review of transfusion reactions and transfusion-transmitted diseases. The review committee may
monitor policies and practices for notifying
recipients of recalled products (look-back
notification) and donors of abnormal test
results. Other assessments important in
transfusion practice include the review of
policies for informed consent, indication
for transfusion, release of directed donor
units, and outpatient or home transfusion.
Additional assessments should include,
where appropriate: therapeutic apheresis,
use of cell-saver devices, procurement and
storage of hematopoietic progenitor cells,
perioperative autologous blood collection,
procurement and storage of tissue, and
evaluation of evolving technologies and
products. Appendix 1-4 lists blood utilization assessment examples.

External Assessments
External assessments include inspections,
surveys, audits, and assessments performed by those not affiliated with the organization, such as the FDA, AABB, CAP,
or JCAHO. Participation in an external
assessment program provides an independent, objective view of the facility’s
performance. External assessors often bring

AABB Technical Manual

broad-based experience and knowledge
of best practices that can be shared. In the
preparation phase of scheduled assessments, there is typically some data gathering and information to submit to the organization performing the assessment.
Coordinated scheduling and planning will
help ensure that adequate time is allotted
for each area to be covered and that adequate staff are available to answer questions and assist in the assessment activities. During the assessment phase, it is
important to know who is responsible for
the assessors or inspectors during the
time they are in the facility. Clear descriptions of what information can be given to
these individuals, and in what form, will
help the facility through the assessment
or inspection process. After the assessment, identified issues must be addressed.
Usually a written response is submitted.

Proficiency Testing for Laboratories
Proficiency testing (PT) is one means for
determining that test systems (including
methods, supplies, and equipment) are
performing as expected. As a condition
for certification, the CMS requires laboratories to participate successfully in an approved PT program for each specialty and
analyte that they routinely test. When no
approved PT program exists for a particular analyte, the laboratory must have another means to verify the accuracy of the
2
test procedure at least twice annually.
Proficiency testing must be performed using routine work processes and conditions
if it is to provide meaningful information.
Handling and testing of PT samples should
be the same as those for patient or donor
specimens. Supervisory review of the summary evaluation report must be documented
along with investigation and corrective
action for results that are unacceptable.

Quality oversight personnel should monitor the proficiency testing program and
verify that test systems are maintained in
a state of control and that appropriate corrective action is taken when indicated.

Process Improvement
Continuous improvement is a fundamental goal in any quality management system. In transfusion medicine, this goal is
tied to patient safety goals and expectations for the highest quality health care.
The importance of identifying, investigating, correcting, and preventing problems
cannot be overstated. The process of developing corrective and preventive action
plans includes identification of problems
and their causes, and identification and
evaluation of solutions to prevent future
problems. It must include a mechanism
for data collection and analysis, as well as
follow-up to evaluate the effectiveness of
the actions taken. Statistical tools and
their applications may be found in publications from the AABB and the American
Society for Quality.27,28 The JCAHO standards for performance improvement are
outlined in Table 1-6.6
Corrective action is defined as the action
taken to eliminate the causes of an existing
nonconformance or other undesirable situation in order to prevent recurrence.1(p94)
Preventive action is defined as the action
taken to eliminate the causes of a potential
nonconformance or other undesirable situation in order to prevent occurrence.1(p97)
Corrective action can be thought of as a reactive approach to reported problems that
includes a preventive component, whereas
preventive action can be thought of as a
proactive approach resulting from the analysis of data and information. In contrast,
remedial action is defined as the action
taken to alleviate the symptoms of existing
nonconformances or any other undesirable

Chapter 1: Quality Systems

Table 1-6. Applicable JCAHO Performance Improvement Standards

■

Data are collected to measure the performance of potentially high-risk processes, including
blood utilization.

■

Performance data are systematically aggregated and analyzed to determine current performance
levels, patterns, and trends over time.

■

Undesirable patterns and trends in performance are evaluated. All confirmed transfusion
reactions are analyzed.

■

There is a defined process for identification and management of serious adverse events.
Root-cause analysis and corrective action are documented.

■

Information from data analysis is used to improve performance and patient safety and minimize
the risk of serious adverse events.

■

The facility defines and implements a program to proactively identify opportunities for
improvement. Preventive actions are implemented and monitored for effectiveness.

27,28

situation.
Remedial action addresses
only the visible indicator of a problem, not
the actual cause (see comparisons in Table
1-7). Effective corrective and preventive actions cannot be implemented until the underlying cause is determined and the process is evaluated in relationship to other
processes. Pending such evaluation, it may
be desirable to implement interim remedial
action.

Identification of Problems and Their Causes
Sources of information for process improvement activities include the following: blood product and other deviations;
nonconforming products and services;
customer complaints; QC records; proficiency testing; internal audits; quality in-

dicators; and external assessments. Active
monitoring programs may be set up to
help identify problem areas. These programs should be representative of the
facility processes, consistent with organizational goals, and reflect customer needs.
Preparation of an annual facility quality
report, in which data from all these sources
are collated and analyzed, can be a valuable tool to identify issues for performance
improvement.
Once identified, problems must be analyzed to determine their scope, potential effects on the quality and operational systems, relative frequency, and the extent of
variation. This analysis is important to
avoid tampering with processes that are
showing normal variation or problems with
little impact.

Table 1-7. Comparison of Remedial, Corrective, and Preventive Action

Action

Problem

Approach

Outcome

Remedial
Corrective
Preventive

Existent
Existent
Nonexistent

Reactive
Reactive
Proactive

Alleviates symptoms
Prevents recurrence
Prevents occurrence

AABB Technical Manual

impossible, or outside the boundaries of
the organization. Use of the “repetitive
why” prevents the mistake of interpreting
an effect as a cause.
The cause-and-effect diagram, also known
as the Ishikawa or fish-bone diagram, employs a specialized form of brainstorming
that breaks down problems into “bite-size”
pieces. An example of a cause-and-effect
diagram is shown in Fig 1-1. It is a method
designed to focus ideas around the component parts of a process, as well as give a pictorial representation of the ideas that are
generated and their interactions. When using the cause-and-effect diagram, one looks
at equipment, materials, methods, environment, and human factors. These tools identify both active and latent failures. Active
failures are those that have an immediate
adverse effect. Latent failures are those
more global actions and decisions with potential for damage that may lie dormant

Identifying underlying causes for an undesirable condition or problem can be accomplished by an individual or a group.
The more complex the problem and the
more involved the process, the greater the
need to enlist a team of individuals and to
formalize the analysis. The three most commonly used tools for identifying underlying
causes in an objective manner are process
flowcharting, use of the “repetitive why,”
and the cause-and-effect diagram. A process flowchart gives a detailed picture of the
multiple activities and important decision
points within the process. By examining
this picture, problem-prone areas may be
identified. The “repetitive why” is used to
work backward through the process. One
repeatedly asks the question “why did this
happen?” until: 1) no new information can
be gleaned, 2) the causal path cannot be
followed because of missing information,
or 3) further investigation is impractical,

Root Cause Analysis of Failed Test Runs
Personnel

Procedure
Inadequate training

SOP not clear
Specimen suitability not defined

Multi-tasking
Inadequate staffing
Too rushed

Inadequate for intended use
Not validated

Failed Test Run
Not scheduled
Wrong lot
Contaminated
Expired

Reagents, Supplies

Wrong calibrators
used
Out of calibration
Mechanical failure
Maintenance
not done

Equipment

Environment

Room temperature too hot
Poor ventilation
Environmental contamination
Cleaning not scheduled
No SOP
Inadequate cleaning

Figure 1-1. Example of a cause-and-effect diagram (SOP = standard operating procedure).

Chapter 1: Quality Systems

and become evident only when triggered
by the presence of localized factors. The key
to successfully determining root cause is
not to stop too soon or get caught in the
trap of placing blame on an individual.
Most problems, particularly those that
are complex, have several root causes. A
method that can be of use when this occurs
is the Pareto analysis. A chart of causes, laid
out in order of decreasing frequency, is prepared. Those that occur most frequently are
considered the “vital few”; the rest are considered the “trivial many.” This method offers direction about where to dedicate resources for maximal impact. An example of
a Pareto chart is shown in Fig 1-2.
Figure 1-2. Example of a Pareto chart.

Identification and Evaluation of Solutions
Potential solutions to problems are identified during the creative phase of process
improvement. Brainstorming and process
flowcharting can be particularly helpful in
this phase. Possible solutions should be
evaluated relative to organizational constraints and narrowed down to those most
reasonable. Individuals who perform the
process are usually the most knowledgeable about what will work. They should be
included when possible solutions are being considered. Individuals with knowledge of the interrelationships of processes
and the more “global” view of the organization should also be included. Solutions
may fail if representatives with these perspectives are not involved.
Potential solutions should be tested before full implementation, with a clear plan
relative to methods, objectives, timelines,
decision points, and algorithms for all possible results of the trial. Large-scale solutions can be tried on a limited basis and
expanded if successful; smaller scale solutions can be implemented pending an effectiveness evaluation. Nonetheless, data
should be collected to evaluate the effec-

tiveness of the proposed change. Data can
be collected by the methods used initially
to identify the problems or by methods
specially designed for the trial. Once solutions have been successfully tested, full implementation can occur. Following implementation, data should be collected, on at
least a periodic basis, to ensure adequate
control of the process.

Work Environment
The facility must provide a safe workplace
with adequate environmental controls
and emergency procedures for the safety
of the employees, donors, patients, and all
other inhabitants or visitors.1(p88) Procedures must be in place to address:
■
General safety
■
Disaster preparedness
■
Biological safety (blood-borne pathogens)
■
Chemical safety
■
Fire safety
■
Radiation safety, if applicable

AABB Technical Manual

■

Discard of blood, components, and
tissue
Current good manufacturing practice
regulations require quality planning and
control of the physical work environment,
including:
Adequate space and ventilation
■
Sanitation and trash disposal
■
Equipment for controlling air quality
■
and pressure, humidity, and temperature
Water systems
■
Toilet and hand-washing facilities
■
An evaluation of the infrastructure and
its limitations before implementation of
procedures or equipment will help to ensure maximum efficiency and safety. A
more thorough discussion of facilities and
safety can be found in Chapter 2.

10.

11.
12.
13.

14.

15.

References
16.
1.

Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.
Code of federal regulations. Title 42 CFR Part
493. Washington, DC: US Government Printing Office, 2004 (revised annually).
Code of federal regulations. Title 21 CFR Parts
606, 610, 630, and 640. Washington, DC: US
Government Printing Office, 2004 (revised
annually).
Code of federal regulations. Title 21 CFR Parts
210 and 211. Washington, DC: US Government Printing Office, 2004 (revised annually).
Food and Drug Administration. Guideline for
quality assurance in blood establishments.
Docket #91N-0450. (July 11, 1995) Rockville,
MD: CBER Office of Communication, Training, and Manufacturers Assistance, 1995.
Hospital accreditation standards. Oakbrook
Terrace, IL: Joint Commission Resources,
Inc., 2004.
College of American Pathologists Laboratory
Accreditation Program checklists. Chicago,
IL: College of American Pathologists, 2003.
A quality system model for health care;
NCCLS approved guideline (HS1-A). Wayne,
PA: National Committee for Clinical Laboratory Standards, 2002.

17.

18.

19.

20.

21.
22.

ANSI/ISO/ASQ Q9000-2000 series—quality
management standards. Milwaukee, WI: ASQ
Quality Press, 2000.
Baldrige National Quality Program. Health
care criteria for performance excellence.
Gaithersburg, MD: National Institute of Standards and Technology, 2004 (revised annually).
Quality program implementation. Association Bulletin 97-4. Bethesda, MD: AABB, 1997.
Juran JM, Godfrey AB. Juran’s quality handbook. 5th ed. New York: McGraw-Hill, 1999.
Food and Drug Administration. Guidance on
general principles of process validation. (May
1, 1987) Rockville, MD: CBER Office of Communication, Training, and Manufacturers
Assistance, 1987.
Food and Drug Administration. Glossary of
computerized system and software development terminology. (August 1995) Rockville,
MD: Division of Field Investigations, Office of
Regional Operations, Office of Regulatory
Affairs, 1995.
Food and Drug Administration. Guidance for
industry: General principles of software validation: Final guidance for industry and FDA
staff. (January 11, 2002) Rockville, MD: CBER
Office of Communication, Training, and
Manufacturers Assistance, 2002.
Clinical laboratory technical procedure manuals. NCCLS approved guideline. 4th ed.
(GP2-A4). Wayne, PA: National Committee for
Clinical Laboratory Standards, 2002.
Code of federal regulations. Title 21 CFR Part
11. Washington, DC: US Government Printing
Office, 2004 (revised annually).
Motschman TL, Santrach PJ, Moore SB. Error/incident management and its practical
application. In: Duckett JB, Woods LL,
Santrach PJ, eds. Quality in action. Bethesda,
MD: AABB, 1996:37-67.
Food and Drug Administration. Biological
products: Reporting of biological product
deviations in manufacturing. Docket No.
97N-0242. (November 7, 2000) Fed Regist
2000;65:66621-35.
Food and Drug Administration. Guidance for
industry: Notifying FDA of fatalities related to
blood collection or transfusion. (September
22, 2003) Rockville, MD: CBER Office of Communication, Training, and Manufacturers
Assistance, 2003.
Reporting donor fatalities. Association Bulletin #04-06. Bethesda, MD: AABB, 2004.
Food and Drug Administration. Draft guidance for industry: Biological product deviation reporting for blood and plasma establishments. (August 10, 2001) Rockville, MD:

Chapter 1: Quality Systems

23.

24.

25.

CBER Office of Communication, Training,
and Manufacturers Assistance, 2001.
Code of federal regulations. Title 21 CFR Part
803. Washington, DC: US Government Printing Office, 2004 (revised annually).
Shulman IA, Lohr K, Derdiarian AK, et al.
Monitoring transfusionist practices: A strategy for improving transfusion safety. Transfusion 1994;34:11-15.
Becker J, Blackall D, Evans C, et al for the Scientific Section Coordinating Committee.
Guidelines for blood utilization review.
Bethesda, MD: AABB, 2001.

26.

27.

28.
29.

Strauss RG, Blanchette VS, Hume H. National
acceptability of American Association of
Blood Banks Hemotherapy Committee guidelines for auditing pediatric transfusion practices. Transfusion 1993;33:168-71.
Anderson TD. Tools for statistical process
control. In: Ziebell LW, Kavemeier K, eds.
Quality control: A component of process control in blood banking and transfusion medicine. Bethesda, MD: AABB Press, 1999:13-48.
Russell JP, Regel T. After the quality audit. 2nd
ed. Milwaukee, WI: ASQ Quality Press, 2000.
Motschman T. Corrective versus preventive
action. AABB News 1999;21(8):5,33.

AABB Technical Manual

Appendix 1-1. Glossary of Commonly Used Quality Terms
Calibration

Comparison of measurements performed by an instrument to those
made by a more accurate instrument or standard for the purpose of detecting, reporting, and eliminating errors in measurement.

Change control

Established procedures for planning, documenting, communicating, and
executing changes to infrastructure, processes, products, or services.
This includes the submission, analysis, decision making, approval, implementation, and postimplementation review of the change. Formal
change control provides a measure of stability and safety and avoids arbitrary changes that might affect quality.

Control chart

A graphic tool used to determine whether the distribution of data values
generated by a process is stable over time. A control chart plots a statistic vs time and helps to determine whether a process is in control or
out of control according to defined criteria, eg, a shift from a central line
or a trend toward upper or lower acceptance limits.

Design output

Documents, records, and evidence in any other format used to verify
that design goals have been met. Design output should identify characteristics of a product or service that are crucial to safety and function
and to meeting regulatory requirements. It should contain or make reference to acceptance criteria. Examples of design output include standard operating procedures, specifications for supplies, reagents and
equipment, identification of quality control requirements, and the results of verification and validation activities.

End-product test and
inspection

Verification through observation, examination, and/or testing that the
finished product or service conforms to specified requirements.

Process capability

Ability of a controlled process to produce a service or product that fulfills requirements. Also, a statistical measure of the inherent process
variability for a given characteristic relative to design specifications. The
most widely accepted formula for process capability is six sigma.

Process control

Activities intended to minimize variation within a process in order to
produce a predictable output that meets specifications.

Qualification

Demonstration that an entity is capable of fulfilling specified requirements. Verification of attributes that must be met or complied with in
order for a person or thing to be considered fit to perform a particular
function. For example, equipment may be qualified for an intended use
by verifying performance characteristics such as linearity, sensitivity, or
ease of use. An employee may be qualified based on technical, academic, and practical knowledge and skills developed through training,
education, and on-the-job performance.

Quality assurance

Activities involving quality planning, control, assessment, reporting,
and improvement necessary to ensure that a product or service meets
defined quality standards and requirements.

Chapter 1: Quality Systems

Appendix 1-1. Glossary of Commonly Used Quality Terms (cont’d)
Quality control

Operational techniques and activities used to monitor and eliminate
causes of unsatisfactory performance at any stage of a process.

Quality indicators

Measurable aspects of processes or outcomes that provide an indication of the condition or direction of performance over time. Used to
monitor progress toward stated quality goals and objectives.

Quality management

The organizational structure, processes, and procedures necessary to
ensure that the overall intentions and direction of an organization’s
quality program are met and that the quality of the product or service is
ensured. Quality management includes strategic planning, allocation of
resources, and other systematic activities such as quality planning, implementation, and evaluation.

Requirement

A stated or obligatory need or expectation that can be measured or observed and is necessary to ensure quality, safety, effectiveness, or customer satisfaction. Requirements can include things that the system or
product must do, characteristics it must have, and levels of performance it must attain.

Specification

Description of a set of requirements to be satisfied by a product, material, or process indicating, if appropriate, the procedures to be used to
determine whether the requirements are satisfied. Specifications are often in the form of written descriptions, drawings, professional standards, and other descriptive references.

Validation

Demonstration through objective evidence that the requirements for a
particular application or intended use have been met. Validation provides assurance that new or changed processes and procedures are capable of consistently meeting specified requirements before implementation.

Verification

Confirmation, by examination of objective evidence, that specified requirements have been met.

AABB Technical Manual

Appendix 1-2. Code of Federal Regulations Quality-Related References
21 CFR Citation

Topic

606.20

Personnel

606.40

Facilities

606.60

Equipment quality control

606.65

Supplies, reagents

606.100

Standard operating procedures

606.140

Laboratory controls

606.160

Records

606.170

Adverse reactions

606.171

Biological product deviations

211.22

Quality control/quality assurance unit responsibilities

211.25

Personnel qualifications

211.28

Personnel responsibilities

211.160

Laboratory controls

211.192

Production record review

211.194

Laboratory records and reviews

Chapter 1: Quality Systems

Appendix 1-3. Statistical Tables for Binomial Distribution* Used to Determine
Adequate Sample Size and Level of Confidence for Validation of Pass/Fail Data
Confidence Levels (%) for Percent Conforming
Requirement for
% Conforming
90%

95%

Sample
Size

No. of
Failures

65.1

–

26.4

Requirement for
% Conforming
90%

95%

Sample
Size

No. of
Failures

99.5

92.3

–

96.6

72.1

87.8

64.2

88.8

45.9

60.8

26.4

75.0

–

32.3

–

56.9

–

95.8

78.5

38.4

–

81.6

44.6

99.8

95.4

58.9

–

98.6

80.8

35.3

–

94.7

58.3

98.5

87.1

86.3

35.3

91.9

60.1

72.9

–

77.7

32.3

56.3

–

57.7

–

39.4

–

37.1

–

% Confidence

This table answers the question, “How confident am I that [90 or 95]% of all products manufactured will meet specifications if I have tested __ number of samples and found __ number to
be nonconforming (failures)?”
*Data from Reliability Analysis Center, http://rac.alionscience.com/Toolbox/.

(cont’d)

AABB Technical Manual

Appendix 1-3. Statistical Tables for Binomial Distribution* Used to Determine
Adequate Sample Size and Level of Confidence for Validation of Pass/Fail Data
(cont'd)
Minimum Sample Size for Percent Conforming
Requirement for Percent Conforming
90%

95%

99%

Confidence Level

90%
No. of
Failures

95%

99%

90%

Sample Size

95%

99%

90%

Sample Size

95%

99%

Sample Size

230

299

459

130

388

467

662

106

125

166

526

625

838

133

180

198

664

773

1002

113

159

208

228

789

913

1157

103

128

184

235

258

926

1049

1307

104

116

142

209

260

288

1051

1186

1453

116

129

158

234

286

317

1175

1312

128

143

170

258

310

344

1297

1441

140

154

184

282

336

370

1418

152

167

197

306

361

397

This table answers the question, “How many samples do I need to test with ___ number of failures if I want to have [90, 95, or 99] % confidence that [90, 95, or 99]% of all products will
meet specifications?”
*Data from Reliability Analysis Center, http://rac.alionscience.com/Toolbox/.

Chapter 1: Quality Systems

Appendix 1-3. Statistical Tables for Binomial Distribution* Used to Determine
Adequate Sample Size and Level of Confidence for Validation of Pass/Fail Data
(cont'd)
Example of Minimum Sample Size and Number of Failures Allowed to Meet AABB Requirements
for Product Validation and Quality Control

Product

Requirement1

Sample
2
Size

Number of
Failures

%
Confidence
Level

Platelets Pheresis

At least 90% of units sampled
contain ≥3 × 1011 platelets
and have a pH ≥6.2 at the
end of allowable storage.

10
10

0
1

65
26

Platelets Pheresis,
Leukocytes
Reduced

At a minimum, 95% of units
sampled shall contain a residual leukocyte count
<5 × 106.

20
20

0
1

64
26

Red Blood Cells
Pheresis

At least 95% of units sampled
shall have >50 g of hemoglobin (or 150 mL red cell
volume) per unit.

20
20

0
1

64
26

Granulocytes
Pheresis

Prepared by a method known
to yield a minimum of
1.0 × 1010 granulocytes in
at least 75% of the units
tested.

4
4

0
1

68
26

From Silva MA, ed. Standards for blood banks and transfusion services. 23rd ed. Bethesda, MD: AABB,
2005. Although the AABB Standards does not require a specific confidence level, the facility may use
this as a way to assess the degree of certainty that each product manufactured will meet
specifications.
Period of time used to define a population for sampling is determined by the facility. (NOTE: The longer
the period, the more difficult it may be to identify causes of failure, and the more products already in
distribution that may be involved in a recall.)

*Data from Reliability Analysis Center, http://rac.alionscience.com/Toolbox/.

AABB Technical Manual

Appendix 1-4. Assessment Examples: Blood Utilization

1,2

A blood usage review committee should consider the following areas of practice and develop specific
measurements for monitoring blood transfusion processes. Some measurements provide data for
several processes.
Ordering of Appropriate Blood Components
1.

Preanalytical errors

Errors in specimen collection, verbal orders, transfusion orders.

Units transfused

Figures for each type of blood component and special preparation.
Use of autologous and directed donor collections. Analyze by clinical service or by prescriber.

Patients transfused

Total number of patients receiving each of the components or
products listed in item 2.

Units transfused
per patient transfused

Average number of units of each component or product given to
patients receiving that component. May be useful to analyze by diagnosis or surgical/medical procedure.

Special components
prepared and transfused

Number and relative percent of leukocyte-reduced, irradiated,
cytomegalovirus-negative units; aliquots prepared and transfused;
outpatient and home transfusions.

Units returned unused

Number and percent of units issued and later returned unused.
Analyze by ward, by clinical service, or by prescriber.

Crossmatch-to-transfusion (C:T) ratio

Number of units crossmatched divided by the number of units
transfused. Analysis could be by institutional total, by emergency
vs routine requests, or by clinical service, surgical procedure, or
prescriber, as needed.

Transfusion guidelines

Verification that guidelines are current, appropriate for the patient
population being treated, and readily available to physicians.

Distributing, Handling, and Dispensing Blood Components
1.

Turnaround time

Interval between the time a transfusion request is received and
time the unit is available for transfusion and/or is transported to
the patient’s bedside. May analyze by emergency, routine, or operative requests.

Emergency requests

Number and percent may be analyzed by clinical service,
prescriber, diagnosis, or time (week, day, shift).

Uncrossmatched units

Number and percent of units issued uncrossmatched or with abbreviated pretransfusion testing. May analyze by clinical service,
or prescriber.

Age distribution
of units

Age of units in inventory and crossmatched by ABO and Rh type,
age of units when received from the supplier, age at the time of
transfusion, age when returned to the supplier.

Chapter 1: Quality Systems

Appendix 1-4. Assessment Examples: Blood Utilization

1,2

(cont'd)

Surgical cancellations
due to unavailability of
blood

Number and percent of cases delayed due to the unavailability of
blood; number of hours or days of delay, analyzed by surgical
procedure and by cause (eg, antibody problem in an individual
patient, general shortage, or shortage of a particular ABO or Rh
type).

Significant type
switches due to
unavailability of blood

Number of Rh-negative patients given Rh-positive red cells or
platelets; transfusions with ABO-incompatible plasma.

Outdate rate

Number of units outdated (expired unused) divided by the number of units received; should be monitored for all blood components and derivatives. Analysis by ABO and Rh type may prove informative.

Wastage rates

Number of units wasted due to breakage, improper preparation,
improper handling or storage; units prepared for a patient but not
used; number of units that failed to meet inspection requirements.

Adequacy of service
from the blood
supplier

Number of orders placed that could be filled as requested; average time between the order and receipt of emergency delivery;
number of orders associated with an error such as improper unit
received or units improperly shipped.

10.

Compatibility testing
requirements

Adequacy, currency, and appropriateness of policies and procedures.

11.

Quality control policies
and procedures

Percent of records of temperature, equipment, component preparation, or testing that are incomplete or have deviations.

Administration of Blood and Components
1.

Blood issue/delivery
errors

Number of wrong units issued; number of units delivered to
wrong patient-care area or improperly transported.

Blood administration
policies and
procedures

Adherence to facility-specific requirements when monitoring patients for signs and symptoms of adverse reactions. Availability of
copies of current policies and procedures and of current Circular
of Information for the Use of Human Blood and Blood Components.

Blood administration
audits

Summary of on-site performance reviews, to include number of
deviations by category (eg, identification of patient and donor
unit, documentation and completeness of medical record). May
include audit for documentation of transfusion order and indication for transfusion or informed consent.

Transfusion
equipment

Review of quality control documentation for equipment, including
blood warmers, infusion pumps, special filters or administration
sets; documentation in the medical record that devices were used;
number of situations where their use was inappropriate.
(cont’d)

AABB Technical Manual

Appendix 1-4. Assessment Examples: Blood Utilization

Special transfusion
situations

1,2

(cont'd)

Review of compliance with policies for out-of-hospital transfusions and perioperative and postoperative collection of autologous blood.

Monitoring Transfusion Results
1.

Compliance with transfusion guidelines

Number and percent of inappropriate transfusions, as determined
by the blood usage review committee; analysis of reasons for inappropriate transfusion.

Transfusion reactions

Number and percent of reported transfusion reactions; turnaround time for complete investigation; documentation of transfusion service and committee review; documentation in the medical
record.

Transfusion-transmitted disease

Number of cases by infectious agent; turnaround time of investigation; completeness of review and recording.

Look-back investigations

Number of cases by infectious agent; turnaround time of investigation; completeness of case-finding, notification, review, and recording.

Review of policies
and procedures

Adequacy, currency, and appropriateness of policies and procedures for detection and reporting of adverse effects of transfusion.

Management Data
1.

Workload and productivity

Evaluation of activities and efficiency of the laboratory; may be
analyzed by day of week and by shift. Hours worked per unit
transfused or patient transfused may be more valuable as an efficiency measure than data obtained from traditional productivity
calculations.

Event reports

Number of events dealing with laboratory processes (eg, labeling,
preparation, testing, issue); procedural events in blood administration; errors, accidents, and recalls by blood supplier(s).

Staff training and
competency

Documentation of training and continuing competency of laboratory and nursing staff to perform transfusion-related procedures
and policies.

1.
2.

Comprehensive accreditation manual for hospitals: The official handbook. Oakbrook Terrace, IL: Joint
Commission Resources, Inc., 2002.
Becker J, Blackall D, Evans C, et al for the Scientific Section Coordinating Committee. Guidelines for
blood utilization review. Bethesda, MD: AABB, 2001.

Chapter 2: Facilities and Safety

Chapter 2

Facilities and Safety

ACILITY DESIGN AND maintenance are critical to ensure that operational needs are met and that
the work environment is safe for both
staff and visitors. The layout of the physical space; management of utilities such as
water and air ventilation; flow of personnel, materials, and waste; and ergonomic
factors should all be considered in the facility management plan.
In addition to providing adequate facilities, the organization must develop and
implement a safety program that defines
policies and procedures for safe work
practices. This includes hazard communication, use of protective equipment, training, and competency assessment in accordance with regulations for emergency and
disaster preparedness, chemical hygiene,
blood-borne pathogens, and radiation
safety when applicable. All employees are
responsible for protecting their own safety

and the safety of others by adhering to policies set forth in the facility safety program.
The AABB requires accredited blood
banks and transfusion services to plan, implement, and maintain a program to minimize risks to the health and safety of donors, patients, volunteers, and employees
from biological, chemical, and radiological
hazards.1(p88) Other professional and accrediting organizations have similar or more detailed safety program requirements, including the College of American Pathologists
(CAP), the Clinical and Laboratory Standards Institute (formerly NCCLS), and the
Joint Commission on Accreditation of
2-5
Healthcare Organizations (JCAHO).
Several federal agencies have issued regulations and recommendations to protect
the safety of workers and the public. Those
relevant to health-care settings are listed in
Appendix 2-1. The contents of these regulations and guidelines are discussed in more
39

AABB Technical Manual

detail in each section of this chapter. Blood
banks and transfusion services should consult with state and local agencies as well to
identify any additional safety requirements.
Trade and professional organizations also
provide safety recommendations that are
relevant to blood banks and transfusion
services. These organizations are also listed
in Appendix 2-1.

Facilities
Facility Design and Workflow
Proper design and maintenance of facilities and organization of work can reduce
or eliminate many potential hazards. Design, maintenance, and organization also
affect efficiency, productivity, error rates,
employee and customer satisfaction, and
the quality of products and services. State
and local building codes should be consulted in the design planning stages for
architectural safety regarding space, furnishings, and storage.
During the design phase for a new space,
the location and flow of personnel, materials, and equipment should be considered
in the context of the processes to be performed. Adequate space must be allotted
for personnel movement, location of supplies and large equipment, and private or
“distraction-free” zones for certain manufacturing tasks (eg, donor interviewing,
record review, and blood component labeling). The facility must be able to accommodate designated “clean” and “dirty” spaces
and provide for controlled movement of
materials and waste in and out of these areas so as to avoid contamination. Chemical
fume hoods and biological safety cabinets
should be located away from drafts and
high-traffic areas. The number and location
of eyewashes and emergency showers must
also be considered. Water sources for reagent preparation must be considered. Staff

handling hazardous materials must have
ready access to hand-washing sinks. For
certain pieces of heavy equipment, such as
irradiators, load-bearing capacity must be
taken into account.
Laboratories must be designed with adequate illumination, electrical power, and
conveniently located outlets. Emergency
backup power sources, such as uninterruptible power supplies and backup generators, should be considered to ensure that
loss of blood products does not occur during power failures. The National Electrical
Code6 is routinely used as a national guideline for the design of essential electrical distribution systems, with modifications approved by the local building authority having
jurisdiction.
Appropriate systems for heating, ventilation, and air conditioning must be used.
Environmental monitoring systems should
be considered for laboratories that require
positive or negative air pressure differentials to be maintained, or where air filtration systems are used to control particle
levels. The nationally accepted specifications for ventilation are published by the
American Society of Heating, Refrigerating,
and Air-Conditioning Engineers, Inc.7

Housekeeping
The workplace should be kept clean and
free of clutter. Work surfaces and equipment should be regularly cleaned and disinfected. Items that may accumulate dust
and debris should not be stored above
clean supplies or work surfaces. Exits and
fire safety equipment must not be blocked or obstructed in any way. Receptacles
and disposal guidelines for nonhazardous
solid waste, biohazardous, chemical, and
radiation waste should be clearly delineated. Housekeeping responsibilities,
methods, and schedules should be defined for every work area. Written proce-

Chapter 2: Facilities and Safety

dures, initial training, continuing education of personnel, and ongoing monitoring
of housekeeping effectiveness are essential to safe operations.

Restricted Areas
Hazardous areas should be clearly and
uniformly identified with warning signs in
accordance with federal Occupational
Safety and Health Administration (OSHA)
and Nuclear Regulatory Commission (NRC)
standards so that personnel entering or
working around them are aware of existing biological, chemical, or radiation dan8-11
Staff not normally assigned to
gers.
these areas should receive adequate training to avoid endangering themselves. Risk
areas can be stratified. For example, “highrisk” areas might include chemical fume
hoods, biological safety cabinets, and storage areas for volatile chemicals or radioisotopes. Technical work areas could be
considered “moderate risk” and restricted
to laboratory personnel. Administrative
and clerical areas could be considered
“low risk” and not restricted.
Whenever possible, functions not requiring special precautions should be separated
from those performed in restricted areas.
Every effort should be made to prevent the
contamination of designated “clean” areas
and common equipment. Work area telephones can be equipped with speakers to
eliminate the need to pick up the receiver.
Computer keyboards and telephones can
be covered with plastic. They should be
cleaned on a regular basis and when visibly
soiled. Employees should remove their personal protective barriers such as gloves and
laboratory coats and wash their hands with
soap and water when leaving a “contaminated” area.
Concerns for safety dictate that there be
no casual visitors in areas where laboratory
hazards may be encountered.12 Children,

especially, should not be allowed in areas
where they could be exposed to hazards
and should be closely supervised in those
areas where their presence is permitted. Facilities should consider establishing specific
safety guidelines for visitors with business
in restricted areas and documenting that this
information was received and understood.

Mobile Sites
Mobile blood collection operations can
present special problems. An individual
trained in safety principles should make an
advance visit to the collection site to ensure
that hazards are minimized. All mobile personnel should be trained to recognize unsafe conditions and understand infection
control policies and procedures, but responsibility for site safety should be assigned to a senior-level employee.
Hand-washing access is essential at all
collection sites. Carpeted or difficult-toclean surfaces may be protected with an
absorbent overlay with waterproof backing
to protect from possible blood spills. Portable screens and ropes are helpful in directing traffic flow to maintain safe work areas.
Food service areas should be physically
separated from areas for blood collection
and storage. Blood-contaminated waste must
be either returned to a central location for
disposal or packaged and decontaminated
using thermal (autoclave, incinerator) or
chemical disinfectant in accordance with
local regulations for medical wastes. Trained staff must perform this decontamination
with particular attention paid to cleanup of
mobile sites after blood collection.

Ergonomics
Consideration in physical design should
be given to ergonomics and accommodations for individuals covered under the
Americans with Disabilities Act (42 U.S.C.

AABB Technical Manual

§ 12101-12213, 1990). Several factors may
contribute to employee fatigue, musculoskeletal disorder syndromes, or injury, including the following13:
Awkward postures—positions that
■
place stress on the body such as
reaching overhead, twisting, bending, kneeling, or squatting.
Repetition—performing the same
■
motions continuously or frequently.
Force—the amount of physical effort
■
used to perform work.
Pressure points—pressing the body
■
against hard or sharp surfaces.
Vibration—continuous or high-intensity
■
hand-arm or whole-body vibration.
Other factors—extreme high or low
■
temperatures; lighting too dark or
too bright.
Both the total time per work shift and
the length of uninterrupted periods of work
can be significant in contributing to problems. Actions to correct problems associated with ergonomics may include:
■
Engineering improvements to reduce
or eliminate the underlying cause,
such as making changes to equipment, workstations, or materials.
■
Administrative improvements, such
as providing variety in tasks; adjusting work schedules and work pace;
providing recovery or relaxation time;
modifying work practices; ensuring
regular housekeeping and maintenance
of work spaces, tools, and equipment;
and encouraging exercise.
■
Provision of safety gear such as gloves,
knee and elbow pads, footwear, and
other items that employees wear to
protect themselves against injury.

Safety Program
An effective safety program starts with a
well-thought-out safety plan. This plan

identifies the applicable regulatory requirements and describes how they will
be met. In general, institutions are required to:
Provide a workplace free of recog■
nized hazards.
Evaluate all procedures for potential
■
exposure risks.
Evaluate each employment position
■
for potential exposure risks.
Identify hazardous areas or materi■
als with appropriate labels and signs.
Educate staff, document training,
■
and monitor compliance.
Apply Standard Precautions (includ■
ing Universal and Blood and Body
Fluid Precautions) to the handling of
blood, body fluids, and tissues.
Dispose of hazardous waste appro■
priately.
Report incidents and accidents and
■
provide treatment and follow-up.
■
Provide ongoing review of safety policies, procedures, operations, and
equipment.
■
Develop facility policies for disaster
preparedness and response.
Safety programs should consider the
needs of all persons affected by the work
environment. Most obvious is the safety of
technical staff, but potential risks for blood
donors, ancillary personnel, volunteers, visitors, housekeeping staff, and maintenance
and repair workers must also be evaluated.
Appropriate provisions must be applied if
these individuals cannot be excluded from
risk areas.
Laboratories should appoint a safety officer who can provide general guidance and
expertise.3 This individual might develop
the safety program, oversee orientation and
training, perform safety audits, survey work
sites, recommend changes, and serve on or
direct the activities of safety committees. It
is recommended that facilities using hazardous chemicals and radioactive materials

Chapter 2: Facilities and Safety

appoint a chemical hygiene officer and radiation safety officer to oversee chemical
and radiation protection programs.8,11 A
general safety officer with sufficient expertise may fill these roles, or separate officers
may be appointed and program oversight
given to a safety committee.
There are five basic elements that must
be addressed for each type of hazard covered in the safety program:
1.
Training.
2.
Hazard identification and communication.
3.
Engineering controls and personal
protective equipment.
4.
Safe work practices, including waste
disposal.
5.
Emergency response plan.
In addition, management controls should
be implemented to ensure that these elements are in place and effective. Management is responsible for:
1.
Developing and communicating the
written plan.
2.
Ensuring implementation and providing adequate resources.
3.
Providing access to employee health
services related to prevention strategies and treatment of exposures.
4.
Monitoring compliance and effectiveness.
5.
Evaluating and improving the safety
plan.

Basic Elements of a Safety Program

Training
Employees must understand the hazards
in their workplace and the appropriate
precautions to take in order to manage
them safely. The mandate for employee
training programs is based on good general practice as well as OSHA requirements.9-11 All persons must be trained to

protect themselves appropriately before
beginning work with hazardous materials.
Supervisors or their designees are responsible for documenting the employee’s
understanding of and ability to apply safety
precautions before independent work is
permitted. Safety training must precede
even temporary work assignments if significant potential for exposure exists. Staff who
do not demonstrate the requisite understanding and skills must undergo retraining. These requirements apply not only to
laboratory staff, but also to housekeeping
and other personnel who may come into
contact with hazardous substances or
waste. Table 2-1 lists topics to cover in
work safety training programs.

Hazard Identification and Communication
Employees must know when they are
working with hazardous substances and
must know where they are located in the
workplace. Employers are required to provide information about workplace hazards to their employees to help reduce the
risk of occupational illnesses and injuries.
This is done by means of signage, labels
on containers, written information, and
training programs.

Engineering Controls and Personal
Protective Equipment
Whenever possible, the physical workspace should be designed to eliminate the
potential for exposure. When this is not
possible, protective gear must be provided to protect the employee. Engineering controls are physical plant controls or
equipment such as sprinkler systems,
chemical fume hoods, and needleless systems that isolate or remove the hazard from
the workplace. Personal protective equipment (PPE) is specialized clothing or
equipment worn by an employee for protection against a hazard, such as gloves,

AABB Technical Manual

Table 2-1. Topics to Cover in a Work Safety Training Program
Work safety training programs should ensure that all personnel:
■

Have access to a copy of pertinent regulatory texts and an explanation of the contents.

■

Understand the employer’s exposure control plan and how to obtain a copy of the written plan.

■

Understand how hepatitis and human immunodeficiency virus (HIV) are transmitted and how
often; be familiar with the symptoms and consequences of hepatitis B virus (HBV), hepatitis C
virus (HCV), and HIV infection.

■

Know that they are offered vaccination against HBV.

■

Recognize tasks that pose infectious risks and distinguish them from other duties.

■

Know what protective clothing and equipment are appropriate for the procedures they will
perform and how to use them.

■

Know and understand the limitations of protective clothing and equipment (eg, different types of
gloves are recommended based on the permeability of the hazardous material to be used).
Employers and staff should be forewarned against a false sense of security.

■

Know where protective clothing and equipment are kept.

■

Are familiar with and understand all requirements for work practices specified in standard
operating procedures (SOPs) for the tasks they perform, including the meaning of signs and
labels.

■

Know how to remove, handle, decontaminate, and dispose of contaminated material.

■

Know the appropriate actions to take and the personnel to contact if exposed to blood or other
biological, chemical, or radiological hazards.

■

Know the corrective actions to take in the event of spills or personal exposure to fluids, tissues,
and contaminated sharps, the appropriate reporting procedures, and the medical monitoring
recommended when parenteral exposure may have occurred.

■

Know their right for access to medical treatment and medical records.

masks, and laboratory coats. General
guidance on the use of engineering controls and PPE is included in Appendix 2-2.

Safe Work Practices
Employees must be trained to know how
to work with hazardous materials in a way
that protects themselves, their co-workers,
and the environment. Safe work practices
are defined as tasks performed in a manner that reduces the likelihood of exposure to workplace hazards. General recommendations for safe work practices are
included in Appendix 2-2.

Emergency Response Plan
When engineering and work practice controls fail, employees must know how to respond. The purpose of advance planning
is to control the hazardous situation as
quickly and safely as possible. Regular
testing of the emergency response plan
will identify areas for improvement and
will also build confidence in staff to respond effectively in a real situation. OSHA
requires a written plan for facilities with
more than 10 employees. Verbal communication of the plan is acceptable for 10 or
fewer employees.14

Chapter 2: Facilities and Safety

Management Controls
Supervisory personnel must monitor safety practices in their areas of responsibility.
Continuing attention to safety issues should
be addressed in routine staff meetings
and training sessions. Periodic audits performed by a safety professional help increase safety awareness. Management
should seek staff input into the design and
improvement of the facility’s safety plan.
The safety program, with its policies,
guidelines, and supporting references to
regulatory documents, should be detailed
in a safety manual and made available to all
personnel at risk. This manual, along with
operational procedures manuals, should be
reviewed at least annually and updated as
technology evolves and new information
becomes available. Work sites and safety
equipment also should be inspected regularly to ensure compliance and response
readiness. Checklists are helpful in documenting these audits and assessing safety
preparedness. Checklist items and essential
elements for safety and environmental
management audits can be obtained from
other sources2,3,15 or can be developed internally.

Employee Health Services

Hepatitis Prophylaxis
All employees routinely exposed to blood
must be offered hepatitis B virus (HBV)
vaccine if they do not already have HBVprotective antibodies (ie, anti-HBs). OSHA
requires that the vaccine be offered at no
cost to the employee, and if the employee
refuses the vaccine, that the refusal be
documented.10

Monitoring Programs
The employer must provide a system for
monitoring exposure to certain substances
as defined in the OSHA standard if there

is reason to believe that exposure levels
routinely exceed the action level.16

Medical First Aid and Follow-up
When requested by a worker who has sustained known or suspected blood exposure, monitoring for HBV, hepatitis C virus
(HCV), and human immunodeficiency virus (HIV) antibodies should be provided
with appropriate counseling. Informed
consent is required for this voluntary testing; rejection of offered testing must be
documented. The usual schedule would
include immediate tests on the worker
and on the source of the potentially infectious material, with follow-up of the worker at intervals after exposure.9,10 All aspects
of accident follow-up should be appropriately documented.
The Centers for Disease Control and Prevention (CDC) has published recommendations for both pre- and postexposure
prophylaxis if the contaminating material is
HBV-positive or if this information is unknown.17 Hepatitis B Immune Globulin is
usually given concurrently with hepatitis B
vaccine in cases of penetrating injuries.
When administered in accordance with the
manufacturer’s directions, both products
are very safe and carry no documented risk
for infection with HBV, HCV, or HIV. Postexposure prophylaxis for HIV is continually evolving; policies are generally based on
Public Health Service recommendations17
and current standards of practice.

Reporting Accidents and Injuries
When an injury occurs, as much relevant
information as possible should be documented (see Table 2-2). In addition, the
supervisor should complete any accident
reports and investigation forms required
by the institution’s insurer and worker’s
compensation agencies. Medical records
for individual employees should be pre-

AABB Technical Manual

Table 2-2. Information to Be Included in Injury Reports
■

Name and address of the injured person.

■

Time of the injury (hour, day, month, year).

■

Specified place where the injury occurred.

■

Details of the injured person’s activities at the time of injury.

■

Nature of the injury (eg, bruise, laceration, burn, etc).

■

Part of the body injured (eg, head, arm, leg, etc).

■

Nature of the known or potential agent, in cases of exposure to pathologic organisms or other
hazardous materials.

■

Nature of medical attention or first aid applied in the workplace.

■

Date the injured person stopped work.

■

Date the injured person returned to work.

■

Estimated cost of damage to property or to equipment.

■

Injured person’s statement of the events leading to the injury.

■

Statements from witnesses.

■

Cause of the injury.

■

Corrective action taken or recommendations for corrective action.

served for the duration of employment
plus 30 years, with few exceptions.17
OSHA requires health service employers
with 11 or more workers to maintain records of occupational injuries and illnesses
that require care that exceeds the capabili18
ties of a person trained in first aid. Records
of first aid provided by a nonphysician for
minor injuries such as cuts or burns do not
have to be retained. Initial documentation
must be completed within 6 days of the incident. All logs, summaries, and supplemental records must be preserved for at
least 5 years beyond the calendar year of
occurrence. Employers must report fatalities and injuries resulting in the hospitalization of three or more employees to
18
OSHA within 8 hours of the accident.

Latex Allergies
With the increased use of gloves, there has
been a rise in the number of health-care

workers with latex allergies. Adverse reactions associated with latex and/or powdered
gloves include contact dermatitis, allergic
dermatitis, urticaria, and anaphylaxis.
Medical devices that contain latex must
bear a caution label. The National Institute for Occupational Safety and Health
offers the following recommendations19:
■

Make nonlatex gloves available as an
alternative to latex. Encourage use of
nonlatex gloves for activities and
work environments where there is
minimal risk of exposure to infectious materials.

■

If latex gloves are used, provide reduced protein, powder-free gloves.
(Note: This is not a requirement, but
a recommendation to reduce exposure.)

■

Use good housekeeping practices to
remove latex-containing dust from
the workplace.

Chapter 2: Facilities and Safety

■

■
■
■

Use work practices that reduce the
chance of reaction, such as hand washing and avoiding oil-based hand lotions.
Provide workers with education programs and training materials about
latex allergy.
Periodically screen high-risk workers
for latex allergy symptoms.
Evaluate current prevention strategies.
If symptoms of latex allergy develop,
avoid direct contact with latex and
consult a physician about allergy
precautions.

pation and understanding should be documented.

Hazard Identification and Communication
Emergency exits must be clearly marked
with an “EXIT” sign. Additional signage
must be posted along the route of egress
to show the direction of travel if it is not
immediately apparent. All flammable materials should be labeled with appropriate
hazard warnings and flammable storage
cabinets should be clearly marked.

Engineering Controls and Personal
Protective Equipment

Fire Prevention
Fire prevention relies on a combination of
facility design based on the National Fire
Protection Association (NFPA) Life Safety
Code,20 defined processes to maintain fire
protection systems in good working order,
and fire safe work practices. The Life
Safety Code includes both active and passive fire protection systems (eg, alarms,
smoke detectors, sprinklers, egress lights
and corridors, and fire-rated barriers).

Training
Fire safety training is recommended at the
start of employment and at least annually
thereafter. Training should emphasize
prevention and an employee’s awareness
of the work environment, including how
to recognize and report unsafe conditions, how to report fires, the locations of
the nearest alarm and fire containment
equipment and their use, and evacuation
policies and routes.
All staff are required to participate in fire
drills at least annually by the CAP and the
JCAHO. 2 , 4 In areas where patients are
housed or treated, the JCAHO requires
quarterly drills on each shift. Staff partici-

Laboratories storing large volumes of
flammable chemicals are usually built
with 2-hour fire separation walls, or with
1-hour separation if there is an automatic
fire extinguishing system.3 Permanent exit
routes must be designed to provide free
and unobstructed egress from all parts of
the facility to an area of safety. Secondary
exits may be required for areas larger than
1000 square feet; consult local safety authority having jurisdiction such as the
local fire marshal and the NFPA. Fire detection and alarm systems should be provided in accordance with federal, state,
and local regulations.

Safe Work Practices
All fire equipment should be inspected on
a regular basis to ensure good working order. Fire extinguishers should be made
readily available and staff should be
trained to use them properly. Nothing
should be stored along emergency exit
routes that would obstruct evacuation efforts. Exit doors cannot be locked from the
inside. Housekeeping and inventory management plans should be designed to
control the accumulations of flammable
and combustible materials stored in the

AABB Technical Manual

facility. In areas where sprinkler systems
are installed, all items should be stored at
least 18 inches below the sprinkler head.
Local fire codes may require greater clearance.

Emergency Response Plan
The fire emergency response plan should
encompass both facility-wide and areaspecific situations. It should describe reporting and alarm systems; location and
use of emergency equipment; roles and
responsibilities for staff during the response; “defend in place” strategies; and
conditions for evacuation, evacuation pro4,14
cedures, and routes of egress. When a
fire occurs, the general sequence for immediate response should be to 1) rescue
anyone in immediate danger, 2) activate
the fire alarm system and alert others in
the area, 3) confine the fire by closing doors
and shutting off fans or other oxygen
sources if possible, and 4) extinguish the
fire with a portable extinguisher if the fire
is small, or evacuate if it is too large to
manage.

Electrical Safety
Electrical hazards, including fire and
shock, may arise from use of faulty electrical equipment, damaged receptacles,
connectors or cords, or unsafe work practices. Proper use of electrical equipment,
periodic inspection and maintenance,
and hazard recognition training are essential to help prevent accidents that may
result in electric shock or electrocution.
The severity of shock depends on the path
that the electrical current takes through
the body, the amount of current flowing
through the body, and the length of time
that it is flowing through the body. Even
low-voltage exposures can lead to serious
injury.21

Training
Safety training should be designed to make
employees aware of electrical hazards associated with receptacles and connectors
and help them recognize potential problems such as broken receptacles and connectors, improper electrical connections,
damaged cords, and inadequate grounding.

Hazard Identification and Communication
The safety plan should address the proper
use of receptacles and connectors. Equipment that does not meet safety standards
should be marked to prevent accidental use.

Engineering Controls and Personal
Protective Equipment
OSHA requires that electrical systems and
equipment be constructed and installed
in a way that minimizes the potential for
workplace hazards. When purchasing
equipment, the facility should verify that
it bears the mark of an OSHA-approved
independent testing laboratory such as
Underwriters Laboratories (UL).22 Adequate working space should be provided
around equipment to allow easy access
for safe operation and maintenance.
Ground-fault circuit interrupters should
be installed in damp or wet areas.

Safe Work Practices
Electrical safety practices are focused
around 1) proper use of electrical equipment and 2) proper maintenance and repair. Staff should not plug or unplug
equipment from an electrical source when
their hands are wet. Overloading circuits
with too many devices may cause the current to heat the wires to a very high temperature and generate a fire. Damaged receptacles and faulty electrical equipment
must be tagged and removed from service

Chapter 2: Facilities and Safety

until they have been repaired and checked for safety. Flexible cords should be secured to prevent tripping and should be
protected from damage from heavy or
sharp objects. Slack in flexible cords
should be kept to prevent tension on electrical terminals and cords should be
checked for cut, broken, or cracked insulation. Extension cords should not be
used in lieu of permanent wiring.

Emergency Response Plan
When it is not possible to decrease the
power or disconnect equipment, the power
supply should be shut off from the circuit
breaker. If it is not possible to interrupt
the power supply, a nonconductive material such as dry wood should be used to
pry a victim from the source of current.21
Victims must not be touched directly.
Emergency first aid for victims of electrical shock must be sought. Water-based
fire extinguishers are not to be used on
electrical fires.

Biosafety
The blood bank or transfusion service must
define and enforce measures to minimize
the risk of exposure to biohazardous materials in the workplace. Requirements
published by OSHA (Bloodborne Pathogen
Standard)10 and recommendations published by the US Department of Health and
Human Services9,12 provide the basis for
an effective biosafety plan.

Bloodborne Pathogen Standard
The OSHA Bloodborne Pathogen Standard is intended to protect employees in
all occupations where there is a risk of exposure to blood and other potentially infectious materials. It requires that the facility develop an Exposure Control Plan
and describes appropriate engineering

controls, personal protective equipment,
and work practice controls to minimize
the risk of exposure. It also requires employers to provide hepatitis B vaccination
for staff with occupational exposure, to
provide medical follow-up in case of accidental exposure, and to keep records related to accidents and exposures.

Standard Precautions
Standard Precautions represent the most
current recommendations by CDC to reduce the risk of transmission of bloodborne and other pathogens in hospitals.
Published in 1996 in the Guidelines for
Isolation Precautions in Hospitals,9 they
build on earlier recommendations, including Body Substance Isolation (1987),
Universal Precautions (1986), and Blood
and Body Fluid Precautions (1983). The
Bloodborne Pathogen Standard refers to
the use of Universal Precautions; however,
OSHA recognizes the more recent guidelines from the CDC and, in Directive CPL
2-2.69, allows hospitals to use acceptable
alternatives, including Standard Precautions, as long as all other requirements in
the standard are met.23
Standard Precautions apply to all patient
care activities regardless of diagnosis where
there is a risk of exposure to 1) blood; 2) all
body fluids, secretions, and excretions, except sweat; 3) nonintact skin; and 4) mucous
membranes.

Biosafety Levels
Recommendations for biosafety in laboratories are based on the potential hazards for specific infectious agents and the
12
activities performed. They include guidance on both engineering controls and
safe work practices. The four biosafety
levels are designated in ascending order,
with increasing protection for personnel,
the environment, and the community.

AABB Technical Manual

Biosafety Level 1 (BSL-1) involves work
with agents of no known or of minimal potential hazard to laboratory personnel and
the environment. Activities are usually conducted on open surfaces and no containment equipment is needed.
Biosafety Level 2 (BSL-2) work involves
agents of moderate potential hazard to personnel and the environment, usually from
contact-associated exposure. Most blood
bank laboratory activities are considered
BSL-2. Precautions described in this section
will focus on BSL-2 requirements. Laboratories should consult the CDC or National
Institutes of Health (NIH) guidelines for
precautions appropriate for higher levels of
containment.
Biosafety Level 3 (BSL-3) includes work
with indigenous or exotic agents that may
cause serious or potentially lethal disease
as a result of exposure to aerosols (eg, Mycobacterium tuberculosis) or by other routes
that would result in grave consequences to
the infected host (eg, HIV). Recommendations for work at BSL-3 are designed to contain biohazardous aerosols and minimize
the risk of surface contamination.
Biosafety Level 4 (BSL-4) applies to work
with dangerous or exotic agents that pose
high individual risk of life-threatening disease from aerosols (eg, agents of hemorrhagic fevers, filoviruses). BSL-4 is not
applicable to routine blood-bank-related
activities.

Training
OSHA requires annual training for all employees whose tasks carry risk of infec10,23
Training programs
tious exposure.
must be tailored to the target group, both
in level and content. General background
knowledge of biohazards, understanding
of control procedures, or work experience
cannot meet the requirement for specific
training, although assessment of such

knowledge is a first step in planning program content. Workplace volunteers require
at least as much safety training as paid
staff performing similar functions.

Hazard Identification and Communication
The facility’s Exposure Control Plan communicates the risks present in the workplace and describes controls to minimize
exposure. BSL-2 through BSL-4 facilities
must have a biohazard sign posted at the
entrance when infectious agents are in
use. It serves to notify personnel and
visitors about the agents used, a point of
contact for the area, and any special protective equipment or work practices required.
Biohazard warning labels must be placed
on containers of regulated waste; refrigerators and freezers containing blood or other
potentially infectious material; and other
containers used to store, transport, or ship
blood or other potentially infectious materials. Blood components that are labeled to
identify their contents and have been released for transfusion or other clinical use
are exempted.

Engineering Controls and Personal
Protective Equipment
OSHA requires that hazards be controlled
by engineering or work practices whenever possible. Engineering controls for
BSL-2 laboratories include limited access
to the laboratory when work is in progress
and biological safety cabinets or other
containment equipment for work that may
involve infectious aerosols or splashes.
Hand-washing sinks and eyewash stations
must be available. The work space should
be designed so that it can be easily cleaned
and bench-tops should be impervious to
water and resistant to chemicals and solvents.

Chapter 2: Facilities and Safety

Biological safety cabinets (BSCs) are primary containment devices for handling
moderate- and high-risk organisms. There
are three types—Class I, II, and III—with
Class III providing the highest protection to
the worker. A comparison of the features
and applications for the three classes of
cabinets is provided in Table 2-3.24 BSCs are
not required for Standard Precautions, but
centrifugation of open blood samples or
manipulation of units known to be positive
for HBsAg or HIV are examples of blood
bank procedures for which a BSC could be
useful. The effectiveness of the BSC is a
function of directional airflow inward and
downward, through a high-efficiency filter.
Efficacy is reduced by anything that disrupts the airflow pattern, eg, arms moving
rapidly in and out of the BSC, rapid movements behind the employee using the BSC,
downdrafts from ventilation systems, or
open laboratory doors. Care should be
taken not to block the front intake and rear
exhaust grills. Performance should be certified annually.25
Injuries from contaminated needles and
other sharps continued to be a major concern in health-care settings even after the
Bloodborne Pathogens Standard went into
effect. In 2001, OSHA revised the standard
to include reference to engineered sharps
injury protections and needleless systems.26
It requires that employers implement appropriate new control technologies and safer
medical devices in their Exposure Control
Plan and that they solicit input from their
employees to identify, evaluate, and select
engineering and work practice controls. Examples of safer devices are needleless systems and self-sheathing needles in which
the sheath is an integral part of the device.

Decontamination
Reusable equipment and work surfaces
that may be contaminated with blood re-

quire daily cleaning and decontamination.
Obvious spills on equipment or work surfaces should be cleaned up immediately;
routine wipe-downs with disinfectant
should occur at the end of each shift or on
a regular basis that provides equivalent
safety. Equipment that is exposed to blood
or other potentially infectious material
must be decontaminated before servicing
or shipping. When decontamination of all
or a portion of the equipment is not feasible, a biohazard label stating which portions remain contaminated should be attached before servicing or shipping.

Choice of Disinfectants
The Environmental Protection Agency
(EPA) maintains a list of chemical products that have been shown to be effective
antimicrobial disinfectants.27 (See http://
www.epa.gov/oppad001/chemreginex.htm
for a current list.) The Association for Professionals in Infection Control and Epidemiology also publishes a guideline to assist health-care professionals in their
decisions involving judicious selection
and proper use of specific disinfectants.28
For facilities covered under the Bloodborne Pathogens Rule, OSHA allows the
use of EPA-registered tuberculocidal disinfectants, EPA-registered disinfectants
that are effective against both HIV and
HBV, and/or a diluted bleach solution to
decontaminate work surfaces.23
Before selecting a product, workers
should consider several factors. Among
them are the type of material or surface to
be treated, the hazardous properties of the
chemical such as corrosiveness, and the
level of disinfection required. After selecting a product, procedures need to be written to ensure effective and consistent
cleaning and treatment of work surfaces.
Some factors to consider for effective de-

Table 2-3. Comparison of Class I, II, and III Biological Safety Cabinets*
Intended Use

Common Applications

Class I

Unfiltered room air is drawn into the cabinet. Inward airflow protects personnel
from exposure to materials inside the
cabinet. Exhaust is HEPA filtered to protect the environment. Maintains airflow at
a minimum velocity of 75 linear feet per
minute (lfpm) across the front opening
(face velocity).

Personal and environmental protection

To enclose equipment (eg, centrifuges) or
procedures that may generate aerosols

Class II (Generalapplies to all
types of Class II
cabinets)

Uses laminar flow (air moving at a constant
velocity in one direction along parallel
lines). Room air is drawn into the front
grille. HEPA filtered air is forced downward in a laminar flow to minimize
cross-contamination of materials in the
cabinet. Exhaust is HEPA filtered.

Personal, environmental, and product protection

Work with microorganisms assigned to
biosafety levels 1, 2, or 3
Handling of products where prevention of
contamination is critical, such as cell culture propagation or manipulation of
blood components in an open system

Class II, A

75% of air is recirculated after passing
through a HEPA filter. Face velocity = 75
lfpm.

See Class II, general

Class II, B1

70% of air exits through the rear grille, is
HEPA filtered, and then discharged from
the building. The other 30% is drawn into
the front grille, HEPA filtered, and
recirculated. Face velocity = 100 lfpm.

See Class II, general

Allows for safe manipulation of small quantities of hazardous chemicals and
biologics

AABB Technical Manual

Main Features

100% of air is exhausted; none is
recirculated. A supply blower draws air
from the room or outside and passes it
through a HEPA filter to provide the
downward laminar flow. Face velocity =
100 lfpm.

See Class II, general

Provides both chemical and biological containment. More expensive to operate because of the volume of conditioned room
air being exhausted.

Class II, B3

Similar in design to Type A, but the system
is ducted and includes a negative pressure system to keep any possible contamination within the cabinet. Face velocity = 100 lfpm.

See Class II, general

Allows for safe manipulation of small quantities of hazardous chemicals and
biologics

Class III

Cabinet is airtight. Materials are handled
with rubber gloves attached to the front
of the cabinet. Supply air is HEPA filtered. Exhaust air is double HEPA filtered
or may have one filter and an air incinerator. Materials are brought in and out of
the cabinet either through a dunk tank or
a double-door pass-through box that can
be decontaminated. Cabinet is kept under
negative pressure.

Maximum protection to
personnel and environment.

Work with biosafety level 4 microorganisms

*Data from the US Department of Health and Human Services. 24

Chapter 2: Facilities and Safety

Class II, B2

AABB Technical Manual

contamination include the contact time,
the type of microorganisms, the presence of
organic matter, and the concentration of
the chemical agent. Workers should review
the basic information on decontamination
and follow the manufacturer’s instructions.

■

Storage
Hazardous materials must be segregated
and areas for different types of storage
must be clearly demarcated. Blood must
be protected from unnecessary exposure
to other materials and vice versa. If transfusion products cannot be stored in a separate refrigerator from reagents, specimens,
and unrelated materials, areas within the
refrigerator must be clearly labeled, and
extra care must be taken to reduce the
likelihood of spills and other accidents.
Storage areas must be kept clean and orderly; food or drink is never allowed where
biohazardous materials are stored.

■

Personal Protective Equipment
Where hazards cannot be eliminated, OSHA
requires employers to provide appropriate
PPE and clothing, and to clean, launder,
or dispose of PPE at no cost to their employees.10 Standard PPE and clothing include uniforms, laboratory coats, gloves,
face shields, masks, and safety goggles. Indications and guidelines for their use are
discussed in Appendix 2-2.

Safe Work Practices
Safe work practices appropriate for Standard Precautions include the following:
■
Wash hands after touching blood,
body fluids, secretions, excretions, and
contaminated items, whether or not
gloves are worn.
■
Wear gloves when touching blood,
body fluids, secretions, excretions, and
contaminated items, and change them
between tasks.

■

Wear a mask and eye protection or a
face shield during activities that are
likely to generate splashes or sprays
of blood, body fluids, secretions, and
excretions.
Wear a gown during activities that
are likely to generate splashes or
sprays of blood, body fluids, secretions, or excretions.
Handle soiled patient-care equipment
in a manner that prevents exposures; ensure that reusable equipment is not used for another patient
until it has been cleaned and reprocessed appropriately; and ensure
that single-use items are discarded
properly.
Ensure that adequate procedures are
defined and followed for the routine
care, cleaning, and disinfection of
environmental surfaces and equipment.
Handle soiled linen in a manner that
prevents exposures.
Handle needles, scalpels, and other
sharp instruments or devices in a
manner that minimizes the risk of
exposure.
Use mouthpieces, resuscitation bags,
or other ventilation devices as an alternative to mouth-to-mouth resuscitation methods.
Place in a private room those patients who are at risk of contaminating the environment or who are not
able to maintain appropriate hygiene (eg, tuberculosis).

Laboratory Biosafety Precautions
Several factors need to be considered when
assessing the risk of blood exposures among
laboratory personnel. Some factors include the number of specimens processed,
personnel behaviors, laboratory techniques, and type of equipment.29 The lab-

Chapter 2: Facilities and Safety

oratory director may wish to institute
BSL-3 practices for procedures that are
considered to be higher risk than BSL-2.
When there is doubt whether an activity is
BSL-2 or BSL-3, the safety precautions for
BSL-3 should be followed. BSL-2 precautions that are applicable to the laboratory
setting are summarized in Appendix 2-3.

Considerations for the Donor Room
The Bloodborne Pathogen Standard acknowledges a difference between hospital
patients and healthy donors, in whom the
prevalence of infectious disease markers
is significantly lower. The employer in a
volunteer blood donation facility may determine that routine use of gloves is not
10
required for phlebotomy as long as :
The policy is periodically reevaluated.
■
Gloves are made available to those
■
who want to use them, and use is not
discouraged.
■
Gloves are required when an employee has cuts, scratches, or breaks
in skin; when there is a likelihood
that contamination will occur; while
drawing autologous units; while performing therapeutic procedures;
and during training in phlebotomy.
Procedures used in the donation of blood
should be assessed for risks of biohazardous exposures and risks inherent in working with a donor or patient. Some procedures are more likely to cause injury than
others, such as using lancets for finger
puncture, handling capillary tubes, crushing vials for arm cleaning, handling any unsheathed needle, cleaning scissors, and giving cardiopulmonary resuscitation.
In some instances, it may be necessary
to collect blood from donors known to pose
a high risk of infectivity (eg, collection of
autologous blood or Source Plasma for the
production of other products such as vaccines). The Food and Drug Administration

(FDA) provides guidance for collecting
blood from such “high-risk” donors.30 The
most recent regulations and guidelines
should be consulted for changes or additions.

Emergency Response Plan

Blood Spills
Every facility handling blood should be
prepared for spills in advance. Table 2-4
lists steps to be taken when a spill occurs.
Cleanup is easier when preparation includes the following elements:
Design work areas so that cleanup is
■
relatively simple.
Prepare a spill kit or cart that con■
tains all necessary supplies and equipment with instructions for their use.
Place it near areas where spills are
anticipated.
Assign responsibility for kit/cart main■
tenance, spill handling, record-keeping, and review of significant incidents.
■
Train personnel in cleanup procedures and reporting of significant incidents.

Biohazardous Waste
Medical waste is defined as any waste
(solid, semisolid, or liquid) generated in
the diagnosis, treatment, or immunization
of human beings or animals in related research, production, or testing of biologics.
Infectious waste includes disposable equipment, articles, or substances that may
harbor or transmit pathogenic organisms
or their toxins. In general, infectious
waste should either be incinerated or decontaminated before disposal in a sanitary landfill. Blood and components,
suctioned fluids, excretions, and secretions may be carefully poured down a
drain connected to a sanitary sewer if
state law allows. Sanitary sewers may also

AABB Technical Manual

Table 2-4. Blood Spill Cleanup
■

Contain the spill if possible.

■

Evacuate the area for 30 minutes if an aerosol has been created.

■

Post warnings to keep the area clear.

■

Remove clothing if it is contaminated.

■

If the spill occurs in the centrifuge, turn the power off immediately and leave the cover closed
for 30 minutes. The use of overwraps helps prevent aerosolization and helps contain the spill.

■

Wear appropriate protective clothing and gloves. If sharp objects are involved, gloves must be
puncture-resistant, and a broom or other instrument should be used during cleanup to avoid
injury.

■

Use absorbent material to mop up most of the liquid contents.

■

Clean the spill area with detergent.

■

Flood the area with disinfectant and use it as described in the manufacturer’s instructions. Allow
adequate contact time with the disinfectant.

■

Wipe up residual disinfectant if necessary.

■

Dispose of all materials safely in accordance with biohazard guidelines. All blood-contaminated
items must be autoclaved or incinerated.

be used to dispose of other potentially infectious wastes that can be ground and
flushed into the sewer. State and local
health departments should be consulted
about laws and regulations on disposal of
biological waste into the sewer.
Laboratories should clearly define what
will be considered hazardous waste. For example, in the blood bank items contaminated with liquid or semiliquid blood are
biohazardous. Items contaminated with
dried blood are considered hazardous if
there is potential for the dried material to
flake off during handling. Contaminated
sharps are always considered hazardous
because of the risk for percutaneous injury.
However, items such as used gloves, swabs,
plastic pipettes with excess liquid removed,
or gauze contaminated with small droplets
of blood may be considered nonhazardous
if the material is dried and will not be
released into the environment during handling.

Guidelines for Biohazardous Waste Disposal. Employees must be trained before
handling or disposing of biohazardous
waste, even if it is packaged. The following
31
disposal guidelines are recommended :
■

Identify biohazardous waste consistently; red seamless plastic bags (at
least 2 mil thick) or containers carrying the biohazard symbol are recommended.

■

Place bags in a protective container
with closure upward to avoid breakage and leakage during storage or
transport.

■

When transported over public roads,
the waste must be prepared and
shipped according to US Department
of Transportation regulations.

■

Discard sharps (eg, needles, broken
glass, glass slides, wafers from sterile
connecting devices) in rigid, puncture-proof, leakproof containers.

Chapter 2: Facilities and Safety

■

Put liquids only in leakproof, unbreakable containers.

■

Do not compact waste materials.
Storage areas for infectious material
must be secured to reduce accident risk.
Infectious waste must never be placed in
the public trash collection system. Most facilities hire private carriers to decontaminate and dispose of infectious or hazardous
waste. Contracts with these companies
should include disclosure of the risks of
handling the waste by the facility, and an
acknowledgment by the carrier that all federal, state, and local laws for biohazardous
(medical) waste transport, treatment, and
disposal are known and followed.
Treating Infectious or Medical Waste.
Facilities that incinerate hazardous waste
must comply with EPA standards of performance for new stationary sources and
emission guidelines for existing sources.32
In this regulation, a hospital/medical/infectious waste incinerator (HMIWI) is any
device that combusts any amount of hospital waste or medical/infectious waste.
Decontamination of biohazardous waste
by autoclaving is another common method
for decontamination/inactivation of blood
samples and blood components. The following elements are considered in determining processing time for autoclaving:
■

Size of load being autoclaved.

■

Type of packaging of item(s) being
autoclaved.

■

Density of items being autoclaved.

■

Number of items in single autoclave
load.

■

Placement of items in the autoclave,
to allow for steam penetration.
It is useful to place a biological indicator
in the center of loads that vary in size and
contents to evaluate optimal steam penetration times. The EPA provides detailed information about choosing and operating
such equipment.31

Longer treatment times are needed for
sterilization, but decontamination requires
a minimum of 1 hour. A general rule is to
process 1 hour for every 10 pounds of waste
being processed. Usually, decontaminated
laboratory wastes can be disposed of as
nonhazardous solid wastes. Staff should
check with the local solid waste authority to
ensure that the facility is in compliance
with the regulations for their area. Waste
containing broken glass or other sharp
items should be disposed of in a method
consistent with policies for the disposal of
other sharp or potentially dangerous materials.

Chemical Safety
One of the most effective preventive measures a facility can take to reduce hazardous chemical exposure is to evaluate the
use of alternative nonhazardous chemicals whenever possible. A review of ordering practices of hazardous chemicals can
result in the purchase of smaller quantities of hazardous chemicals, thus reducing the risk of storing excess chemicals
and later dealing with the disposal of
these chemicals.
OSHA requires that facilities using hazardous chemicals develop a written Chemical Hygiene Plan (CHP) and that the plan
be accessible to all employees. The CHP
should outline procedures, equipment,
personal protective equipment, and work
practices that are capable of protecting employees from hazardous chemicals used in
the facility.11,16 This plan must also provide
assurance that equipment and protective
devices are functioning properly and that
criteria to determine implementation and
maintenance of all aspects of the plan are
in control. Employees must be informed of
all chemical hazards in the workplace and
be trained to recognize chemical hazards,

AABB Technical Manual

to protect themselves when working with
these chemicals, and where to find information on particular hazardous chemicals.
Appendix 2-4 provides an example of a hazardous chemical data safety sheet that may
be used in the CHP. Safety audits and annual reviews of the CHP are important control steps to help ensure that safety practices comply with the policies set forth in
the CHP and that the CHP is up to date.
Establishing a clear definition of what
constitutes hazardous chemicals is sometimes difficult. Generally, hazardous chemicals are those that pose a significant health
risk if an employee is exposed to them or
pose a significant physical risk, such as fire
or explosion, if handled or stored improperly. Categories of health and physical
hazards are listed in Tables 2-5 and 2-6. Appendix 2-5 lists examples of hazardous chemicals that may be found in the blood
bank.
The facility should identify a qualified
chemical hygiene officer to be responsible
for determining guidelines for hazardous
materials.16 The chemical hygiene officer is

also accountable for monitoring and documenting accidents and initiating process
change as needed.

Training
Initial training is required for all employees who may be exposed to hazardous
chemicals—before they begin work in an
area where hazards exist. If an individual
has received prior training, it may not be
necessary to retrain them, depending on
the employer’s evaluation of the new employee’s level of knowledge. New employee
training is likely to be necessary regarding
such specifics as the location of the relevant Material Safety Data Sheets (MSDS),
details of chemical labeling, the personal
protective equipment to be used, and
site-specific emergency procedures.
Training must be provided whenever a
new physical or health hazard is introduced
into the workplace, but not for each new
chemical that falls within a particular hazard class.11 For example, if a new solvent is
brought into the workplace and it has haz-

Table 2-5. Categories of Health Hazards
Hazard

Definition

Carcinogens

Cancer-producing substances

Irritants

Agents causing irritations (edema, burning, etc) to skin or mucous membranes upon contact

Corrosives

Agents causing destruction of human tissue at the site of contact

Toxic or highly toxic agents

Substances causing serious biologic effects following inhalation, ingestion, or skin contact with relatively small amounts

Reproductive toxins

Chemicals that affect reproductive capabilities, including chromosomal damages and effects on fetuses

Other toxins

Hepatotoxins, nephrotoxins, neurotoxins, agents that act on the
hematopoietic systems, and agents that damage the lungs,
skin, eyes, or mucous membranes

Chapter 2: Facilities and Safety

Table 2-6. Categories of Physical Hazards
Hazard

Definition

Combustible or flammable
chemicals

Chemicals that can burn (includes combustible and flammable
liquids, solids, aerosols, and gases)

Compressed gases

A gas or mixture of gases in a container under pressure

Explosives

Unstable or reactive chemicals that undergo violent chemical
change at normal temperatures and pressure

Unstable (reactive) chemicals

Chemicals that could be self-reactive under conditions of
shocks, pressure, or temperature

Water-reactive chemicals

Chemicals that react with water to release a gas that is either
flammable or presents a health hazard

ards similar to existing chemicals for which
training has already been conducted, then
the employer need only make employees
aware of the new solvent’s hazard category
(eg, corrosive, irritant). However, if the
newly introduced solvent is a suspected
carcinogen and carcinogenic hazard training has not been provided before, then new
training must be conducted for employees
with potential exposure. Retraining is advisable as often as necessary to ensure that
employees understand the hazards, particularly the chronic and specific target-organ
health hazards, linked to the materials with
which they work.

Hazard Identification and Communication

Hazard Communication
Employers must prepare a comprehensive
hazard communication program for all
areas using hazardous chemicals to complement the CHP and to “ensure that the
hazards of all chemicals produced or imported are evaluated, and that information
concerning their hazards is transmitted to
employers and employees.” 11 The program should include labeling hazardous
chemicals, when and how to post warning
labels for chemicals, managing MSDS re-

ports for hazardous chemicals in the facilities, and employee training.
Safety materials made available to employees should include:
The facility’s written CHP.
■
■
The facility’s written program for
hazard communication.
■
Identification of work areas where
hazardous chemicals are located.
■
Required list of hazardous chemicals
and their MSDS. (It is the responsibility of the facility to determine
which chemicals may present a hazard to employees. This determination should be based on the quantity
of chemical used; the physical properties, potency, and toxicity of the
chemical; the manner in which the
chemical is used; and the means
available to control the release of, or
exposure to, the chemical.)

Hazardous Chemical Labeling and Signs
The Hazard Communication Standard requires manufacturers of chemicals and
hazardous materials to provide the user
with basic information about the hazards
of these materials through product labeling and Material Safety Data Sheets.11 Em-

AABB Technical Manual

ployers are required to provide the following to employees who are expected to work
with these hazardous materials: information about the hazards of the materials,
how to read the labeling, how to interpret
symbols and signs on the labels, and how
to read and use the MSDS. Table 2-7 lists
the elements to be included in an MSDS.
At a minimum, hazardous chemical container labels must include the name of the
chemical, the name and address of the
manufacturer, hazard warnings, labels,
signs, placards, and other forms of warning
to provide visual reminders of specific hazards. The label may refer to the MSDS for
additional information. Labels applied by
the manufacturer must remain on containers. The user may add storage requirements
and dates of receipt, opening, and expiration. If chemicals are aliquotted into secondary containers, the secondary container
must be labeled with the name of the chemical and appropriate hazard warnings.
Additional information such as precautionary measures, concentration if applicable,
and date of preparation are helpful but not
mandatory. It is a safe practice to label all
containers with the content, even water.

Transfer containers used for temporary
storage need not be labeled if the person
performing the transfer retains control and
intends them for immediate use. Information regarding acceptable standards for
hazard communication labeling is provided
by the NFPA33 and the National Paint and
34
Coatings Association.
Signs meeting OSHA requirements must
be posted in areas where hazardous chemicals are used. Decisions on where to post
warning signs are based on the manufacturer’s recommendations on the chemical
hazards, the quantity of the chemical in the
room or laboratory, and the potency and
toxicity of the chemical.

Material Safety Data Sheets
The MSDS identifies the physical and
chemical properties of a hazardous chemical (eg, flash point, vapor pressure), its
physical and health hazards (eg, potential
for fire, explosion, signs and symptoms of
exposure), and precautions for safe handling and use. Specific instructions in an
individual MSDS take precedence over

Table 2-7. Required Elements of a Material Safety Data Sheet
■

Identity of product as it appears on label

■

Chemical and common name(s) of all hazardous ingredients

■

Physical/chemical characteristics

■

Fire and explosion hazard data

■

Reactivity data

■

Health hazard data, including primary route(s) of entry and exposure limits

■

Precautions for safe handling and use

■

Exposure control measures

■

Emergency and first aid procedures

■

Manufacturer information, MSDS revision date

Chapter 2: Facilities and Safety

generic information in the Hazardous Materials (HAZMAT) program.
Employers must maintain copies of the
required MSDS in the workplace for each
hazardous chemical and must ensure that
they are readily accessible during each
work shift to employees when they are in
their work areas. When household consumer products are used in the workplace
in the same manner that a consumer would
use them, ie, where the duration and frequency of use (and therefore exposure) are
not greater than those the typical consumer
would experience, OSHA does not require
that an MSDS be provided to purchasers.
However, if exposure to such products exceeds that normally found in consumer applications, then employees have a right to
know about the properties of such hazardous chemicals. OSHA does not require or
encourage employers to maintain an MSDS
for nonhazardous chemicals.

Engineering Controls and Personal
Protective Equipment
Guidelines for laboratory areas in which
hazardous chemicals are used or stored
must be established. Physical facilities,
especially ventilation, must be adequate
for the nature and volume of work conducted. Chemicals must be stored according to chemical compatibility (eg, corrosives,
flammables, oxidizers, etc) and in minimal volumes. Bulk chemicals should be
kept outside work areas. NFPA standards
and others provide guidelines for proper
3,33,35
storage.
Chemical fume hoods are recommended
for use with organic solvents, volatile liquids, and dry chemicals with a significant
inhalation hazard.3 Although constructed
with safety glass, most fume hood sashes
are not designed as safety shields. Hoods
should be positioned in an area where
there is minimal foot traffic to avoid dis-

rupting the airflow and compromising the
containment field.
Personal protective equipment that may
be provided depending on the hazardous
chemicals used includes chemical resistant
gloves and aprons, shatterproof safety goggles, and respirators.
Emergency showers should be available
to areas where caustic, corrosive, toxic,
flammable, or combustible chemicals are
used.3,36 There should be unobstructed access, within 10 seconds, from the areas
where hazardous chemicals are used. Safety
showers should be periodically flushed and
tested for function, and associated floor
drains should be checked to ensure that
drain traps remain filled with water.

Safe Work Practices
Hazardous material should not be stored
or transported in open containers. Containers and their lids or seals should be
designed to prevent spills or leakage in all
reasonably anticipated conditions. Containers should be able to safely store the
maximum anticipated volume and should
be easy to clean. Surfaces should be kept
clean and dry at all times. When working
with a chemical fume hood, all materials
should be kept at a distance of at least six
inches behind the face opening; the vertical sliding sash should be positioned at
the height specified on the certification
sticker. The airfoil, baffles and rear ventilation slot must not be blocked. Appendix
2-6 lists specific chemicals and suggestions on how to work with them safely.

Emergency Response
The time to prepare for a chemical spill is
before a spill occurs. A comprehensive
employee training program should provide the employee with all tools necessary
to act responsibly at the time of a chemi-

AABB Technical Manual

cal spill. The employee should know response procedures, be able to assess the
severity of a chemical spill, know or be
able to quickly look up the basic physical
characteristics of the chemicals, and know
where to find emergency response phone
numbers. The employee should be able
to: assess, stop, and confine the spill; either clean up the spill or call for a spill
clean-up team; and follow up on the report of the spill. The employee must know
when to ask for assistance, when to isolate the area, and where to find cleanup
materials.
Chemical spills in the workplace can be
categorized as follows37:
Incidental releases are spills that are
■
limited in quantity and toxicity and
pose no significant safety or health
hazard to the employee. They may
be safely cleaned up by the employees familiar with the hazards of the
chemical involved in the spill. Waste
from the cleanup may be classified
as hazardous and must be disposed
of in the proper fashion. Appendix
2-7 describes appropriate responses
to incidental spills.
■
Releases that may be incidental or
may require an emergency response
are spills that may pose an exposure
risk to the employees depending
upon the circumstances. Considerations such as the hazardous substance properties, the circumstances
of release, and mitigating factors
play a role in determining the appropriate response. The facility’s
emergency response plan should
provide guidance in how to determine whether the spill is incidental
or requires an emergency response.
■
Emergency response releases are
spills that pose a threat to health and
safety regardless of the circumstances surrounding their release.

The spill may require evacuation of
the immediate area. The response typically comes from outside the immediate release area by personnel trained
as emergency responders. These
spills include but are not limited to:
immediate danger to life or health,
serious threat of fire or explosion,
and high levels of toxic substances.
Appendix 2-8 addresses the management of hazardous chemical spills. Spill
cleanup kits or carts tailored to the specific
hazards present should be available in each
area. These may contain the following: rubber gloves and aprons, shoe covers, goggles,
suitable aspirators, general absorbents,
neutralizing agents, broom, dust pan, appropriate trash bags or cans for waste disposal, and cleanup directions. Chemical
absorbents such as clay absorbents or spill
blankets can be used for cleaning up a
number of chemicals and thus may be easier for the employee to use in spill situations.
With any spill of a hazardous chemical,
but especially with a carcinogenic agent, it
is essential to refer to the MSDS and to contact a designated supervisor or designee
trained to handle these spills and hazard3
ous waste disposal. Facility environmental
health and safety personnel also can offer
assistance. The employer must assess the
extent of the employee’s exposure. After an
exposure, the employee must be given an
opportunity for medical consultation to
determine the need for a medical examination.
Another source of a workplace hazard is
the unexpected release of hazardous vapors
into the environment. OSHA has set limits
for exposure to hazardous vapors from
toxic and hazardous substances.38 The potential risk associated with the chemical is
determined by the manufacturer and listed
on the MSDS. See Table 2-8 for a listing of
the limits of exposure.

Chapter 2: Facilities and Safety

Table 2-8. Regulatory Limits for Exposure to Toxic and Hazardous Vapors

Limit

Definition

Permissible exposure
limit

The maximum concentration of vapors in parts per million (ppm) that
an employee may be exposed to in an 8-hour day/40-hour work week.

Short-term exposure
limit

The maximum allowable concentration of vapors that an employee
may be exposed to in a 15-minute period, with a maximum of four exposures per day allowed with at least 1 hour between each.

Ceiling limit

The maximum concentration of vapors that may not be exceeded instantaneously at any time.

Chemical Waste Disposal

Radiation Measurement Units

Most laboratory chemical waste is considered hazardous and is regulated by the
EPA through the Resource Conservation
and Recovery Act (42 U.S.C. § 6901 et seq,
1976). This regulation specifies that hazardous waste can only be legally disposed
of at an EPA-approved disposal facility.
Disposal of chemical waste into a sanitary
sewer is regulated by the Clean Water Act
(33 U.S.C. § 1251 et seq, 1977), and most
states have strict regulations concerning
disposal of chemicals in the water system.
Federal and applicable state regulations
should be consulted when setting up and
reviewing facility waste disposal policies.

The measurement unit quantifying the
amount of energy absorbed per unit mass
of tissue is the Gray (Gy) or rad (radiation
absorbed dose); 1 Gy equals 100 rads.
Dose equivalency measurements are
more useful than simple energy measurements because they take into account the
effectiveness of the different types of radiation to cause biologic effects. The ability of
radiation to cause damage is assigned a
number called a quality factor (QF). For example, exposure to a given amount of alpha
particles (QF = 20) is far more damaging
than exposure to an equivalent amount of
gamma rays (QF = 1). The common unit of
measurement for dose equivalency is the
rem (rad equivalent man). To obtain dose
from a particular type of radiation in rem,
multiply the number of rad by the quality
factor (rad × QF = rem). Because the quality
factor for gamma rays, x-rays, and most
beta particles is 1, the dose in rad is equal
to the dose in rem for these types of radiation.

Radiation Safety
Radiation can be defined as energy in the
form of waves or particles emitted and
propagated through space or a material
medium. Gamma rays are electromagnetic radiation, whereas alpha and beta
emitters are examples of particulate radiation. The presence of radiation in the
blood bank, either from radioisotopes
used in laboratory testing or from selfcontained blood irradiators, requires ad3,39
ditional precautions and training.

Biologic Effects of Radiation
Any harm to tissue begins with the absorption of radiation energy and subsequent disruption of chemical bonds. Mol-

AABB Technical Manual

ecules and atoms become ionized and/or
excited by absorbing this energy. The “direct action” path leads to radiolysis or formation of free radicals that, in turn, alter
the structure and function of molecules in
the cell.
Molecular alterations can cause cellular
or chromosomal changes, depending upon
the amount and type of radiation energy
absorbed. Cellular changes can manifest as
a visible somatic effect, eg, erythema.
Changes at the chromosome level may
manifest as leukemia or other cancers, or
possibly as germ cell defects that are transmitted to future generations.
The type of radiation, the part of the
body exposed, the total absorbed dose, and
the dose rate influence biologic damage.
The total absorbed dose is the cumulative
amount of radiation absorbed in the tissue.
The greater the dose, the greater the potential for biologic damage. Exposure can be
acute or chronic. The low levels of ionizing
radiation likely to occur in blood banks
should not pose any detrimental risk.40-43

Regulations
The NRC controls use of radioactive materials by establishing licensure requirements. States and municipalities may also
have requirements for inspection and/or
licensure. The type of license for using radioisotopes or irradiators will depend on
the scope and magnitude of the use of radioactivity. Facilities should contact the
NRC and appropriate state agencies for license requirements and application as
soon as such activities are proposed.
NRC-licensed establishments must have
a qualified radiation safety officer who is responsible for establishing personnel protection requirements and for proper disposal and handling of radioactive materials.
Specific radiation safety policies and procedures should address dose limits, employee

training, warning signs and labels, shipping
and handling guidelines, radiation monitoring, and exposure management. Emergency procedures must be clearly defined
and readily available to staff.

Exposure Limits
The NRC sets standards for protection
against radiation hazards arising from li8
censed activities, including dose limits.
These limits, or maximum permissible
dose equivalents, are a measure of the radiation risk over time and serve as standards for exposure. The occupational total
effective-dose-equivalent limit is 5 rem/
year. The shallow dose equivalent (skin) is
50 rem/year, the extremity dose equivalent limit is 50 rem/year, and the eye dose
equivalent limit is 15 rem/year.8,41 Dose
limits to an embryo/fetus must not exceed 0.5 rem during the pregnancy.8,41,44
Employers are expected not only to maintain radiation exposure below allowable
limits, but also to keep exposure levels as
far below these limits as can reasonably be
achieved.

Radiation Monitoring
Monitoring is essential for early detection
and prevention of problems due to radiation exposure. It is used to evaluate the
environment, work practices, and procedures, and to comply with regulations and
NRC licensing requirements. Monitoring is
accomplished with the use of dosimeters,
bioassay, survey meters, and wipe tests.3
Dosimeters, such as film or thermoluminescent badges and/or rings, measure
personnel radiation doses. The need for dosimeters depends on the amount and type
of radioactive materials in use; the facility
radiation safety officer will determine individual dosimeter needs. Film badges must

Chapter 2: Facilities and Safety

be changed at least quarterly and in some
instances monthly, protected from high
temperature and humidity, and stored at
work away from sources of radiation.
Bioassay, such as thyroid and whole
body counting or urinalysis, may be used to
determine if there is radioactivity inside the
body and, if so, how much. If necessary,
bioassays are usually performed quarterly
and after an incident where accidental intake may have occurred.
Survey meters are sensitive to low levels
of gamma or particulate radiation and provide a quantitative assessment of radiation
hazard. They can be used to monitor storage areas for radioactive materials or wastes,
testing areas during or after completion of a
procedure, and packages or containers of
radioactive materials. Survey meters must
be calibrated annually by an authorized
NRC licensee. Selection of appropriate meters should be discussed with the radiation
safety officer.
In areas where radioactive materials are
handled, work surfaces, equipment, and
floors that may be contaminated should be
checked regularly with a wipe test. In the
wipe test, a moistened absorbent material
(the wipe) is passed over the surface and
then counted for radiation. Kits are available for this purpose. In most clinical laboratories, exposure levels of radiation are
well below the limits set by federal and
state regulations.

Training
Personnel who handle radioactive materials or work with blood irradiators must receive radiation safety training before beginning work. This training should cover
an explanation of the presence and potential hazards of radioactive materials
found in the employee’s specific work area,
general health protection issues, emergency procedures, and radiation warning

signs and labels in use. Instruction in the
following is also suggested:
NRC regulations and license condi■
tions.
The importance of observing license
■
conditions and regulations and reporting violations or conditions of
unnecessary exposure.
Precautions to minimize exposure.
■
Interpretation of results of monitor■
ing devices.
Requirements for pregnant workers.
■
Employees’ rights.
■
Documentation and record-keeping
■
requirements.
The need for refresher training is determined by the license agreement between
the NRC and the facility.

Engineering Controls and Personal
Protective Equipment
Although self-contained blood irradiators
present little risk to laboratory staff and
film badges are not required for routine
operation, blood establishments with irradiation programs must be licensed by
the NRC.41
The manufacturer of the blood irradiator
usually accepts responsibility for radiation
safety requirements during transportation,
installation, and validation of the unit as
part of the purchase contract. The radiation
safety officer can help oversee the installation and validation processes and confirm
that appropriate training, monitoring systems, procedures, and maintenance protocols are in place before use and reflect the
manufacturer’s recommendations. Suspected malfunctions must be reported immediately to defined facility authorities so
that appropriate actions can be initiated.
Blood irradiators should be located in
secure areas with limited access so that
only trained individuals have access. Fire
protection for the unit must also be consid-

AABB Technical Manual

ered. Automatic fire detection and control
systems should be readily available in the
immediate area. Blood components that
have been irradiated are not radioactive
and pose no threat to staff or the general
public.

If a spill occurs, contaminated skin surfaces must be washed several times and the
radiation safety officer must be notified immediately for further guidance. Others
must not be allowed to enter the area until
emergency response personnel arrive.

Safe Work Practices

Waste Management

Each laboratory should establish policies
and procedures for the safe use of radioactive materials. They should include
requirements for following general laboratory safety principles, appropriate storage of radioactive solutions, and proper
disposal of radioactive wastes. Radiation
safety can be improved with the following:
Minimize time of exposure by work■
ing as efficiently as possible.
Maximize distance from the source
■
of the radiation by staying as far
from the source as possible.
■
Maximize shielding (eg, by using a
self-shielded irradiator or by wearing lead aprons when working with
certain radioactive materials). These
requirements are usually stipulated
in the license conditions.
■
Use good housekeeping practices to
minimize spread of radioactivity to
uncontrolled areas.

Policies for the disposal of radioactive
waste, whether liquid or solid, should be
established, with input from the radiation
safety officer and the disposal contractor,
if an approved company is used.
Liquid radioactive waste may be collected into large sturdy bottles labeled with
an appropriate radiation waste tag. The
rules for separation by chemical compatibility apply. Bottles must be carefully stored
to protect against spillage or breakage. Dry
or solid waste may be sealed in a plastic
bag and tagged as radiation waste. The isotope, activity of the isotope, and date that
the activity was measured should be placed
on the bag. Radiation waste must never be
discharged into the drain system without
prior approval of the radiation safety officer.

Emergency Response Plan

Shipping Hazardous
Materials

Radioactive contamination is the dispersal of radioactive material into or onto
areas where it is not intended; for example, the floor, work areas, equipment,
personnel clothing, or skin. The NRC regulations state that gamma or beta radioactive contamination cannot exceed 2200
2
dpm/100 cm in the posted (restricted)
2
area or 220 dpm/100 cm in an unrestricted area such as corridors; for alpha
emitters, these values are 220 dpm/100
cm2 and 22 dpm/100 cm2, respectively.45

Local surface transport of blood specimens,
components, and biohazardous materials
from one facility (or part thereof ) to another may be made by a local approved
courier service. The safe transport of these
materials requires that they be packaged
in such a way that the possibility of leakage or other release from the package under normal conditions of transport does
not occur. See Method 1.1 for detailed
shipping instructions for diagnostic specimens and infectious substances.

Chapter 2: Facilities and Safety

Waste Management
Those responsible for safety must be concerned with protecting the environment,
as well as staff. Every effort should be
made to establish facility-wide programs
to reduce solid wastes, including nonhazardous and especially hazardous wastes
(ie, biohazardous, chemical, and radiation wastes). A hazardous waste reduction
program instituted at the point of use of
the material achieves several goals. It reduces the institutional risk for occupational exposures to hazardous agents and
“cradle to grave” liability for disposal as
well as enhances compliance with environmental requirements to reduce pollution generated from daily operations of
the laboratory.31,46,47 These requirements
necessitate that a facility minimize pollution of the environment by the “three R’s”
(reduce, reuse, and recycle). Seeking suitable alternatives to the use of materials
that create hazardous waste and separating hazardous waste from nonhazardous
waste can reduce the volume of hazardous waste and decrease costs for its disposal.
A goal of waste management should be
to reduce to a minimum the volume of
hazardous material. Noninfectious waste
should always be separated from infectious
waste. Changes in techniques or materials,
which reduce the volume of infectious
waste or render it less hazardous, should be
carefully considered and employees should
be encouraged to identify safer alternatives
wherever possible.
Facilities should check with state and local health and environmental authorities
for current requirements for storage and
disposal of a particular multihazardous
waste before creating that waste. If the
multihazardous waste cannot be avoided,
the volume generated should be minimized. In some states, copper sulfate con-

taminated with blood is considered a multihazardous waste. The disposal of this waste
poses several problems with transportation
from draw sites to a central facility to disposal of the final containers. State and local
health departments must be involved in the
review of transportation and disposal practices where this is an issue, and procedures
must be developed in accordance with
their regulations as well as those of the US
Department of Transportation.

Disaster Planning
Blood banks and transfusion services should
establish action plans for uncommon but
potential dangers (eg, floods; hurricanes;
tornadoes; earthquakes; fires; explosions;
biological, chemical, or radiation emergencies; structural collapse; hostage situations; bomb threats and other acts of terrorism; or other events in which mass
casualties might occur). These events require a plan to ensure the safety of patients, visitors, workers, and the blood
supply. Such disasters may involve the facility alone, the surrounding area, or both,
and can be categorized by severity level:
minor impact on normal operations;
moderate to substantial reduction in operations; or severe, prolonged loss of operations. The JCAHO requires a plan to
address four phases of activities: mitigation, preparedness, response, and recovery.4 Policies and procedures may address:
■
Notification procedures.
■
Ongoing communication (ie, command center).
■
Evacuation or relocation.
■
Isolation or containment.
■
Personal safety and protection.
■
Provision of additional staffing.
Typically, in a disaster situation, the first
person who becomes aware of the disaster
takes immediate action and notifies others,

AABB Technical Manual

either through an alarm activation system
(eg, fire alarm) or by notifying an individual
in authority, who then implements the initial response steps and contacts the facility’s disaster coordinator. Emergency telephone numbers should be prominently
posted. Employees should be trained in the
facility’s disaster response policies. Because
the likelihood of being involved in an actual
disaster is minimal, drills should be conducted to ensure appropriate responses
and prepare staff to act quickly. Every disaster is a unique occurrence. Modifications
must be made as necessary; flexibility is
important. Once the disaster is under control and recovery is under way, actions
should be evaluated and modifications
made to the disaster plan as needed. However, the single most effective protection a
facility has against unexpected danger is the
awareness that safety-minded employees
have for their surroundings.

6.
7.

10.

11.

12.

13.

14.

References
1.

Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005:88.
Laboratory Accreditation Program Laboratory general checklist. Chicago, IL: College of
American Pathologists Commission on Laboratory Accreditation, 2002.
Clinical laboratory safety; approved guideline. NCCLS document GP17-A. National
Committee for Clinical Laboratory Standards.
Wayne, PA: NCCLS, 1996.
2003 Hospital accreditation standards. Oakbrook Terrace, IL: Joint Commission on Accreditation of Healthcare Organizations, 2003.
2002-2003 Standards for pathology and laboratory services. Oakbrook Terrace, IL: Joint
Commission on Accreditation of Healthcare
Organizations, 2002.
NFPA 70 - National electrical code. Quincy, MA:
National Fire Protection Association, 2002.
ANSI/ASHRAE Standard 62-1999. Ventilation
for acceptable indoor air quality. Atlanta, GA:
American Society of Heating, Refrigerating,
and Air-Conditioning Engineers, Inc., 1999.
Code of federal regulations. Standards for
protection against radiation. Title 10 CFR

15.

16.

17.

18.

19.

Part 20. Washington, DC: US Government
Printing Office, 2004 (revised annually).
Garner JS, for the CDC Hospital Infection Control Practices Advisory Committee. Guidelines for isolation precautions in hospitals.
Infect Control Hosp Epidemiol 1996;17:5380.
Code of federal regulations. Occupational exposure to bloodborne pathogens, final rule.
Title 29 CFR Part 1910.1030. Washington, DC:
US Government Printing Office, 2004 (revised
annually).
Code of federal regulations. Hazard communication standard. Title 29 CFR Part 1910.1200.
Washington, DC: US Government Printing
Office, 2004 (revised annually).
Richmond JY, McKinney RW, eds. Biosafety in
microbiological and biomedical laboratories.
4th ed. Washington, DC: US Government
Printing Office, 1999.
Bernard B, ed. Musculoskeletal disorders and
workplace factors: A critical review of epidemiologic evidence for work-related musculoskeletal disorders of the neck, upper extremity, and low back. NIOSH Publication 97-141.
Cincinnati, OH: US Department of Health
and Human Services, Public Health Service,
Centers for Disease Control and Prevention,
National Institute for Occupational Safety
and Health, 1997.
Code of federal regulations. Emergency action plans. Title 29 CFR Part 1910.38. Washington, DC: US Government Printing Office,
2004 (revised annually).
Wagner KD, ed. Environmental management
in healthcare facilities. Philadelphia: WB
Saunders, 1998.
Code of federal regulations. Occupational exposure to hazardous chemicals in laboratories. Title 29 CFR Part 1910.1450. Washington,
DC: US Government Printing Office, 2004 (revised annually).
Centers for Disease Control. Public Health
Service guidelines for the management of occupational exposures to HBV, HCV, and HIV
and recommendations for post-exposure
prophylaxis. MMWR Morb Mortal Wkly Rep
2001;50:1-52.
Code of federal regulations. Access to employee exposure and medical records. Title 29
CFR Part 1910.1020. Washington, DC: US
Government Printing Office, 2004 (revised
annually).
National Institute for Occupational Safety
and Health. NIOSH Alert: Preventing allergic
reactions to natural rubber latex in the workplace. (June 1997) NIOSH Publication No. 97135. Washington, DC: National Institute for

Chapter 2: Facilities and Safety

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

Occupational Safety and Health, 1997. [Available at http://www.cdc.gov/niosh/latexalt.html.]
NFPA 101: Code for safety to life from fire in
buildings and structures. Quincy, MA: National Fire Protection Association, 2000.
Fowler TW, Miles KK. Electrical safety: Safety
and health for electrical trades student manual. ( January 2002) NIOSH Publication No.
2002-123. Washington, DC: National Institute
for Occupational Safety and Health, 2002.
Occupational Safety and Health Administration. OSHA technical manual: TED 1-0.15A.
Washington, DC: US Department of Labor, 1999.
Occupational Safety and Health Administration. Enforcement procedures for the occupational exposure to bloodborne pathogens,
Directive CPL 2-2.69. Washington, DC: US
Department of Labor, 2001.
US Department of Health and Human Services, CDC, and NIH. Primary containment
for biohazards: Selection, installation and use
of biological safety cabinets. (September,
1995) Bethesda, MD: National Institutes of
Health, 1995. [Available at http://www.niehs.
nih.gov/odhsb/biosafe/bsc/bsc.htm.]
Richmond JY. Safe practices and procedures
for working with human specimens in biomedical research laboratories. J Clin Immunoassay 1988;11:115-9.
Code of federal regulations. Occupational exposure to bloodborne pathogens; needlestick
and other sharps injuries; final rule. Title 29 CFR
Part 1910.1030. Fed Regist 2001;66:5317-25.
Environmental Protection Agency. Registered
hospital disinfectants and sterilants (TS767C).
Washington, DC: Antimicrobial Program
Branch, 1992.
Rutala WA. APIC guideline for selection and
use of disinfectants. Am J Infect Control 1996;
24:313-42.
Evans MR, Henderson DK, Bennett JE. Potential for laboratory exposures to biohazardous
agents found in blood. Am J Public Health
1990;80:423-7.
Food and Drug Administration. Memorandum: Guideline for collection of blood products from donors with positive tests for infectious disease markers (“high risk” donors).
(October 26, 1989) Rockville, MD: CBER Office of Communication, Training, and Manufacturers Assistance, 1989.
Environmental Protection Agency. EPA guide
for infectious waste management. EPA/530SW-86-014. NTIS #PB86-199130. Washington,
DC: National Technical Information Service,
1986.
Code of federal regulations. Standards of performance for new stationary sources and
emission guidelines for existing sources: Hos-

33.

34.
35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

pital/medical/infectious waste incinerators.
Title 40 CFR Part 60. Washington, DC: US
Government Printing Office, 2004 (revised
annually).
NFPA 704—standard for the identification of
the hazards of materials for emergency response. Quincy, MA: National Fire Protection
Association, 2001.
HMIS implementation manual. 3rd ed. Neenah,
WI: JJ Keller and Associates, Inc., 2001.
Lisella FS, Thomasston SW. Chemical safety
in the microbiology laboratory. In: Fleming
DO, Richardson JH, Tulis JJ, Vesley D, eds.
Laboratory safety, principles and practices.
2nd ed. Washington, DC: American Society
for Microbiology Press, 1995:247-54.
American National Standards Institute.
American national standards for emergency
eyewash and shower equipment. ANSI
Z358.1-1998. New York, NY: ANSI, 1998.
Occupational Safety and Health Administration. Inspection procedures for the hazardous waste operations and emergency response standard, 29 CFR 1910.120 and
1926.65, paragraph (q): Emergency response
to hazardous substance releases. OSHA directive CPL 2-2.59A. Washington, DC: US Government Printing Office, 1998.
Code of federal regulations. Air contaminants:
Toxic and hazardous substances. Title 29 CFR
Part 1910.1000. Washington, DC: US Government Printing Office, 2004 (revised annually).
Cook SS. Selection and installation of selfcontained irradiators. In: Butch S, Tiehen A,
eds. Blood irradiation: A user’s guide. Bethesda,
MD: AABB Press, 1996:19-40.
Beir V. Health effects of exposure to low levels
of ionizing radiation. Washington, DC: National Academy Press, 1990:1-8.
Regulatory Guide 8.29: Instruction concerning risks from occupational radiation exposure. Washington, DC: Nuclear Regulatory
Commission, 1996.
NCRP Report No. 115: Risk estimates for radiation protection: Recommendations of the
National Council on Radiation Protection
and Measurements. Bethesda, MD: National
Council on Radiation Protection and Measurements, 1993.
NCRP Report No. 105: Radiation protection
for medical and allied health personnel: Recommendations of the National Council on
Radiation Protection and Measurements.
Bethesda, MD: National Council on Radiation Protection and Measurements, 1989.
NRC Regulatory Guide 8.13: Instruction concerning prenatal radiation exposure. Washington, DC: Nuclear Regulatory Commission, 1999.

45.

46.

47.

AABB Technical Manual

NRC Regulatory Guide 8.23: Radiation surveys
at medical institutions. Washington, DC: Nuclear Regulatory Commission, 1981.
United States Code. Pollution Prevention Act.
42 U.S.C. § 13101 and 13102 et seq. Washington, DC: US Government Printing Office, 1990.
Clinical laboratory waste management. Approved Standard Doc GP5-A. Wayne, PA: National Committee for Clinical Laboratory
Standards, 1993.

Suggested Reading
CDC Office of Biosafety. Radiation safety manual.
Atlanta, GA: Centers for Disease Control, 1992.
Disaster operations handbook: Coordinating the
nation’s blood supply during disasters and biological events. Bethesda, MD: AABB, 2003.
Disaster plan development procedure manual. In:
Developing a disaster plan. Bethesda, MD: AABB,
1998.

Heinsohn PA, Jacobs RR, Concoby BA, eds. Biosafety
reference manual. 2nd ed. Fairfax, VA: American
Industrial Hygiene Association Biosafety Committee, 1995.
Liberman DF, ed. Biohazards management handbook. 2nd ed. New York: Marcel Dekker, Inc, 1995.
NIH guide to waste disposal. Bethesda, MD: National Institutes of Health, 2003. [Available at http://
www.nih.gov/od/ors/ds/wasteguide.]
Prudent practices for handling hazardous chemicals in laboratories. Washington, DC: National
Academy Press, 1981.
Risk management and safety procedure manual.
In: Developing a disaster plan. Bethesda, MD:
AABB, 1998.
Vesley D, Lauer JL. Decontamination, sterilization,
disinfection and antisepsis. In: Fleming DO, Richardson JH, Tulis JJ, Vesley D, eds. Laboratory
safety, principles and practices. 2nd ed. Washington, DC: American Society for Microbiology Press,
1995:219-37.

Handbook of compressed gases. 3rd ed. Compressed
Gas Association. New York: Chapman and Hall, 1990.

Chapter 2: Facilities and Safety

Appendix 2-1. Safety Regulations and Recommendations Applicable to Health-Care
Settings
Agency/Organization

Reference

Title

Federal Regulations and Recommendations
Nuclear Regulatory Commission (NRC)

Department of Labor, Occupational Safety and
Health Administration
(OSHA)

Department of Transportation (DOT)

10 CFR 20

Standards for Protection Against
Radiation

Guide 8.29

Instruction Concerning Risks
from Occupational Radiation
Exposure

29 CFR 1910.1030

Occupational Exposure to
Bloodborne Pathogens

29 CFR 1910.1096

Ionizing Radiation

29 CFR 1910.1200

Hazard Communication Standard

29 CFR 1910.1450

Occupational Exposure to Hazardous Chemicals in Laboratories

49 CFR 171-180

Hazardous Materials Regulations

Environmental Protection
Agency (EPA)

EPA Guide for Infectious Waste
Management

Centers for Disease Control
and Prevention (CDC)

Guideline for Isolation Precautions in Hospitals

Food and Drug Administration (FDA)

21 CFR 606.40, 606.60,
and 606.65

Current Good Manufacturing
Practice for Blood and Blood
Components
Guideline for Collection of Blood
Products from Donors with
Positive Tests for Infectious
Disease Markers

21 CFR 801.437

User Labeling for Devices that
Contain Natural Rubber

(cont’d)

AABB Technical Manual

Appendix 2-1. Safety Regulations and Recommendations Applicable to Health-Care
Settings (cont'd)
Agency/Organization

Reference

Title

Trade and Professional Organizations
National Fire Protection
Association (NFPA)

NFPA 70

National Electrical Code

NFPA 70E

Electrical Safety Requirements
for Employee Workplaces

NFPA 101

Code for Safety to Life from Fire
in Buildings and Structures

NFPA 704

Standard for the Identification of
the Hazards of Materials for
Emergency Response

National Paint and Coatings
Association

Hazardous Materials Identification System (HMIS) Implementation Manual

International Air Traffic
Association (IATA)

Dangerous Goods Regulations

Chapter 2: Facilities and Safety

Appendix 2-2. General Guidelines for Safe Work Practices, Personal Protective
Equipment, and Engineering Controls
Uniforms and Laboratory Coats
Closed laboratory coats or full aprons over long-sleeved uniforms or gowns should be worn
when personnel are exposed to blood, corrosive chemicals, or carcinogens. The material of required coverings should be appropriate for the type and amount of hazard exposure. Plastic disposable aprons may be worn over cotton coats when there is a high probability of large spills or
splashing of blood and body fluids; nitrile rubber aprons may be preferred when pouring caustic
chemicals.
Protective coverings should be removed before leaving the work area and should be discarded or
stored away from heat sources and clean clothing. Contaminated clothing should be removed
promptly, placed in a suitable container, and laundered or discarded as potentially infectious.
Home laundering of garments worn in Biosafety Level 2 areas (see below) is not permitted because unpredictable methods of transportation and handling can spread contamination, and
home laundering techniques may not be effective.1

Gloves
Gloves or equivalent barriers should be used whenever tasks are likely to involve exposure to
hazardous materials. Latex or vinyl gloves are adequate for handling most blood specimens and
chemicals (see latex allergy issues below).

Types of Gloves
Glove type varies with the task:
■ Sterile gloves: for procedures involving contact with normally sterile areas of the body.
■ Examination gloves: for procedures involving contact with mucous membranes, unless otherwise
indicated, and for other patient care or diagnostic procedures that do not require the use of
sterile gloves.
■ Rubber utility gloves: for housekeeping chores involving potential blood contact, for instrument
cleaning and decontamination procedures, for handling concentrated acids and organic solvents.
Utility gloves may be decontaminated and reused but should be discarded if they show signs of
deterioration (peeling, cracks, discoloration) or if they develop punctures or tears.
■ Insulated gloves: for handling hot or frozen material.

Indications for Use
The following guidelines should be used to determine when gloves are necessary1:
■ For donor phlebotomy when the health-care worker has cuts, scratches, or other breaks in his or
her skin.
■ For phlebotomy of autologous donors or patients (eg, therapeutic apheresis procedures,
intraoperative red cell collection).
■ For persons who are receiving training in phlebotomy.
■ When handling “open” blood containers or specimens.
■ When collecting or handling blood or specimens from patients or from donors known to be
infected with a blood-borne pathogen.
■ When examining mucous membranes or open skin lesions.
■ When handling corrosive chemicals and radioactive materials.

(cont’d)

AABB Technical Manual

Appendix 2-2. General Guidelines for Safe Work Practices, Personal Protective
Equipment, and Engineering Controls (cont'd)
■
■

When cleaning up spills or handling waste materials.
When likelihood of exposure cannot be assessed because of lack of experience with a procedure
or situation.

The Occupational Safety and Health Administration (OSHA) does not require routine use of gloves
by phlebotomists working with healthy prescreened donors or the changing of unsoiled gloves
between donors if gloves are worn.1,2 Experience has shown that the phlebotomy process is low
risk because donors have low rates of infectious disease markers. Also, exposure to blood is rare
during routine phlebotomy, and other alternatives can be utilized to provide barrier protection,
such as using a folded gauze pad to control any blood flow when the needle is removed from the
donor’s arm.
The employer whose policies and procedures do not require routine gloving should periodically
reevaluate the potential need for gloves. Employees should never be discouraged from using
gloves, and gloves should always be available.

Guidelines on Use
Guidelines for the safe use of gloves include the following3,4:
■ Securely bandage or cover open skin lesions on hands and arms before putting on gloves.
■ Change gloves immediately if they are torn, punctured, or contaminated; after handling high-risk
samples; or after performing a physical examination, eg, on an apheresis donor.
■ Remove gloves by keeping their outside surfaces in contact only with outside and by turning the
glove inside out while taking it off.
■ Use gloves only where needed and avoid touching clean surfaces such as telephones, door
knobs, or computer terminals with gloves.
■ Change gloves between patient contacts. Unsoiled gloves need not be changed between donors.
■ Wash hands with soap or other suitable disinfectant after removing gloves.
■ Do not wash or disinfect surgical or examination gloves for reuse. Washing with surfactants may
cause “wicking” (ie, the enhanced penetration of liquids through undetected holes in the glove).
Disinfecting agents may cause deterioration of gloves.
■ Use only water-based hand lotions with gloves, if needed; oil-based products cause minute
cracks in latex.

Face Shields, Masks, and Safety Goggles
Where there is a risk of blood or chemical splashes, the eyes and the mucous membranes of the
5
mouth and nose should be protected. Permanent shields, fixed as a part of equipment or bench
design, are preferred, eg, splash barriers attached to tubing sealers or centrifuge cabinets. All
barriers should be cleaned and disinfected on a regular schedule.
Safety glasses alone provide impact protection from projectiles but do not adequately protect
eyes from biohazardous or chemical splashes. Full-face shields or masks and safety goggles are
recommended when permanent shields cannot be used. Many designs are commercially available; eliciting staff input on comfort and selection can improve compliance on use.

Chapter 2: Facilities and Safety

Appendix 2-2. General Guidelines for Safe Work Practices, Personal Protective
Equipment, and Engineering Controls (cont'd)
Masks should be worn whenever there is danger from inhalation. Simple, disposable dust masks
are adequate for handling dry chemicals, but respirators with organic vapor filters are preferred
for areas where noxious fumes are produced, eg, for cleaning up spills of noxious materials. Respirators should be fitted to their specific wearers and checked annually.

Hand Washing
Frequent effective hand washing is the first line of defense in infection control. Blood-borne
pathogens generally do not penetrate intact skin, so immediate removal reduces the likelihood of
transfer to a mucous membrane or broken skin area or of transmission to others. Thorough
washing of hands (and arms) also reduces the risks from exposure to hazardous chemicals and
radioactive materials.
Hands should always be washed before leaving a restricted work area, before using a biosafety
cabinet, between medical examinations, immediately after becoming soiled with blood or hazardous materials, after removing gloves, and after using the toilet. Washing hands thoroughly before
touching contact lenses or applying cosmetics is essential.
OSHA allows the use of waterless antiseptic solutions for hand washing as an interim method.2
These solutions are useful for mobile donor collections or in areas where water is not readily
available for cleanup purposes. If such methods are used, however, hands must be washed with
soap and running water as soon as feasible thereafter. Because there is no listing or registration
of acceptable hand wipe products similar to the one the Environmental Protection Agency maintains for surface disinfectants, consumers should request data from the manufacturer to support
advertising claims.

Eye Washes
3,6

Laboratory areas that contain hazardous chemicals must be equipped with eye wash stations.
Unobstructed access, within 10 seconds from the location of chemical use, must be provided for
these stations. Eye washes must operate so that both of the user’s hands are free to hold open
the eyes. Procedures and indications for use must be posted and routine function checks must be
performed. Testing eye wash fountains weekly helps ensure proper function and flushes out the
stagnant water. Portable eye wash systems are allowed only if they can deliver flushing fluid to
the eyes at a rate of at least 1.5 liters per minute for 15 minutes. They should be monitored routinely to ensure the purity of their contents.
Employees should be trained in the proper use of eye wash devices, although prevention, through
consistent and appropriate use of safety glasses or shields, is preferred. If a splash occurs, the
employee should be directed to keep the eyelids open and use the eye wash according to procedures, or go to the nearest sink and direct a steady, tepid stream of water into the eyes. Solutions
other than water should be used only upon a physician’s direction.
After adequate flushing (many facilities recommend 15 minutes), follow-up medical care should
be sought, especially if pain or redness develops. Whether eye washing is effective in preventing
infection has not been demonstrated but it is considered desirable when accidents occur.
(cont’d)

AABB Technical Manual

Appendix 2-2. General Guidelines for Safe Work Practices, Personal Protective
Equipment, and Engineering Controls (cont'd)
1.
2.
3.
4.
5.
6.

Code of federal regulations. Occupational exposure to bloodborne pathogens, final rule. Title 29 CFR
Part 1910.1030. Fed Regist 1991;56(235):64175-82.
Occupational Safety and Health Administration. Enforcement procedures for the occupational
exposure to bloodborne pathogens. OSHA Instruction CPL2-2.44D. Washington, DC: US Government
Printing Office, 1999.
Clinical and Laboratory Standards Institute. Clinical laboratory safety; approved guideline. NCCLS
document GP17-A. Wayne, PA: CLSI, 1996.
Food and Drug Administration. Medical glove powder report. (September 1997) Rockville, MD: Center
for Devices and Radiological Health, 1997. [Available at http://www.fda.gov/cdrh/glvpwd.html.]
Inspection checklist: General laboratory. Chicago, IL: College of American Pathologists, 2001.
American National Standards Institute. American national standards for emergency eyewash and
shower equipment. ANSI Z358.1-1998. New York: ANSI, 1998.

Chapter 2: Facilities and Safety

Appendix 2-3. Biosafety Level 2 Precautions
Biosafety Level 2 precautions as applied in the blood establishment setting include at least the
following1,2:
■

High-risk activities are appropriately segregated from lower risk activities, and the boundaries
are clearly defined.

■

Bench tops are easily cleaned and are decontaminated daily with a hospital disinfectant
approved by the Environmental Protection Agency.

■

Laboratory rooms have closable doors and sinks. An air system with no recirculation is
preferred, but not required.

■

Workers are required to perform procedures that create aerosols (eg, opening evacuated tubes,
centrifuging, mixing, or sonicating) in a biologic safety cabinet or equivalent, or to wear masks
and goggles in addition to gloves and gowns during such procedures. (Note: Open tubes of
blood should not be centrifuged. If whole units of blood or plasma are centrifuged,
overwrapping is recommended to contain leaks.)

■

Gowns and gloves are used routinely and in accordance with general safety guidelines. Face
shields or their equivalent are used where there is a risk from splashing.

■

Mouth pipetting is prohibited.

■

No eating, drinking, smoking, application of cosmetics, or manipulation of contact lenses occurs
in the work area. All food and drink are stored outside the restricted area, and laboratory
glassware is never used for food or drink. Personnel are instructed to avoid touching their face,
ears, mouth, eyes, or nose with their hands or other objects, such as pencils and telephones.

■

Needles and syringes are used and disposed of in a safe manner. Needles are never bent,
broken, sheared, replaced in sheath, or detached from syringe before being placed in
puncture-proof, leakproof containers for controlled disposal. Procedures are designed to
minimize exposure to sharp objects.

■

All blood specimens are placed in well-constructed containers with secure lids to prevent
leaking during transport. Blood is packaged for shipment in accordance with regulatory agency
requirements for etiologic agents or clinical specimens, as appropriate.

■

Infectious waste is not compacted and is decontaminated before its disposal in leakproof
containers. Proper packaging includes double, seamless, tear-resistant, orange or red bags
enclosed in protective cartons. Both the carton and the bag inside display the biohazard symbol.
Throughout delivery to an incinerator or autoclave, waste is handled only by suitably trained
persons. If a waste management contractor is used, the agreement should clearly define
respective responsibilities of the staff and the contractor.

■

Equipment to be repaired or submitted for preventive maintenance, if potentially contaminated
with blood, must be decontaminated before its release to a repair technician.

■

Accidental exposure to suspected or actual hazardous material is reported to the laboratory
director or responsible person immediately.

1.
2.

Clinical and Laboratory Standards Institute. Clinical laboratory safety; approved guideline. NCCLS
document GP17-A. Wayne, PA: CLSI, 1996.
Fleming DO. Laboratory biosafety practices. In: Fleming DO, Richardson JH, Tulis JJ, Vesley D, eds.
Laboratory safety, principles and practices. 2nd ed. Washington, DC: American Society for
Microbiology Press, 1995:203-18.

AABB Technical Manual

Appendix 2-4. Sample Hazardous Chemical Data Sheet
The following information should be a part of the procedures for use of hazardous chemicals.
Facility Identification

: ________________________________________________________

Laboratory Name

: ________________________________________________________

Room Number

: ________________________________________________________

Name of Chemical

: ________________________________________________________

Synonyms

: ________________________________________________________

Chemical Abstract No.
(Case #)

: ________________________________________________________

Common Name

: ________________________________________________________

Primary Hazard

Carcinogen: _______________________________________________
Reproductive toxin: _________________________________________
High acute toxicity: _________________________________________
Other health hazard: ________________________________________
Safety hazard: _____________________________________________
MSDS or other reference available: ____________________________
Is prior approval required for use of the chemical; if so,
by whom? ________________________________________________

General and Special Precautions:
Signs Required (Warning signs indicating presence of hazardous chemicals/operations):
____________________________________________________________________________
Storage (Secondary containment, temperature-sensitive, incompatibilities, water-reactive,
etc): ________________________________________________________________________
____________________________________________________________________________
Special Controls and Location (Fume hood, glove box, etc): ____________________________
____________________________________________________________________________

Chapter 2: Facilities and Safety

Appendix 2-4. Sample Hazardous Chemical Data Sheet (cont'd)
Special Equipment and Location (Vacuum line filter, liquid or other traps, special shielding):
____________________________________________________________________________
Personal Protective Equipment (Glove type, eye protection, special clothing, etc): __________
____________________________________________________________________________
Emergency Procedures:
Spill or release: _______________________________________________________________
Fire:_________________________________________________________________________
Decontamination procedures: ____________________________________________________
Disposal procedures: ___________________________________________________________
____________________________________________________________________________
____________________________________________________________________________

AABB Technical Manual

Appendix 2-5. Sample List of Hazardous Chemicals in the Blood Bank
Chemical

Hazard

Ammonium chloride

Irritant

Bromelin

Irritant, sensitizer

Calcium chloride

Irritant

Carbon dioxide, frozen (dry ice)

Corrosive

Carbonyl iron powder

Oxidizer

Chloroform

Toxic, suspected carcinogen

Chloroquine

Irritant, corrosive

Chromium-111 chloride hexahydrate

Toxic, irritant, sensitizer

Citric acid

Irritant

Copper sulfate (cupric sulfate)

Toxic, irritant

Dichloromethane

Toxic, irritant

Digitonin

Toxic

Dimethyl sulfoxide (DMSO)

Irritant

Dry ice (carbon dioxide, frozen)

Corrosive

Ethidium bromide

Carcinogen, irritant

Ethylenediaminetetraacetic acid (EDTA)

Irritant

Ethyl ether

Highly flammable and explosive, toxic, irritant

Ficin (powder)

Irritant, sensitizer

Formaldehyde solution (34.9%)

Suspected carcinogen, combustible, toxic

Glycerol

Irritant

Hydrochloric acid

Highly toxic, corrosive

Imidazole

Irritant

Isopropyl (rubbing) alcohol

Flammable, irritant

Liquid nitrogen

Corrosive

Lyphogel

Corrosive

2-Mercaptoethanol

Toxic, stench

Mercury

Toxic

Mineral oil

Irritant, carcinogen, combustible

Papain

Irritant, sensitizer

Polybrene

Toxic

Potassium hydroxide

Corrosive, toxic

Saponin

Irritant

Sodium azide

Toxic, irritant, explosive when heated

Sodium ethylmercurithiosalicylate (thimerosal)

Highly toxic, irritant

Sodium hydrosulfite

Toxic, irritant

Sodium hydroxide

Corrosive, toxic

Sodium hypochlorite (bleach)

Corrosive

Chapter 2: Facilities and Safety

Appendix 2-5. Sample List of Hazardous Chemicals in the Blood Bank (cont'd)
Chemical

Hazard

Sodium phosphate

Irritant, hygroscopic

Sulfosalicylic acid

Toxic, corrosive

Trichloroacetic acid (TCA)

Corrosive, toxic

Trypsin

Irritant, sensitizer

Xylene

Highly flammable, toxic, irritant

AABB Technical Manual

Appendix 2-6. Specific Chemical Categories and How to Work Safely with These
Chemicals
Chemical
Category

Hazard

Precautions

Special Treatment

Acids, alkalis,
and corrosive
compounds

Irritation
Severe burns
Tissue damage

During transport, protect
large containers with
plastic or rubber bucket
carriers
During pouring, wear eye
protection and chemicalresistant-rated gloves
and gowns as recommended
Always ADD ACID TO
WATER, never water to
acid
When working with large
jugs, have one hand on
the neck and the other at
the base, and position
them away from the face

Store concentrated acids in
acid safety cabinets
Limit volumes of concentrated acids to 1 liter
Post cautions for materials
in the area
Report changes in appearance (perchloric acid
may be explosive if it becomes yellowish or
brown) to chemical
safety officer

Acrylamide

Neurotoxic
Carcinogenic
Adsorbed
through the
skin

Wear chemically rated
gloves
Wash hands immediately
after exposure

Store in a chemical cabinet

Compressed
gases

Explosive

Label as to contents
Transport using hand trucks
Leave valve safety covers on
or dollies
until use
Place cylinders in a stand or
Open valves slowly for use
secure them to prevent
Label empty tanks
falling
Store in well-ventilated separate rooms
Oxygen should not be
stored close to combustible gas or solvents
Check connections for leaks
with soapy water

Liquid nitrogen

Freeze injury
Severe burns to
skin or eyes

Use heavy insulated gloves The tanks should be seand goggles when workcurely supported to avoid
ing with liquid nitrogen
being tipped over
The final container of liquid
nitrogen (freezing unit)
must be securely supported to avoid tipping
over

Chapter 2: Facilities and Safety

Appendix 2-6. Specific Chemical Categories and How to Work Safely with These
Chemicals (cont'd)
Chemical
Category
Flammable
solvents

Hazard

Precautions

Special Treatment

Classified accord- Use extreme caution when Make every attempt to rehandling
place hazardous materiing to flash
Post NO SMOKING signs in
als with less hazardous
point—see
materials
working area
MSDS
Classified accord- Have a fire extinguisher and Store containers larger than
ing to volatility
solvent cleanup kit in the
1 gallon in a flammable
solvent storage room or
room
in a fire safety cabinet
Pour volatile solvents under
suitable hood
Ground metal containers by
connecting the can to a
Use eye protection when
water pipe or ground
pouring and chemical-reconnection; if recipient
sistant neoprene gloves
No flame or other source of
container is also metal, it
should be electrically
possible ignition should
connected to the delivery
be in or near areas where
container while pouring
flammable solvents are
being poured
Label as FLAMMABLE

Appendix 2-7. Incidental Spill Response*
Hazards

PPE

Control Materials

Acids
Acetic
Hydrochloric
Nitric
Perchloric
Sulfuric
Photographic
chemicals
(acid)

Severe irritant if inhaled
Contact causes burns to skin
and eyes
Corrosive
Fire or contact with metal may
produce irritating or poisonous gas
Nitric, perchloric, and sulfuric
acids are water-reactive oxidizers

Acid-resistant gloves
Apron and coveralls
Goggles and face shield
Acid-resistant foot
covers

Acid neutralizers/
absorbent
Absorbent boom
Leakproof containers
Absorbent pillow
Mat (cover drain)
Shovel or paddle

Bases and caustics
Potassium hydroxide
Sodium hydroxide
Photographic chemicals
(basic)

Corrosive
Fire may produce irritating or
poisonous gas

Gloves; impervious apron or
coveralls
Goggles or face shield; impervious foot covers

Base control/neutralizer
Absorbent pillow
Absorbent boom
Drain mat
Leakproof container
Shovel/paddle

Chlorine
Bleach
Sodium hypochlorite

Inhalation can cause respiratory
irritation
Liquid contact can produce irritation of the eyes or skin
Toxicity due to alkalinity, possible chlorine gas generation,
and oxidant properties

Gloves (double set 4H
undergloves and butyl or
nitrile overgloves);
impervious apron
or coveralls
Goggles or face shield
Impervious foot covers (neoprene boots for emergency
response releases)
Self-contained breathing apparatus (emergency response
releases)

Chlorine control powder
Absorbent pillow
Absorbent
Absorbent boom
Drain mat
Vapor barrier
Leakproof container
Shovel or paddle

AABB Technical Manual

Chemicals

Contact with liquid
nitrogen can produce
frostbite
Asphyxiation (displaces oxygen)
Anesthetic effects
(nitrous oxide)

Full face shield or goggles; neoprene boots; gloves (insulated to protect from the cold)

Hand truck (to transport cylinder
outdoors if necessary)
Soap solution (to check for
leaks)
Putty (to stop minor pipe and
line leaks)

Flammable gases
Acetylene
Oxygen gases
Butane
Propane

Simple asphyxiate
(displaces air)
Anesthetic potential
Extreme fire and explosion
hazard
Release can create an oxygen-deficient
atmosphere

Face shield and goggles; neoprene boots; double set of
gloves; coveralls with hood
and feet

Hand truck (to transport cylinder
outdoors if needed)
Soap solution (to check for
leaks)

Flammable liquids
Acetone
Xylene
Methyl alcohol toluene
Ethyl alcohol
Other alcohols

Vapors harmful if inhaled
(central nervous system
depressants)
Harmful via skin absorption
Extreme flammability
Liquid evaporates to form flammable vapors

Gloves (double 4H undergloves
and butyl or nitrile
overgloves); impervious
apron or coveralls; goggles or
face shield; impervious foot
covers

Absorbent
Absorbent boom
Absorbent pillow
Shovel or paddle (nonmetal,
nonsparking)
Drain mat
Leakproof containers

Formaldehyde and
glutaraldehyde
4% formaldehyde
37% formaldehyde
10% formalin
2% glutaraldehyde

Harmful if inhaled or absorbed
through skin;
Irritation to skin, eyes, and
respiratory tract
Formaldelyde is a suspected
human carcinogen
Keep away from heat, sparks,
and flame (37% formaldehyde)

Gloves (double set 4H
undergloves and butyl or
nitrile overgloves); impervious apron or coveralls; goggles; impervious foot covers

Aldehyde neutralizer/absorbent
Absorbent boom
Absorbent pillow
Shovel or pallet (nonsparking)
Drain mat
Leakproof container

(cont’d)

Chapter 2: Facilities and Safety

Cryogenic gases
Carbon dioxide
Nitrous oxide
Liquid nitrogen

Chemicals

Hazards

PPE

Control Materials

Mercury
Cantor tubes
Thermometers
Barometers
Sphygmomanometers
Mercuric chloride

Mercury and mercury vapors are
rapidly absorbed in respiratory tract, GI tract, skin
Short-term exposure may cause
erosion of respiratory/GI
tracts, nausea, vomiting,
bloody diarrhea, shock, headache, metallic taste
Inhalation of high concentrations
can cause pneumonitis, chest
pain, dyspnea, coughing
stomatitis, gingivitis, and salivation
Avoid evaporation of mercury
from tiny globules by quick
and thorough cleaning

Gloves (double set 4H
underglove and butyl or nitrile
overglove); impervious apron
or coveralls; goggles; impervious foot covers

Mercury vacuum or spill kit
Scoop
Aspirator
Hazardous waste containers
Mercury indicator powder
Absorbent
Spatula
Disposable towels
Sponge with amalgam
Vapor suppressor

*This list of physical and health hazards is not intended as a substitute for the specific MSDS information. In the case of a spill or if any questions arise, always refer to the chemical-specific MSDS for more complete information. GI = gastrointestinal; MSDS = material safety data sheet; PPE = personal protective equipment.

AABB Technical Manual

Appendix 2-7. Incidental Spill Response* (cont'd)

Chapter 2: Facilities and Safety

Appendix 2-8. Managing Hazardous Chemical Spills
Actions

Instructions for Hazardous Liquids, Gases, and Mercury

De-energize

Liquids: For 37% formaldehyde, de-energize and remove all sources of ignition within 10 feet of spilled hazardous material. For flammable liquids, remove all sources of ignition.
Gases: Remove all sources of heat and ignition within 50 feet for flammable gases.
Remove all sources of heat and ignition for nitrous oxide release.

Isolate, evacuate,
and secure the
area

Isolate the spill area and evacuate everyone from the area surrounding the
spill except those responsible for cleaning up the spill. (For mercury,
evacuate within 10 feet for small spills, 20 feet for large spills.) Secure
area.

Have the appropriate See Appendix 2-2 for recommended PPE.
PPE
Contain the spill

Liquids or mercury: Stop the source of spill if possible.
Gases: Assess the scene; consider the circumstances of the release (quantity, location, ventilation). If circumstances indicate it is an emergency
response release, make appropriate notifications; if release is determined to be incidental, contact supplier for assistance.

Confine the spill

Liquids: Confine spill to initial spill area using appropriate control equipment and material. For flammable liquids, dike off all drains.
Gases: Follow supplier’s suggestions or request outside assistance.
Mercury: Use appropriate materials to confine the spill (see Appendix 2-2).
Expel mercury from aspirator bulb into leakproof container, if applicable.

Neutralize the spill

Liquids: Apply appropriate control materials to neutralize the chemical—
see Appendix 2-2.
Mercury: Use mercury spill kit if needed.

Spill area cleanup

Liquids: Scoop up solidified material, booms, pillows, and any other materials. Put used materials into a leakproof container. Label container with
name of hazardous material. Wipe up residual material. Wipe spill area
surface three times with detergent solution. Rinse areas with clean water. Collect supplies used (goggles, shovels, etc) and remove gross
contamination; place into separate container for equipment to be
washed and decontaminated.
Gases: Follow supplier’s suggestions or request outside assistance.
Mercury: Vacuum spill using a mercury vacuum or scoop up mercury
paste after neutralization and collect it in designated container. Use
sponge and detergent to wipe and clean spill surface three times to remove absorbent. Collect all contaminated disposal equipment and put
into hazardous waste container. Collect supplies and remove gross contamination; place them into a separate container for equipment that will
be thoroughly washed and decontaminated.

(cont’d)

AABB Technical Manual

Appendix 2-8. Managing Hazardous Chemical Spills (cont'd)
Actions

Instructions for Hazardous Liquids, Gases, and Mercury

Disposal

Liquids: For material that was neutralized, dispose of it as solid waste. Follow facility’s procedures for disposal. For flammable liquids, check with
facility safety officer for appropriate waste determination.
Gases: The manufacturer or supplier will instruct facility on disposal if applicable.
Mercury: Label with appropriate hazardous waste label and DOT diamond
label.

Report

Follow appropriate spill documentation and reporting procedures. Investigate the spill; perform root cause analysis if needed. Act on opportunities for improving safety.

DOT = Department of Transportation; PPE = personal protective equipment.

Chapter 3: Blood Utilization Management

Chapter 3

Blood Utilization
Management
3

HE GOAL OF BLOOD utilization
management is to ensure effective
use of limited blood resources. It
includes the policies and practices related
to inventory management and blood usage review. Although regional blood centers and transfusion services approach
utilization management from different
perspectives, they share the common goal
of providing appropriate, high-quality
blood products with minimum waste. This
chapter reviews the elements of utilization management, emphasizing the transfusion service.

Minimum and Ideal
Inventory Levels
Transfusion services should establish both
minimum and ideal inventory levels. Inventory levels should be evaluated periodically and adjusted if needed. Important
indicators of performance include, but are

not limited to, outdate rates, the frequency
of emergency blood shipments, and delays
in scheduling elective surgery. Inventory
levels should also be reevaluated whenever a significant change is planned or
observed. Examples of significant change
may include adding more beds; performing new surgical procedures; or changing
practices in oncology, transplantation, neonatology, or cardiac surgery.

Determining Inventory
Levels
The ideal inventory level provides adequate supplies of blood for routine and
emergency situations and minimizes outdating. Forecasting is an attempt to predict future blood product use from data
collected about past usage. The optimal
number of units to keep in inventory can
be estimated using mathematical formulas, computer simulations, or empirical
89

AABB Technical Manual

calculations. Three less complicated methods of estimating minimum inventory are
described below. When the minimum inventory level has been calculated, a buffer
margin for emergencies should be added
to obtain an ideal inventory level.

required to be on hand (this may be 3,
5, or 7 days depending on the blood
supplier’s delivery schedule).
A transfusion service may find the average daily use calculation more helpful
when blood shipments are made once or
more per day.

Average Weekly Use Estimate
This method gives an estimate of the average weekly blood usage of each ABO
group and Rh type.
1.
Collect weekly blood and product
usage data over a 26-week period.
2.
Record usage by ABO group and Rh
type for each week.
3.
Disregard the single highest usage
for each type to correct for unusual
week-to-week variation (eg, a large
volume used for an emergency).
4.
Total the number of units of each
ABO group and Rh type, omitting
the highest week in each column.
5.
Divide each total by 25 (total number of weeks minus the highest week).
This gives an estimate of the average
weekly blood usage of each ABO
group and Rh type.

Average Daily Use Estimate
Facilities that transfuse on a daily basis
may calculate daily blood usage by the
following method.
1.
Determine the total use over several
months.
2.
Divide the total use by the number
of days in the period covered.
3.
Determine the percentage of each of
the blood types used during one or
more representative months.
4.
Multiply the average blood use per
day by the percentage of blood use
by type.
5.
Determine the minimum inventory
level by multiplying the daily use by
the number of days of blood supply

Moving Average Method
The moving average method can be useful in facilities with any level of activity.
1.
Determine the preferred recording
period (such as day or week).
2.
Add the number of units used in each
period to obtain the total use.
3.
Divide the total number of units by
the number of recording periods.
4.
Delete old data as new data are added.
This method tends to minimize variation
from one period to another.

Factors that Affect Outdating
Benchmark data on component outdating
have been published by the National Blood
Data Resource Center (NBDRC).1 The data
from this report showed that approximately 2,190,000 total components outdated in 2001, a 6.6% decrease from 1999.
Whole-blood-derived platelet concentrates accounted for more than half of the
outdated components (49%), or 1,074,000
units. Outdated allogeneic RBCs (directed
and nondirected) accounted for 26.8% of
all outdated components, or 588,000 units.
The outdate rate for each component is
shown in Table 3-1. Outdating continues to
be a problem, particularly for autologous
units and platelets. The outdate rate is affected by many factors other than inventory level (eg, the size of the hospital, the
extent of services provided, the shelf life
of the products, the shipping distance
and frequency, and ordering policies).

Chapter 3: Blood Utilization Management

Table 3-1. Blood Component Units Processed, Transfused, and Outdated in United
1
States in 2001
Units
Processed

Units
Transfused

Units
Outdated

14,259,000

13,361,000

576,000

4.0

RBCs (autologous)

619,000

359,000

263,000

42.5

RBCs (directed)

169,000

95,000

12,000

7.1

Platelets
(whole-bloodderived)

4,164,000

2,614,000

1,074,000

25.8

Platelets Pheresis

1,456,000

1,264,000

160,000

11.0

FFP

4,437,000

3,926,000

77,000

1.7

Cryoprecipitated AHF

1,068,000

898,000

28,000

2.6

Component
RBCs (allogeneic,
nondirected)

Percent
Outdated

FFP = Fresh Frozen Plasma; RBCs = Red Blood Cells.

In addition to establishing both minimum and ideal inventory levels, maximum
inventory levels may assist staff in determining when to arrange for return or transfer of in-date products to avoid outdating.
Both transfusion services and donor centers should establish record-keeping systems that allow personnel to determine the
number of units ordered and the number of
units received or shipped. The responsibility for ordering may be centralized, and orders should be based on established policies for minimal and maximal levels.
Standing orders can simplify inventory
planning for both transfusion services and
blood centers. Blood centers may send a
predetermined number of units on a regular schedule or may keep the transfusion
service inventory at established levels by
replacing all units reported as transfused.
Optimal inventory management requires
distribution and transfusion of the oldest
blood first and this requires clearly written
policies on blood storage and blood selec-

tion. Technologists generally find it easier
to select, crossmatch, and issue the oldest
units first when inventories are arranged by
expiration date.
Policies on blood selection must be flexible, to allow use of fresher blood when indicated (eg, for infants). Generally, however,
oldest units are crossmatched for patients
most likely to need transfusion.

Improving Transfusion
Service Blood Ordering
Practices
The shelf life decreases each time a unit is
held or crossmatched for a patient who
does not use it. When physicians order more
blood than needed, it is unavailable for
other patients, which may increase the
outdate rate. Providing testing guidelines,
such as type and screen (T/S) policies and

AABB Technical Manual

maximum surgical blood order schedules
(MSBOS),2 as well as monitoring crossmatch-to-transfusion (C:T) ratios may be
helpful. A C:T ratio greater than 2.0 usually indicates excessive crossmatch requests. In some situations, it may be useful to determine C:T ratios by service to
identify areas with the highest ratio.
Some institutions define those procedures that normally do not use blood in a
“type and screen” guideline. Both the T/S
guideline and the MSBOS use data about
past surgical blood use to recommend a
T/S order or a maximum number of units
that should be ordered initially for common
elective surgical procedures. With the
MSBOS, physicians may order the number
of units believed to be appropriate for the
patient since the MSBOS is intended to be a
guideline for appropriate patient care.
Some institutions have modified the MSBOS
concept into a “standard” blood order (SBO)
system for surgical procedures.3
Ordering guidelines such as those in Table 3-2 are derived by reviewing a facility’s
blood use over a suitable period. Conclusions can then be drawn about the likelihood of transfusion and probable blood
use for each surgical procedure. A T/S order
is a recommended SBO for procedures that
require on average less than 0.5 unit of
blood per patient per procedure. An SBO
often represents the average number of
units transfused for each procedure, whereas the MSBOS often defines the number of
units needed to meet the needs of 80% to
90% of patients undergoing a specific surgical procedure.3
An institution’s guidelines must reflect
local patterns of surgical practice and patient population. These may be compared
to published guidelines to ensure that local
practice does not markedly deviate from
generally accepted standards of care.
(Transfusion audits are discussed in Chapter 1.) Once the T/S, MSBOS, SBO, or other

Table 3-2. Example of a Maximum
Surgical Blood Order Schedule
Procedure

Units*

General Surgery
Breast biopsy
Colon resection
Exploratory laparotomy
Gastrectomy
Laryngectomy
Mastectomy, radical
Pancreatectomy
Splenectomy
Thyroidectomy

T/S
2
T/S
2
2
T/S
4
2
T/S

Cardiac-Thoracic
Aneurism resection
Redo coronary artery bypass graft
Primary coronary artery bypass graft
Lobectomy
Lung biopsy

6
4
2
T/S
T/S

Vascular
Aortic bypass with graft
Endarterectomy
Femoral-popliteal bypass with graft

4
T/S
2

Orthopedics
Arthroscopy
Laminectomy
Spinal fusion
Total hip replacement
Total knee replacement

T/S
T/S
3
3
T/S

OB-GYN
Abdomino-perineal repair
Cesarean section
Dilation and curettage
Hysterectomy, abdominal/
laparoscopic
Hysterectomy, radical

T/S
T/S
T/S
T/S
2

Urology
Bladder, transurethral resection
Nephrectomy, radical
Radical prostatectomy, perineal
Prostatectomy, transurethral
Renal transplant
*Numbers may vary with institutional practice.

T/S
3
2
T/S
2

Chapter 3: Blood Utilization Management

schedule is accepted, inventory levels often
can be reduced. Ordering guidelines should
be periodically reviewed to keep pace with
changing methods and practices. A change
in the C:T ratio might signal a significant
modification in clinical practice.
The T/S, MSBOS, or SBO systems are intended for typical circumstances. Surgeons
or anesthesiologists may individualize specific requests and override the system to accommodate special needs. The transfusion
service must give special consideration to
patients with a positive antibody screen.
The antibody should be identified and, if it
is potentially clinically significant, an appropriate number of antigen-negative units
should be identified (eg, two, if the original
order was a type and screen).
Facilities using the immediate spin or
computer crossmatch can provide additional crossmatched units more rapidly if
required. This capability can allow such facilities to adjust their T/S, MSBOS, and SBO
schedules accordingly. More procedures
can be safely handled as type and screens
and fewer crossmatched units may be necessary.

Routine vs Emergency Orders
Transfusion services should establish procedures that define ideal stocking levels for
each blood type and critical levels at which
emergency orders are indicated. Transfusion service staff should have institutional
policies identifying the following:
■
Who monitors inventory levels?
■
Who is responsible for placing orders?
■
When and how are orders to be
placed (by telephone or facsimile)?
■
How are orders documented?
The addresses and telephone numbers of
approved blood suppliers and any needed
courier or cab services should be immediately available. Transfusion services need to
establish guidelines for handling blood

shortages and unexpected emergencies.
Equally important is specifying the actions
to take if transfusion requests cannot immediately be met.
Transfusion services should develop policies defining the following:
When ABO-compatible units may be
■
given instead of ABO-identical units.
When Rh-positive units may be given
■
to Rh-negative recipients.
When units crossmatched for a sur■
gical procedure may be released before the standard interval.
If units may be crossmatched for more
■
than one patient at a time.
What resources are available for trans■
fer of inventory.
Mechanisms to notify physicians of
■
critical blood shortages.
When cancellation of elective proce■
dures should be considered.
■
Methods to notify staff and patients
of surgery cancellations.

Inventory Counts and Inspection
On-hand units may be counted once or
several times a day to determine ordering
needs; computerized facilities may prefer
to take inventory electronically. Individual units must be visually inspected for
signs of contamination or atypical appearance before issue or shipping. Units
that do not meet inspection criteria must
be quarantined for further evaluation.
An organizational format for storage
should be established and followed. Unprocessed or incompletely processed units,
autologous units, and unsuitable units
must be clearly segregated (quarantined)
from routine stock.4(p15) Most institutions organize their blood inventory by status
(quarantined, retype unconfirmed, retype
confirmed, available, crossmatched, etc), by
product, by ABO group and Rh type, and,
within these categories, by expiration date.

AABB Technical Manual

Attention to detail in placing blood into
storage is necessary because a placement
error could be critical if a quarantined unit
is issued or a group O Rh-positive unit, incorrectly placed among group O Rh-negative units, is issued without careful checking in an emergency situation.

products. The formulas described in the
beginning of the chapter may be used to
estimate ideal inventory ranges.
Platelet usage often increases the day after a holiday because elective procedures
and oncology transfusions will have been
postponed. Planning ahead to stock platelets helps meet postholiday demand.

Frozen Plasma Products

Special Product Concerns
Platelets
Few articles address the management of
platelet inventory. Optimal levels are difficult to determine because demand is episodic and the shelf life is short. Often, the
effective shelf life is 3 days because, of the
allowable 5 days after phlebotomy, day 1
may be taken for testing and day 2 for
shipment. Planning is further complicated
by requests for special products, such as
leukocyte-reduced, crossmatched, HLAmatched, or cytomegalovirus (CMV)-seronegative platelets.
Platelet inventory management requires
good communication and cooperation
among patient care providers, transfusion
services, and blood centers. Information
about patients’ diagnoses and expected
transfusion schedules helps the blood center plan how many platelets to prepare and
which donors to recruit for plateletpheresis.
Transfusion services with low platelet
use usually order platelets only when they
receive a specific request. If the transfusion
service staff follows daily platelet counts
and special transfusion requirements of
known platelet users, they can often anticipate needs and place orders in advance.
Transfusion services with high use may find
it helpful to maintain platelets in inventory.
Transfusion services should define selection and transfusion guidelines for ABO
group, Rh type, irradiation, CMV serologic
testing, and leukocyte reduction of platelet

Because plasma components can be stored
frozen up to 1 year, these inventories are
easier to manage. Optimal inventory levels are determined by assessing statistics
on patient populations and usage patterns. Production goals and schedules can
then be established. Most centers find it
best to maintain consistent production
levels throughout the year, to achieve
evenly distributed expiration dates.
Some facilities prefer to freeze plasma
from group AB and A donors because these
units will be ABO-compatible with most
potential recipients. Plasma can be collected by apheresis to increase general
stock and provide for special needs.
Cryoprecipitated AHF is a labor-intensive product to prepare and supplies cannot easily be increased to meet large acute
needs. It is prudent to maintain inventories
at close-to-maximum levels.

Autologous and Directed Units
If autologous and directed donor units
constitute an increasing fraction of inventory, their management becomes a significant and controversial issue both for the
intended recipients and for institutions.
An extended discussion of autologous
blood collection and transfusion methods
can be found in Chapter 5.
Autologous and directed units should be
stored in separate designated areas in the
blood refrigerator. Such units are often arranged alphabetically by the intended re-

Chapter 3: Blood Utilization Management

cipient’s last name. Available units must be
clearly identified and monitored to ensure
issue in the proper sequence. Autologous
blood should always be used first, followed
by directed donor blood, and, finally, allogeneic units from general stock. Policies
about the reservation period for directed
donor units and possible release to other
recipients should be established at both the
transfusion service and donor center and
should be made known to laboratory staff,
to potential recipients, and to their physicians.

Special Inventories
Donor centers and transfusion services
are faced with requests for specialty products such as CMV-reduced-risk units,
HLA-matched platelets, antigen-matched
red cells, or irradiated components. The
appropriate use of these products is discussed in other chapters. Depending on
how and when they were prepared, these
products may have shortened expiration
dates.

If demand and inventory levels are very
high, a transfusion service may need to
keep separate inventories of these products
to make them easier to locate and monitor.
These special units can be rotated into general stock as they near their outdate because they can be given safely to others.

References
1.

Comprehensive report on blood collection
and transfusion in the United States in 2001.
Bethesda, MD: National Blood Data Resource
Center, 2003.
Friedman BA, Oberman HA, Chadwick AR, et
al. The maximum surgical blood order schedule and surgical blood use in the United States.
Transfusion 1976;16:380-7.
Devine P, Linden JV, Hoffstadter L, et al. Blood
donor-, apheresis-, and transfusion-related
activities: Results of the 1991 American Association of Blood Banks Institutional Membership Questionnaire. Transfusion 1993;33:77982.
Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005:15.

Chapter 4: Allogeneic Donor Selection and Blood Collection

Chapter 4

Allogeneic Donor Selection
and Blood Collection

LOOD CENTERS AND transfusion
services depend on volunteer donors to provide the blood necessary to meet the needs of the patients
they serve. To attract volunteer donors
and encourage their continued participation, it is essential that conditions surrounding blood donation be as pleasant,
safe, and convenient as possible. To protect donors and recipients, donors are
questioned about their medical history
and are given a miniphysical examination
to help blood center staff determine
whether they are eligible donors. The
phlebotomy is conducted carefully to
minimize any potential donor reactions
or bacterial contamination of the unit.

Blood Donation Process
The donor area should be attractive, accessible, and open at hours convenient
for donors. It must be well lighted, com-

fortably ventilated, orderly, and clean.
Personnel should be friendly, understanding, professional, and well trained. The area
must provide adequate space for private
and accurate examinations of individuals
to determine their eligibility as blood donors, and for the withdrawal of blood
from donors with minimum risk of contamination or exposure to activities and
equipment unrelated to blood collection.

Registration
The information obtained from the donor
during registration must fully identify the
donor and link the donor to existing records.1(p13) Some facilities require photographic identification. Current information must be obtained and recorded for
each donation. Selected portions of donation records must be kept indefinitely and
must make it possible to notify the donor
of any information that needs to be con1(p69)
The following information should
veyed.
be included:
97

AABB Technical Manual

1.
2.

Date and time of donation.
Name: Last, first (and middle initial
if available).
3.
Address: Residence and/or business.
4.
Telephone: Residence and/or business.
5.
Gender.
6.
Age or date of birth. Blood donors must
be at least 17 years of age or the age
stipulated by applicable state law.
7.
A record of reasons for previous deferrals, if any. Persons who have been
placed on a deferral or surveillance
list must be identified before any
unit drawn from them is made available for release. Ideally, a donor deferral registry should be available to
identify ineligible donors before
blood is drawn. If such a registry is
not available, there must be a procedure to review prior donation records and/or deferral registries before releasing the components from
quarantine.2
The following information may also be
useful:
1.
Additional identification such as social security or driver’s license number or any other name used by the
donor on a previous donation. The
Social Security Act specifically allows the use of the social security
number for this purpose. These data
are required for information retrieval
in some computerized systems. Identification of other names used by a
donor is particularly important to
ensure that the appropriate donor
file is accessed or that a deferral status is accurate.
2.
Name of patient or group to be acknowledged.
3.
Race. Although not required, this information can be particularly useful
when blood of a specific phenotype
is needed for patients who have un-

expected antibodies. Care should be
taken to be sure that minority populations understand the medical importance and scientific applications
of this information.3,4
Unique characteristics of the donor.
Certain information about the donor
may enable the blood bank to make
optimal use of the donation. For example, blood from donors who are
seronegative for cytomegalovirus
(CMV), or who are group O, Rh negative, is often designated for neonatal patients. The blood center may
specify that blood from these individuals be drawn routinely into collection bags suitable for pediatric
transfusion. Individuals known to
have clinically significant antibodies
may be identified so that their blood
can be processed into components
that contain only minimal amounts
of plasma.
A record of special communications
to a donor, special drawing of blood
samples for studies, etc.
If the donation is directed to a specific patient, information about when
and where the intended recipient will
be hospitalized should be obtained.
An order from the intended recipient’s physician should be provided
to the blood center staff. The intended recipient’s date of birth, social security number, or other identifiers may be required by the transfusion
service. If the donor is a blood relative
of the intended recipient, this information must be noted so that cellular
1(p43)
components can be irradiated.

Information Provided to the Prospective
Donor
All donors must be given educational materials informing them of significant risk

Chapter 4: Allogeneic Donor Selection and Blood Collection

of the procedure, the clinical signs and
symptoms associated with human immunodeficiency virus (HIV ) infection and
AIDS, high-risk activities for transmission,
and the importance of refraining from donating blood if they have engaged in these
activities or experienced associated signs
or symptoms. Before donating, the prospective donors must document that they
have read the material and have been
given the opportunity to ask questions
about the information. This information
must include a list of activities defined by
the Food and Drug Administration (FDA)
that increase the risk of exposure to HIV. A
description of HIV-associated clinical signs
and symptoms, including the following,
must be provided5:
1.
Unexplained weight loss.
2.
Night sweats.
3.
Blue or purple spots on or under the
skin or on mucous membranes.
4.
Swollen lymph nodes lasting more
than 1 month.
5.
White spots or unusual sores in the
mouth.
6.
Temperature greater than 100.5 F for
more than 10 days.
7.
Persistent cough and shortness of
breath.
8.
Persistent diarrhea.
The donor should be provided with
information about the tests to be done on
his or her blood, the existence of registries
of ineligible donors, and regulations or local standard operating procedures (SOPs)
that require notification to government
agencies of the donor’s infectious disease
status. The requirement to report positive
test results may differ from state to state;
they may include HIV, syphilis, and hepatitis testing. Prospective donors must also be
informed if there are routine circumstances
in which some tests for disease markers are
not to be performed.1(p15) The donor should
be told that he or she will be notified when

abnormal test results are recorded and the
donor has been placed on a deferral list.
When applicable, the donor must also be
informed that his or her blood is to be
tested with an investigational test such as a
nucleic acid amplification test. The possibility that testing may fail to identify infective individuals in an early seronegative
stage of infection should be included as
well.6 The same educational material can
be used to warn the prospective donor of
possible reactions and provide suggestions
for postphlebotomy care.
This information should be presented in
a way that the donor will understand.5 Provisions should be made for the hearing- or
vision-impaired, and interpreters should be
available for donors not fluent in English.
The use of interpreters known to the donor
should be discouraged. If such a practice is
necessary, a signed confidentiality statement should be obtained. In some locations, it may be helpful to have brochures
in more than one language. It is also helpful
to provide more detailed information for
first-time donors. Information about alternative sites or other mechanisms to obtain
HIV tests should be available to all prospective donors.

Donor Selection
The donor screening process is one of the
most important steps in protecting the
safety of the blood supply. The process is
intended to identify elements of the medical history and behavior or events that
put a person at risk for transmissible disease or at personal medical risk. It is,
therefore, imperative that proper guidelines and procedures be followed to make
the donor screening process effective.
A qualified physician must determine
the eligibility of donors. The responsibility
may be delegated to a designee working

100

AABB Technical Manual

under the physician’s direction after appropriate training.7 Donor selection criteria are
established through regulations, recommendations, and standards of practice.
When a donor’s condition is not covered or
addressed by any of these, a qualified physician should determine the eligibility.
Donor selection is based on a medical
history and a limited physical examination
done on the day of donation to determine
if giving blood will harm the donor or if
transfusion of the unit will harm a recipient.1(p17) The medical history questions (including questions pertaining to risk behavior associated with HIV infections) may be
asked by a qualified interviewer or donors
may complete their own record, which
must then be reviewed with the donor and
initialed by a trained knowledgeable staff
member of the donor service according to
local SOPs, state, and FDA approval.5,8 Some
donor centers have instituted an FDA-approved computer-generated questionnaire.
The interview and physical examination
must be performed in a manner that ensures adequate auditory and visual privacy,
allays apprehensions, and provides time for
any necessary discussion or explanation.
Details explaining a donor’s answers that
require further investigation should be documented by the staff on the donor form.
Results of observations made when a physical examination is given and when tests
are performed must be recorded concurrently.
Donors must understand the information that is presented to them in order to
make an informed decision to donate their
blood. Effective communication is vital for
conveying important information and eliminating ineligible donors from the donor
pool. Of equal importance is the training of
donor center staff. Screening can be effective only if the staff members are proficient
in their jobs and understand thoroughly the
technical information required to perform

the job. Good interpersonal and public relations skills are essential for job
competency. Because donor center staff are
in constant contact with donors, knowledgeable personnel and effective communication contribute to positive public
perception and to the success of donor
screening programs.

Medical History
While the medical history is obtained,
some very specific questions are necessary to ensure that, to the greatest extent
possible, it is safe for the donor to donate
and for the blood to be transfused. The interviewer should review and evaluate all
responses to determine eligibility for donation and document the decision. To be
sure that all the appropriate questions are
asked and that donors are given a consistent message, use of the most recent FDAapproved AABB donor history questionnaire is recommended. The most recent
FDA-approved version is found on the AABB
Web site. (See Appendices 4-1 through 4-3,
which were current at the time of this writing.)
One area of the medical history—medications and drugs taken by the donor—often requires further investigation. New prescription drugs and over-the-counter
medications enter the marketplace daily,
and donors may report use of a drug not
specifically noted in the facility’s SOP manual. Although there is consensus on those
drugs that are always or never a cause for
deferral, many drugs fall into a category
over which disagreement exists. In these
cases, the reason for taking the drug (rather
than the drug itself) is usually the cause for
deferral. Appendix 4-4 lists drugs that many
blood banks do consider acceptable without approval from a donor center physician.
Prospective donors who have taken isotretinoin (Accutane) or finasteride (Proscar

Chapter 4: Allogeneic Donor Selection and Blood Collection

or Propecia) within the 30 days preceding
donation; dutasteride (Avodart) within the
6 months preceding donation; acitretin
(Soriatane) within the 3 years preceding donation; or etretinate (Tegison) at any time
must be deferred.1(p62) The Armed Services
Blood Program Office makes its drug deferral list available to the public.9
Deferring or rejecting potential donors
often leaves those persons with negative
feelings about themselves as well as the
blood donation process. Donors who are
deferred must be given a full explanation of
the reason and be informed whether or
when they can return to donate. It may be
prudent to document this notification.

Confidential Unit Exclusion
Donors may be given the opportunity to
indicate confidentially whether their blood
is or is not suitable for transfusion to others. This should be done by a mechanism
that allows the donor to avoid face-toface admission of risk behaviors.
The donor must be given instructions to
the effect that he or she may call the blood
bank after the donation and ask that the
unit collected not be used. A mechanism
should exist to allow retrieval of the unit
without obtaining the donor’s identity (eg,
use of Whole Blood number).
If the donor indicates that blood collected should not be used for transfusion,
he or she should be informed that the
blood will be subjected to testing and that
there will be notification of any positive

101

results. Counseling or referral must be provided for positive HIV test results, or if any
other medically significant test results have
been detected.

Physical Examination
The following variables must be evaluated
for each donor. The donor center physician must approve exceptions to routinely
acceptable findings. For special donor
categories, the medical director may provide policies and procedures to guide decisions. Other donors may require individual evaluation.
1.
General appearance: If the donor
looks ill, or is excessively nervous, it
is best to defer the donation.
2.
Weight: No more than 10.5 mL of
whole blood per kilogram of body
weight shall be collected at a donation.1(p61) This amount shall include
samples for testing. If it is necessary
to draw a smaller amount than appropriate for a standard collection
container, then the amount of anticoagulant in the container must be
adjusted appropriately. The formula
in Table 4-1 may be used to determine the amount of anticoagulant to
remove. The volume of blood drawn
must be measured carefully and accurately.
3.
Temperature: The donor’s temperature must not exceed 37.5 C (99.5 F)
if measured orally, or its equivalent if
measured by another method. Lower

Table 4-1. Calculations for Drawing Donors Weighing Less than 50 kg (110 lb)
A. Volume to draw* = (Donor’s weight in kg/50) × 450 mL
B. Amount of anticoagulant† needed = (A/100) × 14
C. Amount of anticoagulant to remove from collection bag = 63 mL – B
*Approximately 12% of total blood volume.
†
CPD or CPDA-1 solutions for which the desired anticoagulant:blood ratio is 1.4:10.

102

AABB Technical Manual

than normal temperatures are usually of no significance in healthy individuals; however, they should be
repeated for confirmation.
Pulse: The pulse rate should be counted
for at least 15 seconds. It should exhibit no pathologic irregularity, and
the frequency should be between 50
and 100 beats per minute. If a prospective donor is a known athlete
with high exercise tolerance, a pulse
rate below 50 may be noted and
should be acceptable. A donor center physician should evaluate marked
abnormalities of pulse and recommend acceptance, deferral, or referral for additional evaluation.
Blood pressure: The blood pressure
should be no higher than 180 mm Hg
systolic and 100 mm Hg diastolic. Prospective donors whose blood pressure is above these values should not
be drawn without individual evaluation by a qualified physician.
Hemoglobin or packed cell volume
(hematocrit): Before donation, the
hemoglobin or hematocrit must be
determined from a sample of blood
obtained at the time of donation.
Although this screening test is intended to prevent collection of blood
from a donor with anemia, it does
not ensure that the donor has an adequate store of iron. Table 4-2 gives

the lower limits of hemoglobin for
accepting allogeneic donors. Individuals with unusually high hemoglobin or hematocrit levels may need
to be evaluated by a physician because the elevated levels may reflect
pulmonary, hematologic, or other
abnormalities. Methods to evaluate
hemoglobin concentration include
1) specific gravity determined by copper sulfate (see Method 6.1), 2) spectrophotometric measurement of hemoglobin or determination of the
hematocrit, or 3) alternate accepted
methods to rule out erroneous results that may lead to rejection of a
donor. Earlobe puncture is not an
acceptable source for a blood sample.1(p61),10
Skin lesions: The skin at the site of
venipuncture must be free of lesions.
Both arms must be examined for
signs of repeated parenteral entry,
especially multiple needle puncture
marks and/or sclerotic veins as seen
with drug use. Such evidence is reason for indefinite exclusion of a prospective donor. Mild skin disorders
or the rash of poison ivy should not
be cause for deferral unless unusually extensive and/or present in the
antecubital area. Individuals with
boils, purulent wounds, or severe
skin infections anywhere on the

Table 4-2. Minimum Levels of Hemoglobin, Hematocrit, and Red Cell Density for
Accepting an Allogeneic Blood Donor
Donor Test Method

Minimal Acceptable Value

Hemoglobin
Hematocrit
Copper sulfate

12.5 g/dL
38%
1.053 sp gr

Chapter 4: Allogeneic Donor Selection and Blood Collection

103

body should be deferred, as should
anyone with purplish-red or hemorrhagic nodules or indurated plaques
suggestive of Kaposi’s sarcoma.
The record of the physical examination
and the medical history must identify and
contain the examiner’s initials or signature.
Any reasons for deferral must be recorded
and explained to the donor. A mechanism
must exist to notify the donor of clinically
significant abnormal findings in the physical examination, medical history, or postdonation laboratory testing.11 Abnormalities
found before donation may be explained
verbally by qualified personnel. Test results
obtained after donation that preclude further donation may be reported in person,
by telephone, or by letter. Donors should be
asked to report any illness developing within a few days after donation and, especially,
to report a positive HIV test or the occurrence of hepatitis or AIDS that develops
within 12 months after donation.

plasma. If I am potentially at risk for
spreading the virus known to cause AIDS, I
agree not to donate blood or plasma for
transfusion to another person or for further
manufacture. I understand that my blood
will be tested for HIV and other disease
markers; however, there may be unforeseen
circumstances when infectious disease testing may not be performed. If this testing indicates that I should no longer donate blood
or plasma because of the risk of transmitting
an infectious disease, my name will be entered on a list of permanently deferred donors. I understand that I will be notified of a
positive laboratory test result(s). If, instead,
the results of the testing are not clearly negative or positive, my blood will not be used
and my name may be placed on a deferral
list.”
If units are occasionally used for reasons
other than transfusion, such as research,
then the informed consent should address
such occasions.

Donor Consent

Special Donor Categories

Written consent that allows donor center
personnel to collect and use blood from
the prospective donor is required.1(p15) The
consent form is part of the donor record
and must be completed before donation.
The procedure must be explained in
terms that donors can understand, and
there must be an opportunity for the prospective donor to ask questions. The
signed donor record or consent form
should also indicate that the donor has
read and understood the information
about infectious diseases transmissible by
transfusion and has given accurate and
truthful answers to the medical history
questions. Wording equivalent in meaning to the following is suggested:
“I have read and understand the information provided to me regarding the spread
of the AIDS virus (HIV ) by blood and

Exceptions to the usual eligibility requirements may be made for special donor categories:
1.
Autologous donors: The indications
for collection and variations from
usual donor procedures are discussed in Chapter 5.
2.
Hemapheresis: Special requirements
and recommendations for cytapheresis donors or for donors in a plasmapheresis program are detailed in
Chapter 6.
3.
Recipient-specific “designated” donations: Under certain circumstances,
it may be important to use blood or
components from a specific donor
for a specific patient. Examples include the patient with an antibody
to a high-incidence antigen or a
combination of antibodies that makes

104

AABB Technical Manual

it difficult to find compatible blood;
the infant with neonatal alloimmune
thrombocytopenia whose mother
can provide platelets; the patient
awaiting a kidney transplant from a
living donor; or the multitransfused
patient whose family members can
provide components.
The repeated use of a single donor
to supply components needed for a
single patient is allowed, provided it
is requested by the patient’s physician and approved by the donor center physician. The donor must meet
all the usual requirements for donation, except that the frequency of
donation can be as often as every 3
days, as long as the predonation hemoglobin level meets or exceeds the
minimum value for routine allogeneic blood donation.
The blood must be processed according to AABB Standards for Blood
Banks and Transfusion Services.1(pp24-32)
Special tags identifying the donor
unit number and the intended recipient must be affixed to the blood or
component bag, and all such units
must be segregated from the normal
inventory. A protocol for handling
such units must be included in the
SOP manual.
Directed donors: The public’s concern about the safety of transfusion
has generated demands from potential recipients to choose the donors
to be used for their transfusions.
Several states have laws establishing
this as an acceptable procedure that
must be offered by a donor service
in nonemergency situations, if requested by a potential blood recipient or ordered by a physician. Despite
logistic and philosophic problems
associated with these “directed” donations, most blood centers and

hospitals provide this service. The
selection and testing of directed donors should be the same as for other
allogeneic donors, although special
exemptions to the 56-day (or 112
days for double red cell donation)
waiting period between donations
may be made. Federal regulations
state that a person may serve as a
source of Whole Blood more than
once in 8 weeks only if at the time of
donation the donor is examined and
certified by a physician to be in good
health. 12 To avoid misunderstandings, it is important to establish SOPs
that define the interval required between collection of the blood and its
availability to the recipient; the policy
about determining ABO group before
collection; and the policy for releasing units for use by other patients.

Collection of Blood
Blood is to be collected only by trained
personnel working under the direction of
a qualified licensed physician. Blood collection must be by aseptic methods, using
a sterile closed system. If more than one
skin puncture is needed, a new container
and donor set must be used for each additional venipuncture unless the SOP allows
the use of an FDA-approved device to attach a new needle while preserving sterility. The phlebotomist must sign or initial
the donor record, even if the phlebotomy
did not result in the collection of a full
unit.

Blood Containers
Blood must be collected into an FDA-approved container that is pyrogen-free and
sterile and contains sufficient anticoagulant for the quantity of blood to be col-

Chapter 4: Allogeneic Donor Selection and Blood Collection

lected. The container label must state the
type and amount of anticoagulant and the
approximate amount of blood collected.
Blood bags may be supplied in packages
containing more than one bag. The manufacturer’s directions should be followed for
the length of time unused bags may be
stored in packages that have been opened.

Identification
Identification is essential in each step
from donor registration to final disposition of each component. A numeric or alphanumeric system must be used that
identifies, and relates to, the source donor, the donor record, the specimens used
for testing, the collection container, and
all components prepared from the unit.
Extreme caution is necessary to avoid any
mix-up or duplication of numbers. All records and labels should be checked before use for printing errors. If duplicate
numbers are found, they must be removed and may be investigated to ascertain the reason for the duplication (eg,
supplier error, etc). A record must be kept
of all voided numbers.
Before beginning the collection, the
phlebotomist should:
1.
Identify the donor record (at least by
name) with the donor and ask the
donor to state or spell his or her
name.
2.
Attach numbered labels to the donor
record and ensure that it matches
the blood collection container, attached satellite bags, and tubes for
donor blood samples. Attaching the
numbers at the donor chair, rather
than during the examination procedures, helps reduce the likelihood of
identification errors.
3.
Be sure that the processing tubes are
correctly numbered and that they
accompany the container during the

105

collection of blood. Tubes may be attached in any convenient manner to
the primary bag or integral tubing.
Recheck all numbers.

Preparation of the Venipuncture Site
Blood should be drawn from a large firm
vein in an area (usually the antecubital
space) that is free of skin lesions. Both
arms must be inspected for evidence of
drug use, skin disease, or scarring. A tourniquet or a blood pressure cuff inflated to
40 to 60 mm Hg makes the veins more
prominent. Having the donor open and
close the hand a few times is also helpful.
Once the vein is selected, the pressure device should be released before the skin
site is prepared.
There is no way to make the venipuncture site completely aseptic, but surgical cleanliness can be achieved to provide
the best assurance of an uncontaminated
unit. Several acceptable procedures exist
(see Method 6.2). After the skin has been
prepared, it must not be touched again to
repalpate the vein. The entire site preparation must be repeated if the cleansed skin is
touched.

Phlebotomy and Collection of Samples
A technique for drawing a donor unit and
collecting samples for testing appears in
Method 6.3. The unit should be collected
from a single venipuncture after the pressure device has again been inflated. During collection, the blood should be mixed
with the anticoagulant. The amount of
blood collected should be monitored carefully so that the total, including samples,
does not exceed 10.5 mL per kilogram of
1(p61)
donor weight per donation.
When the
appropriate amount has been collected,
segments and specimen tubes must be
filled. The needle and any blood-contaminated waste must be disposed of safely in

106

AABB Technical Manual

accordance with universal precaution
guidelines. The needle must not be recapped unless a safety recapping device is
used. Disposal of the needle must be in a
puncture-proof container. After collection, there must be verification that the
identifiers on the unit, the donor history,
and the tubes are the same. Gloves must
be available for use during phlebotomy
and must be worn by phlebotomists when
collecting autologous blood and when individuals are in training.

Care of the Donor After Phlebotomy
After removing the needle from the vein,
the phlebotomist should:
1.
Apply firm pressure with sterile gauze
over the point of entry of the needle
into vein. (The donor may be instructed to continue application of
pressure for several minutes.) Check
arm and apply a bandage only after
all bleeding stops.
2.
Have donor remain reclining on the
bed or in the donor chair for a few
minutes under close observation by
staff.
3.
Allow the donor to sit up under observation until his or her condition
appears satisfactory. The donor should
be observed in the upright position
before release to the observation/refreshment area. Staff should monitor
donors in this area. The period of observation and provision of refreshment
should be specified in the SOP manual.
4.
Give the donor instructions about
postphlebotomy care. The medical
director may wish to include some
or all of the following recommendations or instructions:
a.
Eat and drink something before
leaving the donor site.
b.
Do not leave until released by a
staff member.

Drink more fluids than usual in
the next 4 hours.
d.
Avoid consuming alcohol until
something has been eaten.
e.
Do not smoke for 30 minutes.
f.
If there is bleeding from the
phlebotomy site, raise arm and
apply pressure to the site.
g.
If fainting or dizziness occurs,
either lie down or sit with the
head between the knees.
h.
If any symptoms persist, either
telephone or return to the donor center or see a doctor.
i.
Resume all normal activities if
asymptomatic. Donors who work
in certain occupations (eg, construction workers, operators of
machinery) or persons working
at heights should be cautioned
that dizziness or faintness may
occur if they return to work immediately after giving blood.
j.
Remove bandage after a few
hours.
k.
Maintain high fluid intake for
several days to restore blood
volume.
Thank the donor for an important
contribution and encourage repeat
donation after the proper interval.
All personnel on duty throughout
the donor area, volunteer or paid,
should be friendly and qualified to
observe for signs of a reaction such
as lack of concentration, pallor, rapid
breathing, or excessive perspiration.
Donor room personnel should be
trained and competent to interpret
instructions, answer questions, and
accept responsibility for releasing
the donor in good condition.
Note on the donor record any adverse reactions that occurred. If the
donor leaves the area before being
released, note this on the record.

Chapter 4: Allogeneic Donor Selection and Blood Collection

Adverse Donor Reactions
Most donors tolerate giving blood very
well, but adverse reactions occur occasionally. Personnel must be trained to recognize adverse reactions and to provide
initial treatment.
Donor room personnel should be trained in cardiopulmonary resuscitation (CPR).
Special equipment to handle emergency
situations must be available.
Syncope (fainting or vasovagal syndrome) may be caused by the sight of
blood, by watching others give blood, or by
individual or group excitement; it may also
happen for unexplained reasons. Whether
caused by psychologic factors or by neurophysiologic response to blood donation, the
symptoms may include weakness, sweating, dizziness, pallor, loss of consciousness,
convulsions, and involuntary passage of feces or urine. On occasion, the skin feels
cold and blood pressure falls. Sometimes,
the systolic blood pressure levels fall as low
as 50 mm Hg or cannot be heard with the
stethoscope. The pulse rate often slows significantly. This can be useful in distinguishing between vasovagal attack and cardiogenic or hypovolemic shock, in which cases
the pulse rate rises. This distinction, although characteristic, is far from absolute.
Rapid breathing or hyperventilation may
cause the anxious or excited donor to lose
excessive amounts of carbon dioxide. This
may cause generalized sensations of suffocation or anxiety, or localized problems
such as tingling or twitching.
The donor center physician must provide written instructions for handling donor reactions, including a procedure for obtaining emergency medical help. Sample
instructions might be as follows:
1.
General.
a.
Remove the tourniquet and withdraw the needle from the arm if

107

signs of a reaction occur during
the phlebotomy.
b.
If possible, remove any donor
who experiences an adverse reaction to an area where he or
she can be attended in privacy.
c.
Apply the measures suggested
below and, if they do not lead
to rapid recovery, call the blood
bank physician or the physician
designated for such purposes.
Fainting.
a.
Apply cold compresses to the
donor’s forehead or the back of
the neck.
b.
Place the donor on his or her
back, with their legs raised above
the level of the head.
c.
Loosen tight clothing.
d.
Be sure the donor has an adequate airway.
e.
Monitor blood pressure, pulse,
and respiration periodically until the donor recovers.
Note: Some donors who experience prolonged hypotension may respond to an infusion of normal saline. The decision to initiate such
therapy should be made by the donor center physician either on a caseby-case basis or in a policy stated in
the facility’s SOP manual.
Nausea and vomiting.
a.
Make the donor as comfortable
as possible.
b.
Instruct the donor who is nauseated to breathe slowly and
deeply.
c.
Apply cold compresses to the
donor’s forehead and/or back
of neck.
d.
Turn the donor’s head to the side.
e.
Provide a suitable receptacle if
the donor vomits and have cleansing tissues or a damp towel
ready. Be sure the donor’s head

108

AABB Technical Manual

is turned to the side because of
the danger of aspiration.
f.
After vomiting has ended, give
the donor some water to rinse
out his or her mouth.
Twitching or muscular spasms. Extremely nervous donors may hyperventilate, causing faint muscular
twitching or tetanic spasm of their
hands or face. Donor room personnel should watch closely for these
symptoms during and immediately
after the phlebotomy.
a.
Divert the donor’s attention by
engaging in conversation, to
interrupt the hyperventilation
pattern.
b.
Have the donor cough if he or
she is symptomatic. Do not give
oxygen.
Hematoma during or after phlebotomy.
a.
Remove the tourniquet and the
needle from the donor’s arm.
b.
Place three or four sterile gauze
squares over the venipuncture
site and apply firm digital pressure for 7 to 10 minutes, with
the donor’s arm held above the
heart level. An alternative is to
apply a tight bandage, which
should be removed after 7 to 10
minutes to allow inspection.
c.
Apply ice to the area for 5 minutes, if desired.
d.
Should an arterial puncture be
suspected, immediately withdraw needle and apply firm
pressure for 10 minutes. Apply
pressure dressing afterwards.
Check for the presence of a radial pulse. If pulse is not palpable or is weak, call a donor center physician.
Convulsions.
a.
Call for help immediately. Prevent the donor from injuring him-

self or herself. During severe
seizures, some people exhibit
great muscular power and are
difficult to restrain. If possible,
hold the donor on the chair or
bed; if not possible, place the
donor on the floor. Try to prevent injury to the donor and to
yourself.
b.
Be sure the donor has an adequate airway. A padded device
should separate the jaws after
convulsion has ceased.
c.
Notify the donor center physician.
7.
Serious cardiac difficulties.
a.
Call for medical aid and/or an
emergency care unit immediately.
b.
If the donor is in cardiac arrest,
begin CPR immediately and
continue it until help arrives.
The nature and treatment of all reactions
should be recorded on the donor’s record
or a special incident report form. This should
include a notation of whether the donor
should be accepted for future donations.
The medical director should decide what
emergency supplies and drugs should be in
the donor area. The distance to the nearest
emergency room or emergency care unit
heavily influences decisions about necessary supplies and drugs. Most donor centers maintain some or all of the following:
1.
Emesis basin or equivalent.
2.
Towels.
3.
Oropharyngeal airway, plastic or hard
rubber.
4.
Oxygen and mask.
5.
Emergency drugs: Drugs are seldom
required to treat a donor’s reaction.
If the donor center physician wishes
to have any drugs available, the kind
and amount to be kept on hand must
be specified in writing. In addition,
the medical director must provide
written policies stating when and by

Chapter 4: Allogeneic Donor Selection and Blood Collection

whom any of the above medical supplies or drugs may be used.

References
1.

10.

11.

12.

109

Code of federal regulations. Title 21 CFR
640.3(f ). Washington, DC: US Government
Printing Office, 2004 (revised annually).

Suggested Reading

Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.
Code of federal regulations. Title 21 CFR
606.160(e). Washington, DC: US Government
Printing Office, 2004 (revised annually).
Beattie KM, Shafer AW. Broadening the base
of a rare donor program by targeting minority
populations. Transfusion 1986;26:401-4.
Vichinsky EP, Earles A, Johnson RA, et al. Alloimmunization in sickle cell anemia and
transfusion of racially unmatched blood. N
Engl J Med 1990;322:1617-21.
Food and Drug Administration. Memorandum: Revised recommendations for the prevention of human immunodeficiency virus
(HIV) transmission by blood and blood products. (April 23, 1992) Rockville, MD: CBER Office of Communication, Training, and Manufacturers Assistance, 1992.
Centers for Disease Control. Update: Universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus, and other bloodborne pathogens
in health-care settings. JAMA 1988;260:528-31.
Code of federal regulations. Title 21 CFR
640.4(a). Washington, DC: US Government
Printing Office, 2004 (revised annually).
Food and Drug Administration. Guidance for
Industry: Streamlining the donor interview
process: Recommendations for self-administered questionnaires. (July 3, 2003) Rockville,
MD: CBER Office of Communication, Training, and Manufacturers Assistance, 2003.
Armed Services Blood Program Office. Drugs
and medications. [Available at http://www.
tricare.osd.mil/asbpo/librar y/policies/
downloads/medication_list.doc.]
Newman B. Blood donor suitability and
allogeneic whole blood donation. Transfus
Med Rev 2001;15:234-44.
Food and Drug Administration. General requirements for blood, blood components, and
blood derivatives; donor notification. Title 21
CFR 630.6. Fed Regist 2001;66:31165-77.

Code of federal regulations. Title 21 CFR 640.3.
Washington, DC: US Government Printing Office,
2004 (revised annually). [History of viral hepatitis
before the 11th birthday.]
Food and Drug Administration. Memorandum:
Revised recommendations for the prevention of
human immunodeficiency virus (HIV ) transmission by blood and blood products. (April 23, 1992)
Rockville, MD: CBER Office of Communication,
Training, and Manufacturers Assistance, 1992.
Food and Drug Administration. Guidance for industry: Revised preventive measures to reduce the
possible risk of transmission of Creutzfeldt-Jakob
disease (CJD) and new variant Creutzfeldt-Jakob
disease (nvCJD) by blood and blood products.
( January 9, 2002) Rockville, MD: CBER Office of
Communication, Training, and Manufacturers
Assistance, 2002.
Infectious disease testing for blood transfusions.
NIH Consensus Statement 13:1, January 1995.
Bethesda, MD: National Institutes of Health, 1995.
Kasprisin C, Laird-Fryer B, eds. Blood donor collection practices. Bethesda, MD: AABB, 1993.
Linder J, ed. Practical solutions to practical problems in transfusion medicine and tissue banking.
[Supplement 1 to Am J Clin Pathol 1997;107(4).]
Chicago, IL: American Society of Clinical Pathologists, 1997.
Schmuñis GA. Trypanosoma cruzi, the etiologic
agent of Chagas’ disease: Status in the blood supply in endemic and nonendemic countries. Transfusion 1991;31:547-57.
Smith KJ, Simon TL. Recruitment and evaluation
of blood and plasma donors. In: Rossi EC, Simon
TL, Moss GS, eds. Principles of transfusion medicine. 2nd ed. Baltimore, MD: Williams and Wilkins,
1995:871-9.
Tan L, Williams MA, Khan MK, et al. Risk of transmission of bovine spongiform encephalopathy to
humans in the United States. JAMA 1999;281:2330-8.

110

AABB Technical Manual

Appendix 4-1. Full-Length Donor History Questionnaire*

Chapter 4: Allogeneic Donor Selection and Blood Collection

Appendix 4-1. Full-Length Donor History Questionnaire (cont’d)*

111

112

AABB Technical Manual

Appendix 4-1. Full-Length Donor History Questionnaire (cont’d)*

*Downloaded from http://www.aabb.org on April 19, 2005. Check web site for updates.

Chapter 4: Allogeneic Donor Selection and Blood Collection

Appendix 4-2. Medication Deferral List*

*Downloaded from http://www.aabb.org on April 19, 2005. Check web site for updates.

113

114

AABB Technical Manual

Appendix 4-3. Blood Donor Education Materials*

*Downloaded from http://www.aabb.org on April 19, 2005. Check web site for updates.

Chapter 4: Allogeneic Donor Selection and Blood Collection

115

Appendix 4-4. Some Drugs Commonly Accepted in Blood Donors
In many blood centers, blood donation may be allowed by individuals who have taken the following drugs:
■
■
■

■
■
■
■
■

Tetracyclines and other antibiotics taken to treat acne.
Topical steroid preparations for skin lesions not at the venipuncture site.
Blood pressure medications, taken chronically and successfully so that pressure is at or below
allowable limits. The prospective donor taking antihypertensive drugs should be free from side
effects, especially episodes of postural hypotension, and should be free of any cardiovascular
symptoms.
Over-the-counter bronchodilators and decongestants.
Oral hypoglycemic agents in well-controlled diabetics without any vascular complications of the
disease.
Tranquilizers, under most conditions. A physician should evaluate the donor to distinguish
between tranquilizers and antipsychotic medications.
Hypnotics used at bedtime.
Marijuana (unless currently under the influence), oral contraceptives, mild analgesics, vitamins,
replacement hormones, or weight reduction pills.

Note: Acceptance of donors must always be with the approval of the blood bank’s medical director.

Chapter 5: Autologous Blood Donation and Transfusion

Chapter 5

Autologous Blood Donation
and Transfusion

UTOLOGOUS BLOOD TRANSFUsion is an alternative therapy for
many patients anticipating transfusion. Different categories of autologous
transfusion are:
1.
Preoperative collection (blood is drawn
and stored before anticipated need).
2.
Perioperative collection and administration.
a.
Acute normovolemic hemodilution (blood is collected at the
start of surgery and then infused during or at the end of
the procedure).
b.
Intraoperative collection (shed
blood is recovered from the
surgical field or circulatory device and then infused).
c.
Postoperative collection (blood
is collected from drainage devices and reinfused to the patient).
Each type of autologous transfusion
practice offers potential benefits and risks

depending on the type of surgery, condition
of the patient, and technology available.
Each facility must analyze its own transfusion practices, transfusion practices of
other similarly situated institutions, and its
own capabilities to determine the appropriate services to be offered.
However, it is generally accepted that,
when feasible, the patient should have the
option to use his or her own blood. The US
Supreme Court has ruled that asymptomatic infection with HIV is a disability protected under the Americans with Disabilities Act.1 Therefore, if institutions offer
autologous services to any patient, they
should consider offering such services to
HIV-positive patients.2 Patients who are
likely to require transfusion therapy and
who also meet the donation criteria should
be told about the options for autologous
transfusion therapies. Patients considering
autologous transfusion therapy should be
informed about the risks and benefits of both
the autologous donation and the auto117

118

AABB Technical Manual

logous transfusion process. Specific issues
unique to the use of autologous transfusion
in the anticipated surgical procedure should
be identified, including the possibility of
administrative error. In addition, patients
need information about any special fees for
autologous services, the level of infectious
disease testing that will be performed, and
the possibility that additional, allogeneic,
units may be used.

Preoperative Autologous
Blood Collection
Frequently cited advantages and disadvantages of preoperative autologous blood
donation (PAD) are summarized in Table
5-1. Candidates for preoperative collection are stable patients scheduled for surgical procedures in which blood transfusion is likely. For procedures that are
unlikely to require transfusion (ie, a maximal surgical blood ordering schedule
does not suggest that crossmatched blood
be available), the use of preoperative blood
collection is not recommended.

In selected patient subgroups, preoperative collection of autologous blood can significantly reduce exposure to allogeneic
blood. PAD collections should be considered
for patients likely to receive transfusion,
such as patients undergoing major orthopedic procedures, vascular surgery, and
cardiac or thoracic surgery.3,4 The most
common surgical procedures for which
autologous blood is donated are total joint
replacements.4 Autologous blood should not
be collected for procedures that seldom
(less than 10% of cases) require transfusion,
such as cholecystectomy, herniorrhaphy,
vaginal hysterectomy, and uncomplicated
obstetric delivery.5

Special Patient Categories
In special circumstances, preoperative
autologous blood collection can be performed for patients who would not ordinarily be considered for allogeneic donation. The availability of medical support is
important in assessing patient eligibility.
With suitable volume modification, parental cooperation, and attention to preparation and reassurance, pediatric patients

Table 5-1. Autologous Blood Donation
Advantages

Disadvantages

1. Prevents transfusion-transmitted disease.

3. Supplements the blood supply.

1. Does not eliminate risk of bacterial contamination.
2. Does not eliminate risk of ABO incompatibility error.
3. Is more costly than allogeneic blood.

4. Provides compatible blood for patients with
alloantibodies.
5. Prevents some adverse transfusion reactions.
6. Provides reassurance to patients concerned about blood risks.

4. Results in wastage of blood that is not
transfused.
5. Increased incidence of adverse reactions to
autologous donation.
6. Subjects patients to perioperative anemia
and increased likelihood of transfusion.

2. Prevents red cell alloimmunization.

Chapter 5: Autologous Blood Donation and Transfusion

can participate in preoperative collection
programs.6 The successful use of autologous blood in a patient with sickle cell
disease has been reported,7 and it may be
particularly useful for a sickle cell patient
with multiple alloantibodies; however, the
patient may derive greater benefit from
allogeneic transfusions that provide hemoglobin A. Red cells containing hemoglobin S require special handling during
the cryopreservation process.8
Patients with significant cardiac disease
are considered poor risks for autologous
blood donation. Despite reports of safety in
small numbers of patients who underwent
autologous blood donation,9 the risks that
are associated with autologous blood donation10 in these patients are probably greater
than the current estimated risks of allogeneic transfusion.11,12 Table 5-2 summarizes the contraindications to a patient’s
participation in an autologous blood donation program.13
The collection of autologous blood from
women during routine pregnancy is unwar-

Table 5-2. Contraindications to
Participation in Autologous Blood
Donation Programs
1. Evidence of infection and risk of
bacteremia.
2. Scheduled surgery to correct aortic stenosis.
3. Unstable angina.
4. Uncontrolled seizure disorder.
5. Myocardial infarction or cerebrovascular
accident within 6 months of donation.
6. Patients with significant cardiac or pulmonary disease who have not yet been cleared
for surgery by their treating physician.
7. High-grade left main coronary artery disease.
8. Cyanotic heart disease.
9. Uncontrolled hypertension.

119

ranted, because blood is so seldom needed.
Many centers give serious consideration to
autologous collection for women with alloantibodies to multiple or high-incidence
antigens, placenta previa, or other conditions
placing them at high risk for ante- or intrapartum hemorrhage.5 A policy should be
developed for situations in which maternal
red cells are considered for transfusion to
the infant.

Voluntary Standards
AABB Standards for Blood Banks and Transfusion Services offers uniform standards to
be followed in determining patient eligibility; collecting, testing, and labeling the
unit; and pretransfusion testing.15(pp18,39,51)
These AABB standards apply to preoperative autologous blood collection. Standards for Perioperative Autologous Blood
Collection and Administration have been
established to enhance the quality and
safety of perioperative autologous transfusion activities (intra- and postoperative
blood recovery, perioperative autologous
component production, and intraoperative acute normovolemic hemodilution).16

Compliance Considerations
Food and Drug Administration (FDA) requirements have evolved over time. The
FDA first issued guidance for autologous
blood and blood components in March of
1989.17 This guidance was clarified in a
second memorandum issued in February
of 1990.18 Much of the information in previous guidance has been superseded by
regulations. The FDA included requirements
regarding autologous blood in regulations
issued June 11, 2001.19,20

Testing
The FDA requires tests for evidence of infection resulting from the following com-

120

AABB Technical Manual

municable diseases: HIV-1, HIV-2, HBV,
HCV, HTLV-I, and HTLV-II (21 CFR 610.40)
and a serologic test for syphilis [21 CFR
610.40(a)(1) and 610.40(I)]. Such tests in21
clude nucleic acid tests for HCV and HIV.
Testing of autologous donations is not
required unless the donations are to be
used for allogeneic transfusion [21 CFR
610.40(d)(1)]. Autologous donations that
are to be shipped to another facility that
does allow autologous units to be used for
allogeneic transfusion must be tested [21
CFR 610.40(d)(2)]. For autologous donations shipped to another establishment
that does not allow autologous donations
to be used for allogeneic transfusion, the
first donation in each 30-day period must
be tested [21 CFR 610.40(d)(3)].15(p34)
Autologous donations found to be reactive by a required screening test must be retested whenever a supplemental (additional,
more specific) test has been approved by
the FDA. At a minimum, the first reactive
donation in each 30-day period must be
tested unless a record exists for a positive
supplemental test result for that donor [21
CFR 610.40(e)(1,2)]. Both the AABB Standards15(p34) and the FDA requirements [21
CFR 630.6(d)] state that the patient and the
patient’s physicians must be notified of any
medically significant abnormalities.

Donor Deferral
If an autologous donor has a reactive
screening test for a communicable disease agent or a reactive screening test for
syphilis (21 CFR 610.41), the donor must
be deferred from making future allogeneic
donations. Within 8 weeks, the patient
and referring physician must be notified
of the reason for deferral, including the
results of supplemental testing and, where
appropriate, the types of donation that the
autologous donor should not make in the
future (21 CFR 630.6).

Special Labeling Considerations
Each autologous unit must be labeled
“Autologous Donor.” Another special label,
“BIOHAZARD,” is required for any unit
that is reactive in the current collection or
reactive in the last 30 days. Autologous
units that are untested must be labeled
“DONOR UNTESTED.” If the autologous
unit tested negative within the last 30
days, it must be labeled “DONOR TESTED
WITHIN THE LAST 30 DAYS” [21 CFR
610.40(d)(4)].

Shipping
Blood or components (including reactive
donations) intended for autologous use
may be shipped provided that the units
have been tested as required and are labeled appropriately [21 CFR 610.40(d)]. If
distributed on a common carrier not under the direct control of the collection facility, the transportation of the product
must meet provisions for shipping an infectious agent.22

Establishing a Preoperative Autologous
Blood Collection Program
Each blood center or hospital that decides
to conduct an autologous blood collection
program must establish its own policies,
processes, and procedures. Guidelines exist for establishing a new program, monitoring utilization, or improving an existing one.13,23,24

Physician Responsibility
A successful autologous program requires
cooperation and communication among
all the physicians involved. Responsibility
for the health and safety of the patient
during the collection process rests with the
medical director of the collecting facility;
during the transfusion, responsibility rests

Chapter 5: Autologous Blood Donation and Transfusion

with the patient’s physician and the medical director of the transfusion service. The
patient’s physician initiates the request
for autologous services, which must be
approved by the transfusion service physician. There should be a transfusion
medicine physician available to help assess patients whose medical history suggests a risk for complications if a donor
reaction occurs during blood collection.

Supplemental Iron
The patient should be advised about taking supplemental iron. Ideally, supplemental iron is prescribed by the requesting
physician before the first blood collection,
in time to allow maximum iron intake.
Iron-restricted erythropoiesis is one of the
limiting factors in collecting multiple
units of blood over a short interval. Oral
iron is commonly provided but may be
insufficient to maintain iron stores.25 The
dose and administration schedule should
be adjusted to minimize gastrointestinal
side effects.

Collection
The collection of autologous blood has
many elements in common with collection from regular volunteer donors, but
numerous special considerations exist.
Requests for autologous blood collection
are made in writing by the patient’s physician; a request form (which may be a simple prescription or a form designed for
the purpose) is kept by the collecting facility. The request should include the
patient’s name, a unique identification
number, the number of units and kind of
component requested, the date of scheduled surgery, the nature of the surgical
procedure, and the physician’s signature.
It is important to establish guidelines for
the appropriate number of units to be collected. A sufficient number of units should

121

be drawn, whenever possible, so that the
patient can minimize exposure to allogeneic blood. However, excessive collection
and/or collection close to the date of surgery increases the patient’s likelihood of requiring transfusion. A hospital’s surgical
blood order schedule can provide estimates
of transfusion levels for specific procedures.
Two-unit collections via an automated red
cell apheresis system may be an option. The
collection of units for liquid blood storage
should be scheduled as far in advance of
surgery as possible, in order to allow compensatory erythropoiesis to minimize anemia.
A schedule for blood collections should
be established with the patient. A weekly
schedule is often used. Table 5-3 details the
value of beginning autologous blood donation early in the known preoperative interval, in order to allow optimal compensatory
erythropoiesis (shown here as equivalent
RBC units).26 Ordinarily, the last collection
should occur no sooner than 72 hours before the scheduled surgery and preferably
longer, to allow time for adequate volume
repletion. Programs should notify the requesting physician of the total number of
units donated when the requested number
of units cannot be collected. Each program
should establish a policy regarding rescheduling of surgery beyond the expiration date of autologous units and whether
discarding or freezing the unit are options.

Donor Screening
Because of the special circumstances regarding autologous blood transfusion,
rigid criteria for donor selection are not
required. In situations where requirements for allogeneic donor selection or
collection are not applied, alternative requirements must be established by the
medical director and recorded in the procedures manual. The hemoglobin concentration of the donor should be no less

122

AABB Technical Manual

Table 5-3. Timing and Red Cell Regeneration During Preoperative Autologous
26
Donation
Time from Donation to
Surgery (days)

No. of
Patients

Mean RBC Units
Regenerated

5% CI
of Mean

6-13
14-20
21-27
28-34
35-41

39
127
128
48
30

0.52
0.54
0.75
1.16
1.93

0.25-0.79
0.40-0.68
0.61-0.90
0.96-1.36
1.64-2.2

than 11.0 g/dL and the hematocrit, if substituted, should be no less than 33%. Individual deviations from the alternate requirements must be approved by the blood
bank medical director, usually in consultation with the donor-patient’s physician.

Medical Interview
The medical interview should be structured to meet the special needs of autologous donors. For example, more attention
should be given to questions about medications, associated medical illnesses, and
cardiovascular risk factors.10 Questions
should elicit any possibility of intermittent
bacteremia. Because crossover is not routinely permitted, a substantially shortened
set of interview questions can be used for
autologous donations; for example, questions related to donor risks for transfusiontransmitted diseases are not necessary.

plasma ratio. Under-collected units (<300
mL) may be suitable for autologous use
with approval of the medical director. For
patients weighing <50 kg, there should be
a proportional reduction in the volume of
blood collected. Regardless of donor weight,
the volume collected should not exceed
10.5 mL/kg of the donor’s estimated body
weight, including the samples for testing.

Serologic Testing
The collecting facility must determine
ABO and Rh type on all units. Transfusing
facilities must retest ABO and Rh type on
units drawn at other facilities, unless the
collecting facility tests segments from the
unit according to AABB Standards.15(p37)
Testing for ABO and Rh type must be
performed on a properly labeled blood
sample from the patient. An antibody
screen should be performed to provide for
the possible need for allogeneic blood.

Volume Collected
For autologous donors weighing >50 kg,
the 450-mL collection bag is usually used
instead of the 500-mL bag, in case the donor cannot give a full unit. If a low-volume
(300-405 mL) unit is collected, the red cells
are suitable for storage and subsequent
autologous transfusion. The plasma from
low-volume units cannot be transfused
because of the abnormal anticoagulant/

Labeling
Units should be clearly labeled with the
patient’s name and an identifying number, the expiration date of the unit, and, if
available, the name of the facility where
the patient is to be transfused. The unit
should be clearly marked “For Autologous
Use Only” if intended for autologous use
only. If components have been prepared,

Chapter 5: Autologous Blood Donation and Transfusion

the container of each component must be
similarly labeled. A biohazard label must
be applied when indicated by FDA requirements (see Compliance Considerations).
Labeling requirements for autologous units
are detailed in the AABB Standards,15(p51)
which parallels the FDA regulations.

Storage
Collection should be scheduled to allow
for the longest possible shelf life for collected units. This increases flexibility for
the patient and the collecting facility and
allows time for the patient to rebuild red
cell mass during the interval between
blood collection and surgery. Liquid storage is feasible for up to 6 weeks. Some
programs store autologous units as Whole
Blood for 35 days rather than as RBCs;
Whole Blood is simpler to store, and the
risk of volume overload subsequent to
transfusion is low. The collection of autologous units more than 6 weeks before
scheduled surgery has been described,
but requires that the red cells be frozen.
Although this provides more time for the
donor to recover lost red cell mass, freezing and thawing add to the cost of the
program, reduce the volume of red cells
through processing losses, and complicate blood availability during the perioperative period.

Transfusion of Autologous Units
Autologous transfusion programs should
have a system to ensure that if autologous
blood is available, it be issued and used
before allogeneic components are given.
A special “autologous” label may be used
with numbering to ensure that the oldest
units are issued first. Anesthesiologists,
surgeons, and physicians should be educated about the importance of selecting
autologous components before allogeneic
units are given, and a policy should be in

123

place regarding the issue of autologous,
allogeneic, and/or directed units to the
operating room.

Records
AABB standards for the proper issue and
return of unused autologous units are the
same as for allogeneic units.15(pp45,71) Records must be maintained that identify
the unit and all components made from
it, from collection and processing through
their eventual disposition.

Adverse Reactions
The investigation of suspected adverse
transfusion events should be the same for
autologous and allogeneic units. Autologous transfusions have a lower risk of infectious and immune complications but
carry a similar risk of bacterial contamination, volume overload, and misadministration compared with volunteer allogeneic
units. For these reasons, autologous blood
should not be transfused without a clear
indication for transfusion.

Continuous Quality Improvement
Several quality improvement issues have
5
been identified for PAD practices. The
most important indicator for autologous
blood practice is how effectively it reduces
allogeneic transfusions to participating
patients. The “wastage” rate of autologous
units for surgical procedures can also be
monitored. However, even for procedures
such as joint replacement or radical prostatectomy, a well-designed program may
result in 50% of collected units being un5,27
used (Fig 5-1). Nevertheless, as much as
25% of autologous blood is collected for
procedures that seldom require transfusion, such as vaginal hysterectomies and
normal vaginal deliveries. Up to 90% of
units collected for these procedures are
wasted.14 The additional costs associated

124

AABB Technical Manual

Percent of Total RBCs that Were PAD RBCs
Collected
Transfused

1980

1986

1989

1992

1994

1997

1999

2001

0.3

1.5

4.8

8.5

7.8

4.9

4.7

4.0

<1.0

3.1

5.0

4.3

3.7

3.0

2.6

Figure 5-1. Autologous RBC collection and transfusion data from 1980 to 2001 in the United States illustrate the rise and fall of interest in PAD. The dashed line in the chart indicates the percent (right
axis) of collected PAD units transfused. (Modified with permission from Brecher and Goodnough. 27 )

with the collection of autologous units,
along with advances in the safety of allogeneic blood, have altered the cost-effectiveness of PAD in many situations.28 Such
cost-effectiveness analyses do not consider
an immunomodulatory effect of avoiding
allogeneic leukocytes, which remains
29,30
controversial.
Criteria can be established to monitor
the appropriateness of autologous transfusions. These criteria may be the same as, or
different from, those established for allogeneic units.24 As with allogeneic blood,
transfusion of preoperatively donated autologous blood carries the same risks associated with administrative error or bacterial
contamination. Autologous programs should
be monitored for unavailability of autologous blood when needed, the transfusion
of allogeneic blood before autologous
blood, and identification errors.

Evolving Issues in Preoperative
Autologous Services

Selection of Patients
Attempts to stratify patients into groups
at high and low risk for needing transfusion based on the baseline level of hemoglobin and on the type of procedure show
some promise. In a Canadian study using
a point score system, 80% of patients undergoing orthopedic procedures were
identified to be at low risk (<10%) for
transfusion, so autologous blood procurement for these patients would not be rec31
ommended. However, one problem with
algorithms that consider the estimated
blood loss and preoperative hematocrit is that
blood losses are difficult to measure32,33 or
predict because specific surgical procedures performed even by the same sur-

Chapter 5: Autologous Blood Donation and Transfusion

geon can be accompanied by a wide range
of blood losses.

The Role of Aggressive Phlebotomy and the
Use of Erythropoietin
The efficacy of PAD is dependent on the
degree to which the patient’s erythropoiesis increases the production of red
cells.25,34-36 The endogenous erythropoietin
response and compensatory erythropoiesis are suboptimal under “standard” conditions of one blood unit donated weekly.
Weekly PAD is accompanied by an 11%
(with no oral iron supplementation) to 19%
(with oral iron supplementation) expansion in red cell volume over a 3-week period, which is not sufficient to prevent increasing anemia in patients undergoing
PAD.34,35 If the erythropoietic response to
autologous blood phlebotomy is not able
to maintain the patient’s hematocrit level
during the donation interval, the donation of autologous blood may be harmful.37 This outcome was confirmed in a
study of patients undergoing hysterectomies,38 in which it was shown that PAD
resulted in perioperative anemia and an
increased likelihood of any blood transfusion. A published mathematical model37
illustrates the relationship between anticipated surgical blood losses, the level of
hematocrit that the physician may want
to maintain perioperatively, and the need
for autologous blood donation for individual patients (Fig 5-2). Models such as
this may be helpful in designing autologous procurement programs or monitoring their value through quality assurance.
In contrast to autologous blood donation
under “standard” conditions, studies of “aggressive” autologous blood phlebotomy
(twice weekly for 3 weeks, beginning 25 to
35 days before surgery) have demonstrated
that endogenous erythropoietin levels do
increase, along with enhanced erythropoie-

125

sis representing 19% to 26% red cell volume
expansion.39-41 Exogenous (pharmacologic)
erythropoietin therapy to further stimulate
erythropoiesis (up to 50% red cell volume
expansion39-41) during autologous phlebotomy has been approved in Canada and Ja42
pan but not in the United States.

Transfusion Trigger
Disagreement exists about the proper hemoglobin/hematocrit level (“transfusion
trigger”) at which autologous blood should
be given.23 Autologous blood transfusion
is not without risks to the recipient; these
include misidentification of patients or
units, bacterial contamination of stored
units, and volume overload. The case can
be made that autologous and allogeneic
blood transfusion triggers should be similar because the additional mortality risks
of allogeneic blood now approach the risks
of mortality from administrative errors associated with both autologous and allogeneic blood.43 Data from a well-designed
clinical trial indicate that even critical care
patients can tolerate substantial anemia
(to hemoglobin ranges of 7 to 9 g/dL) with
no apparent benefit from more aggressive
transfusion therapy.44

Cost-Effectiveness
Although autologous blood collections have
become popular, the costs associated with
their collection are usually higher than
those associated with the collection of
allogeneic blood. The continued need for
autologous blood programs has been questioned because of the reduced risk of
allogeneic blood transfusions and pressure
to reduce health-care costs.27 Table 5-4 lists
suggestions for improving the efficiency
of hospital-based autologous blood programs without sacrificing safety.45

126

AABB Technical Manual

Figure 5-2. Relationship of estimated blood loss (EBL) and minimum (nadir) hematocrit during hospitalization at various initial hematocrit levels (30%,35%,40%,45%) in a surgical patient with a whole
blood volume of 5000 mL. = 30%; = 35%;
= 40%; + = 45%.(Reprinted with permission from Cohen and Brecher.37 )

Preoperative Collection of Components
Some workers believe that preoperative or
intraoperative collection of platelet-rich
plasma during cardiopulmonary bypass
surgery may improve hemostasis and decrease allogeneic exposures, but others
have found no benefit.46 Preoperative collection of autologous platelets, especially
in cardiac surgery, is often impractical because patients may be taking antiplatelet
drugs; surgery is often scheduled on an
emergency basis; and relatively low numbers of platelets are harvested.
Recently, autologous platelet collection
using commercial point-of-care collection
systems has been advocated to produce a
platelet gel for topical use.47 Platelet gel is
created by adding calcium chloride and
thrombin to a platelet concentrate. The
platelet gel serves as a rich source of pla-

telet-derived growth factors, which have
been reported in small studies to enhance
tissue repair and wound healing.48,49 Larger
randomized clinical trials are needed to establish the clinical efficacy of this product.
Platelet gel is not FDA-approved yet.

Acute Normovolemic
Hemodilution
Acute normovolemic hemodilution (ANH)
is the removal of whole blood from a patient, with concurrent restoration of the
circulating blood volume with an acellular
fluid shortly before an anticipated significant surgical blood loss. To minimize the
manual labor associated with hemodilution, the blood should be collected in
standard blood bags containing anticoag-

Chapter 5: Autologous Blood Donation and Transfusion

127

Table 5-4. Suggestions for Making Autologous Blood Transfusion Protocols
Cost-Effective
1. Use standardized indications for preoperative autologous blood collection and transfusion.
2. Streamline the autologous blood donor interview.
3. Discontinue serologic tests for infectious disease markers following autologous blood
collections.
4. Simplify the donation process for uncomplicated patients.
5. Limit the use of frozen blood.
6. Store autologous whole blood, rather than components.
7. Use standardized indications and appropriate technology for intraoperative and postoperative
autologous blood recovery.
8. Share intraoperative blood recovery resources among institutions.
9. Cautiously adopt new research applications for autologous blood techniques.

ulant on a tilt-rocker with automatic cutoff via volume sensors. Then, the blood is
stored at room temperature and reinfused
during surgery after major blood loss has
ceased, or sooner if indicated. Simultaneous
infusions of crystalloid (3 mL crystalloid
for each 1 mL of blood withdrawn) and
colloid (dextrans, starches, gelatin, albumin, 1 mL for each 1 mL of blood withdrawn) have been recommended.50
Subsequent intraoperative fluid management is based on the usual surgical requirements. Blood units are reinfused in the
reverse order of collection. The first unit
collected, and therefore the last unit transfused, has the highest hematocrit and concentration of coagulation factors and platelets. Although this technique has been
primarily developed and used in Europe,
increasing interest in the United States has
led to data that show promise as an alternative method of autologous blood procurement.51 Augmented hemodilution (replacement of ANH collected or surgical blood
lost in part by oxygen therapeutics) has the
advantage of not being limited by anemia.
Its use is restricted to the investigational
setting until these solutions are approved
by the FDA.

Physiologic Considerations

Conserved Red Cell Mass
The chief benefit of ANH is the reduction
of red cell losses when whole blood is shed
perioperatively at lower hematocrit levels
after ANH has been completed.52 Mathematical modeling has suggested that severe ANH to preoperative hematocrit levels of less than 20%, accompanied by
substantial blood losses, would be required before the red cell volume “saved”
by ANH would become clinically impor53
tant. However, the equivalent of one blood
54
55
unit can be “saved” by ANH, which approaches the red cell volume expansion
generated by PAD under standard conditions (Table 5-3).

Improved Oxygenation
Withdrawal of whole blood and replacement with crystalloid or colloid solution
decrease arterial oxygen content, but compensatory hemodynamic mechanisms and
the existence of surplus oxygen-delivery
capacity make ANH safe. A sudden decrease in red cell concentration lowers
blood viscosity, thereby decreasing peripheral resistance and increasing cardiac

128

AABB Technical Manual

output. If cardiac output can effectively
compensate, oxygen delivery to the tissues at a hematocrit of 25% to 30% is as
good as, but no better than, oxygen delivery at a hematocrit of 30% to 35%.56

Preservation of Hemostasis
Because blood collected by ANH is stored
at room temperature and is usually returned to the patient within 8 hours of
collection, there is little deterioration of
platelets or coagulation factors. The
hemostatic value of blood collected by
ANH is of questionable benefit for orthopedic or urologic surgery because plasma
and platelets are rarely indicated in this
setting. Its value in protecting plasma and
platelets from the acquired coagulopathy
of extracorporeal circulation in cardiac
surgery is better established.46,57

Clinical Studies
Prospective randomized studies in radical
prostatectomy,58 knee replacement,59 and
60
hip replacement suggest that ANH can
be considered equivalent to PAD as a
method of autologous blood procurement. Additional, selected clinical trials of
ANH are summarized in Table 5-5.61-67 Reviews68,69 and commentaries70 on the merits of ANH have been published. However,
a recently published meta-analysis of 42
clinical trials of ANH found only a modest
benefit with unproven safety.71 When ANH
and reinfusion are accomplished in the
operating room by on-site personnel, the
procurement and administration costs
are minimized. Blood obtained during
ANH does not require the commitment of
the patient’s time, transportation, costs,
and loss of work time that can be associated with PAD. The wastage of PAD units
(approximately 50% of units collected)
also is eliminated with ANH. Additionally,
autologous blood units procured by ANH

require no inventory or testing costs. Because the blood never leaves the patient’s
room, ANH minimizes the possibility of
an administrative or a clerical error that
could lead to an ABO-incompatible blood
transfusion and death, as well as bacterial
contamination associated with prolonged
storage at 4 C.

Practical Considerations
The following considerations are important in establishing an ANH program:
1.
Decisions about ANH should be
based on the surgical procedure and
on the patient’s preoperative blood
volume and hematocrit, target hemodilution hematocrit, and other physiologic variables.
2.
The institution’s policy and procedures and the mechanisms for educating staff should be established
and periodically reviewed.
3.
There should be careful monitoring
of the patient’s circulating volume
and perfusion status during the procedure.
4.
Blood must be collected in an aseptic manner, ordinarily into standard
blood collection bags with citrate
anticoagulant.
5.
Units must be properly labeled and
stored. The label must contain, at a
minimum, the patient’s full name,
medical record number, date, and
time of collection, and the statement
“For Autologous Use Only.” Room
temperature storage should not exceed 8 hours. Units maintained at
room temperature should be reinfused in the reverse order of collection to provide the maximum
number of functional platelets and
coagulation factors in the last units
infused. If more time elapses between collection and transfusion,

Postoperative
Hematocrit (%)

Estimated Blood Loss (mL)

Allogeneic RBC-Containing
Units or Liters Transfused

Surgery

Control

ANH

p Value

Control

ANH

p Value

Control

ANH

p Value

Vascular
Liver resection
Hip arthroplasty
Spinal fusion
Colectomy
Prostate
Prostate

2250
1479
1800
5490
NR
1246
1717

2458
1284
2000
1700
NR
1106
1710

NS
NS
NS
<0.005
NR
NS
NS

NR
37.9
38.4
NR
37.0
35.5
29.5

33.0
33.8
32.4
28.7
35.0
31.8
27.9

NR
<0.01
NS
NR
NR
<0.001
<0.5

6.0
3.8
(2.1)
8.6
2.4
0.16
0.30

2.6
0.4
(0.9)
<1.0
0
0
0.13

<0.01
<0.001
NR
<0.001
NR
NS
NS

Modified from Brecher and Rosenfeld. NR = not reported; NS = not significant.

Reference
61
62
63
64
65
66
67

Chapter 5: Autologous Blood Donation and Transfusion

Table 5-5. Selected Clinical Trials of Acute Normovolemic Hemodilution (ANH)

129

130

AABB Technical Manual

the blood should be stored in a
monitored refrigerator. ANH blood
collected from an open system (eg,
from a central venous line or an arterial catheter) may be stored for up
to 8 hours at room temperature or 24
hours in a monitored refrigerator.
Policies, procedures, and guidelines
must be developed for ANH by an
experienced group of anesthesiologists in conjunction with the operating room’s nursing staff and the hospital’s blood bank and transfusion
services.68 These will include indications for ANH, monitoring requirements, endpoints for blood withdrawal and transfusion, types and
amounts of replacement fluids (ie,

colloid/crystalloid ratios), and full
adherence to AABB guidelines. Some
practical considerations are listed in
Table 5-6. Suggested criteria for patient selection are listed in Table 5-7.

Intraoperative Blood
Collection
The term intraoperative blood collection
or recovery describes the technique of
collecting and reinfusing blood lost by a
patient during surgery. The oxygen-transport properties of recovered red cells are
equivalent to stored allogeneic red cells.
The survival of recovered blood cells appears to be at least comparable to that of

Table 5-6. Practical Considerations for Acute Normovolemic Hemodilution (ANH)
■

■
■
■

■

There must be a physician responsible for the perioperative blood recovery program.
Responsibilities shall include compliance with AABB standards,16 the establishment of written
policies and procedures, and periodic review of those policies and procedures.
The blood bank or transfusion service should participate in the development of policies and
procedures related to the perioperative blood recovery program.
Blood collected perioperatively shall not be transfused to other patients.
Methods for perioperative blood collection and reinfusion shall be safe and aseptic and ensure
accurate identification of all blood and components collected. The equipment used shall be
pyrogen-free, shall include a filter capable of retaining particles potentially harmful to the
recipient, and must preclude air embolism. If the blood is warmed before infusion, warming
protocols apply.
A complete written protocol of all perioperative collection procedures should be maintained,
including selection of anticoagulants and solutions used for processing, labeling of collected
blood or components, and procedures for the prevention and treatment of adverse reactions.
All facilities regularly collecting blood by perioperative procedures should establish a program of
quality control and quality assurance. Written procedures should include criteria for acceptable
performance. Records of results should be reviewed and retained. Quality control measurements
should address the safety and quality of the blood or components collected for the recipient.
Units collected for ANH shall be stored under one of the following conditions before the start of
transfusion:
— At room temperature, for up to 8 hours
— At 1 to 6 C for up to 24 hours, provided that storage at 1 to 6 C is begun within 8 hours of
initiating the collection.

Chapter 5: Autologous Blood Donation and Transfusion

131

Table 5-7. Criteria for Selection of Patients for Acute Normovolemic Hemodilution
1. Likelihood of transfusion exceeds 10% (ie, blood requested for crossmatch according to
maximum surgical blood order schedule).
2. Preoperative hemoglobin level of at least 12 g/dL.
3. Absence of clinically significant coronary, pulmonary, renal, or liver disease.
4. Absence of severe hypertension.
5. Absence of infection and risk of bacteremia.

transfused allogeneic red cells. Intraoperative collection is contraindicated
when certain procoagulant materials (eg,
topical collagen) are applied to the surgical field because systemic activation of
coagulation may result. Microaggregate
filters (40 microns) are used most often
because recovered blood may contain tissue debris, small blood clots, or bone
fragments.
Cell washing devices can provide the
equivalent of 12 units of banked blood per
hour to a massively bleeding patient.72 Data
regarding adverse events of reinfusion of
recovered blood have been published.73 Air
embolus is a potentially serious problem.
Three fatalities from air embolus were reported over a 5-year interval to the New
York State Department of Public Health, for
an overall fatality risk of one in 30,000.43
Hemolysis of recovered blood can occur
during suctioning from the surface instead
of from deep pools of shed blood. For this
reason, manufacturers’ guidelines recommend a maximum vacuum setting of no
more than 150 torr. One study found that
vacuum settings as high as 300 torr could
be used when necessary, without causing
74
excessive hemolysis. The clinical importance of free hemoglobin in the concentrations usually seen has not been established,
although excessive free hemoglobin may
indicate inadequate washing. Positive bacterial cultures from recovered blood are
sometimes observed; however, clinical in75
fection is rare.

Most programs use machines that collect shed blood, wash it, and concentrate
the red cells. This process typically results
in 225-mL units of saline-suspended red
cells with a hematocrit of 50% to 60%. Patients exhibit a level of plasma-free hemoglobin that is usually higher than after
allogeneic transfusion. Sodium and chloride concentrations are the same as in the
saline wash solution, and potassium concentration is low. The infusate contains
minimal coagulation factors and platelets.

Clinical Studies
As with PAD and ANH, collection and recovery of intraoperative autologous blood
should undergo scrutiny with regard to
both safety and efficacy. Controlled studies in cardiothoracic surgery have reported conflicting results when transfusion requirements and clinical outcome
were followed.75,76 Although the collection
of a minimum of one blood unit equivalent is possible for less expensive (with
unwashed blood) methods, it is generally
agreed that at least two blood unit equivalents need to be recovered using a cell-recovery instrument (with washed blood) in
77
order to achieve cost-effectiveness. The
value of intraoperative blood collection
has been best defined for vascular surgeries with large blood losses, such as aortic
aneurysm repair and liver transplanta78
tion. However, a prospective randomized

132

AABB Technical Manual

trial of intraoperative recovery and reinfusion in patients undergoing aortic aneurysm repair showed no benefit in the
reduction of allogeneic blood exposure. A
mathematical model of cell recovery suggests that when it is combined with normovolemic anemia, the need for allogeneic
transfusion can be avoided—even with
large blood loss, eg, 5 to 10 liters.80 The
value of this technology may rest on cost
savings and blood inventory considerations in patients with substantial blood
losses.

Medical Controversies
Collection devices that neither concentrate
nor wash shed blood before reinfusion increase the risk of adverse effects. Shed
blood has undergone varying degrees of
coagulation/fibrinolysis and hemolysis,
and infusion of large volumes of washed
or unwashed blood has been described in
association with disseminated intravascular
81
coagulation. Factors that affect the degree
of coagulation and clot lysis include:
1.
Whether the patient had received
systemic anticoagulation.
2.
The amount and type of anticoagulant used.
3.
The extent of contact between blood
and serosal surfaces.
4.
The extent of contact between blood
and artificial surfaces.
5.
The degree of turbulence during collection.
In general, blood collected at low flow rates
or during slow bleeding from patients who
are not systemically anticoagulated will
have undergone coagulation and fibrinolysis and will not contribute to hemostasis
upon reinfusion.
The high suction pressure and surface
skimming during aspiration and the turbulence or mechanical compression that oc-

curs in roller pumps and plastic tubing
make some degree of hemolysis inevitable.
High concentrations of free hemoglobin
may be nephrotoxic to patients with impaired renal function. Many programs limit
the quantity of recovered blood that may be
reinfused without processing.

Practical Considerations
Collection and recovery services require
the coordinated efforts of surgeons, anesthesiologists, transfusion medicine specialists, and specific personnel trained in
the use of special equipment. Equipment
options may include:
1.
Devices that collect recovered blood
for direct reinfusion.
2.
Devices that collect recovered blood,
which is then concentrated and washed in a separate cell washer.
3.
High-speed machines that automatically concentrate and wash recovered red cells.
Some hospitals develop their own programs, whereas others contract with outside
services. Each hospital’s needs should dictate whether blood collection and recovery
are used and how they are achieved.

Processing Before Reinfusion
Several devices automatically process recovered blood before reinfusion. Vacuum
suction and simultaneous anticoagulation are used for collection. To minimize
hemolysis, the vacuum level should ordinarily not exceed 150 torr, although higher
levels of suction may occasionally be
needed during periods of rapid bleeding.
Either citrate (ACD) or heparin may be
used as an anticoagulant. Blood is held in
a reservoir until centrifuged and washed
with a volume of saline that varies between 500 and 1500 mL. If not infused immediately, the unit must be labeled with
the patient’s name and identification

Chapter 5: Autologous Blood Donation and Transfusion

number, the date and time collection was
initiated, and the statement “For Autologous Use Only.”
An alternative approach is to collect
blood in a canister system designed for direct reinfusion and then concentrate and
wash the recovered red cells in a blood
bank cell washer. Intraoperatively collected
and recovered blood must be handled in
the transfusion service laboratory like any
other autologous unit. The unit should be
reinfused through a filter.

Direct Reinfusion
Systems are available that collect recovered
blood and return it directly. These systems generally consist of a suction catheter attached to a disposable collection bag
or rigid plastic canister, to which anticoagulant (citrate or heparin) may have
been added. Blood is suctioned into the
holding canister before being reinfused
through a microaggregate filter. Low-vacuum suction and minimal hemolysis are
preferred in nonwashed systems.

Requirements and Recommendations
The AABB requires a process that includes
patient and storage bag identification and
time collected with expiration date.16(p10)
Units collected intraoperatively should be
labeled with the patient’s first and last
name, hospital identification number, the
date and time of collection and expiration, and the statement “For Autologous
Use Only.”16(p10)
Conditions for storage and expiration of
autologous components collected in the
operating room are listed in Table 5-8.16(p14) If
the blood leaves the patient for washing or
storage in a remote location, there must be
appropriate procedures to ensure proper
labeling of the blood according to AABB
standards.16(pp9,10)

133

Hospitals with collection and recovery
programs should establish written policies
and procedures that are regularly reviewed
by a physician who has been assigned responsibility for the program. Transfusion
medicine specialists should play an active
role in design, implementation, and operation of the program. Written policies must
be in place for the proper collection, labeling, and storage of intraoperative autologous blood. Equipment and techniques for
collection and infusion must ensure that
the blood is aseptic. Quality management
should include evaluation of the appropriate use of blood collection and recovery
services and adequate training of personnel. Written protocols, procedure logs, machine maintenance, procedures for handling adverse events, and documentation
are recommended.24

Postoperative Blood
Collection
Postoperative blood collection denotes
the recovery of blood from surgical drains
followed by reinfusion, with or without
processing. In some programs, postoperative shed blood is collected into sterile
canisters and reinfused, without processing, through a microaggregate filter. Recovered blood is dilute, partially hemolyzed
and defibrinated, and may contain high
concentrations of cytokines. For these
reasons, most programs set an upper limit
on the volume (eg, 1400 mL) of unprocessed
blood that can be reinfused. If transfusion
of blood has not begun within 6 hours of
initiating the collection, the blood must be
discarded. Hospitals should establish
written policies, procedures, labeling requirements, quality assurance, and review
consistent with AABB standards.16(p2)

134

AABB Technical Manual

Table 5-8. Handling, Storage, and Expiration of Intraoperative Blood Collections
Collection
Type

Storage
Temperature

Acute normovolemic
hemodilution

Room temperature
1-6 C

8 hours from start
of collection
24 hours from start
of collection

None

Intraoperative blood
recovered with
processing

Room temperature
1-6 C

4 hours from end of
collection
24 hours from start
of collection

None

Intraoperative blood
recovered without
processing

Room temperature or 1-6 C

4 hours from end of
collection

None

Shed blood under postoperative or posttraumatic conditions
with or without processing

N/A

6 hours from start
of collection

None

Non-red-cell component
preparation

Room temperature

Shall be used before
leaving the operating room

None

Clinical Studies
The evolution of cardiac surgery has been
accompanied by a broad experience in
postoperative conservation of blood. Postoperative autologous blood transfusion is
practiced widely, but not uniformly. Prospective and controlled trials have disagreed over the efficacy of postoperative
blood recovery in cardiac surgery patients; at least three such studies have
demonstrated lack of efficacy,82-84 but at
85,86
least two studies have shown benefit.
The disparity of the results in these studies may be explained, in part, by differences

Expiration

Special
Conditions

Storage at 1-6 C shall
begin within 8 hours
of start of collection

Storage at 1-6 C shall
begin within 4 hours
of start of collection

in transfusion practices. Modification of
physician transfusion practices may have
been an uncredited intervention in these
blood conservation studies.
In the postoperative orthopedic surgical
setting, several reports have similarly described the successful recovery and reinfusion of washed87 and unwashed88,89 wound
drainage from patients undergoing arthroplasty. Red cells recovered in this setting
appear to have normal survival in the circulation.90 The volume of reinfused drainage
blood has been reported to be as much as
3000 mL and averages more than 1100 mL
in patients undergoing cementless knee re-

Chapter 5: Autologous Blood Donation and Transfusion

placement. Because the red cell content of
the fluid collected is low (hematocrit levels
of 20%), the volume of red cells reinfused is
often small.91 A prospective randomized
study of postoperative recovery and reinfusion in patients undergoing total knee or
hip replacement found no differences in
perioperative hemoglobin levels or allogeneic blood transfusions between patients
who did or did not have joint drainage de92
vices. The safety of reinfused unwashed
orthopedic wound drainage has been controversial. Theoretical concerns have been
expressed regarding infusion of potentially
harmful materials in recovered blood, including free hemoglobin, red cell stroma,
marrow fat, toxic irritants, tissue or methacrylate debris, fibrin degradation products,
activated coagulation factors, and complement. Although two small studies have
93,94
reported complications,
several larger
studies have reported no serious adverse
effects when drainage was passed through
a standard 40-micron blood filter.88,89,95

Patient Selection

References
1.
2.

10.

The potential for decreasing exposure to
allogeneic blood among orthopedic patients
undergoing postoperative blood collection
(whether washed or unwashed) is greatest
for cementless bilateral total knee replacement, revision hip or knee replacement,
and long segment spinal fusion. As in the
case of intraoperative recovery, blood loss
must be sufficient to warrant the additional cost of processing technology.96,97 As
in the selection of patients who can benefit from PAD and ANH, prospective identification of patients who can benefit from
intra- and postoperative autologous blood
recovery is possible if anticipated surgical
blood losses and the perioperative “transfusion trigger” are taken into account (Fig
5-2).

135

11.

12.

13.

14.

15.

16.

Pub. Law No. 101-336, 104 Stat. 327 (1990).
Codified at 42 USC §12101-213.
The ADA, HIV, and autologous blood donation. Association Bulletin 98-5. Bethesda, MD:
AABB, 1998.
Goodnough LT. Preoperative autologous donation. In: Spence RK, ed. Problems in general surgery. Philadelphia: Lippincott Williams & Wilkins, 2000;17:25-31.
Bierbaum BE, Callaghan JJ, Galante JO,
Rubash HE. An analysis of blood management in patients having total hip or knee
arthroplasty. J Bone J Surg 1999;81A:2-10.
Renner SW, Howanitz PJ, Bachner P. Preoperative autologous blood donation in 612 hospitals. Arch Pathol Lab Med 1992;116:613-9.
Silvergleid AJ. Safety and effectiveness of predeposit autologous transfusions in preteen
and adolescent children. JAMA 1987;257:
3403-4.
Chaplin H, Mischeaux JR, Inkster MD,
Sherman LA. Frozen storage of 11 units of
sickle cell red cells for autologous transfusion
of a single patient. Transfusion 1986;26:341-5.
Meryman HT, Hornblower M. Freezing and
deglycerolizing sickle trait red blood cells.
Transfusion 1976;16:627-32.
Mann M, Sacks HJ, Goldfinger D. Safety of
autologous blood donation prior to elective
surgery for a variety of potentially high risk
patients. Transfusion 1983;23:229-32.
Popovsky MA, Whitaker B, Arnold NL. Severe
outcomes of allogeneic and autologous blood
donation: Frequency and characterization.
Transfusion 1995;35:734-7.
Goodnough LT, Brecher ME, Kanter MH,
AuBuchon JP. Transfusion medicine. First of
two parts. Blood transfusion. N Engl J Med
1999;340:438-47.
Dodd RY, Notari EP, Stramer SL. Current prevalence and incidence of infectious disease
markers and estimated window-period risk in
the American Red Cross blood donor populations. Transfusion 2002;42:975-9.
Thomas MJG, Gillon J, Desmond MJ. Preoperative autologous blood donation. Transfusion 1996;36:633-9.
Sayers MH. Controversies in transfusion medicine. Autologous blood donation in pregnancy: Con. Transfusion 1990;30:172-4.
Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.
Santrach P, ed. Standards for perioperative
autologous blood collection and administration. 1st ed. Bethesda, MD: AABB, 2001.

136

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

AABB Technical Manual

Food and Drug Administration. Memorandum:
Guidance for autologous blood and blood
components. (March 15, 1989) Rockville, MD:
CBER Office of Communication, Training,
and Manufacturers Assistance, 1989.
Food and Drug Administration. Memorandum:
Autologous blood collection and processing
procedures. (February 12, 1990) Rockville,
MD: CBER Office of Communication, Training, and Manufacturers Assistance, 1990.
Food and Drug Administration. General requirements for blood, blood components,
and blood derivatives; donor notification. 21
CFR 630.6. Fed Regist 2001;66(112):31165-77.
Food and Drug Administration. Requirements for testing human blood donors for evidence of infection due to communicable disease agents. 21 CFR 610.40. Fed Regist 2001;
66(112):31146-65.
Food and Drug Administration. Guidance for
industry: Use of nucleic acid tests on pooled
and individual samples from donors of whole
blood and blood components (including
Source Plasma and Source Leukocytes) to adequately and appropriately reduce the risk of
transmission of HIV-1 and HCV. (October 21,
2004) Rockville, MD: CBER Office of Communication, Training, and Manufacturers Assistance, 2004.
Code of federal regulations. Title 42 CFR Part
72.3. Washington, DC: US Government Printing Office, 2004 (revised annually).
National Heart, Lung, and Blood Institute
Autologous Transfusion Symposium Working
Group. Autologous transfusion: Current trends
and research issues. Transfusion 1995;35:52531.
Becker J, Blackall D, Evans C, et al for the Scientific Section Coordinating Committee.
Guidelines for blood utilization review.
Bethesda, MD: AABB, 2001:20-4.
Goodnough LT, Skikne B, Brugnara C. Erythropoietin, iron, and erythropoiesis. Blood
2000;96:823-33.
Toy P, Ahn D, Bacchetti P. When should the
first of two autologous donations be made?
(abstract) Transfusion 1994;34(Suppl):14S.
Brecher ME, Goodnough LT. The rise and fall
of preoperative autologous blood donation
(editorial). Transfusion 2001;41:1459-62.
Etchason J, Petz L, Keeler E, et al. The cost-effectiveness of preoperative autologous blood
donations. N Engl J Med 1995;332:719-24.
Vamvakas EC. Meta-analysis of randomized
controlled trials comparing the risk of postoperative infection between recipients of
allogeneic and autologous blood. Vox Sang
2002;83:339-46.

30.
31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

Vanderlinde E, Heal JM, Blumberg N. Autologous transfusion. Br Med J 2002;324:772-5.
Larocque BJ, Gilbert K, Brien WF. Prospective
validation of a point score system for predicting blood transfusion following hip or knee
replacement. Transfusion 1998;38:932-7.
Brecher MA, Monk TG, Goodnough LT. A
standardized method for calculating blood
loss. Transfusion 1997;37:1070-4.
Rosencher N. Orthopedic surgery transfusion
hemoglobin over view (OSTHEO) study.
Transfusion 2003;43:459-69.
Kasper SM, Gerlich W, Buzello W. Preoperative red cell production in patients undergoing weekly autologous blood donation.
Transfusion 1997;37:1058-62.
Kasper SM, Lazansky H, Stark C, et al. Efficacy of oral iron supplementation is not enhanced by additional intravenous iron during
autologous blood donation. Transfusion
1998;38:764-70.
Weisbach V, Skoda P, Rippel R, et al. Oral or
intravenous iron as an adjunct to autologous
blood donation in elective surgery: A randomized, controlled study. Transfusion 1999;
39:465-72.
Cohen JA, Brecher ME. Preoperative autologous blood donation: Benefit or detriment? A
mathematical analysis. Transfusion 1995;35:640-4.
Kanter MH, Van Maanen D, Anders KH, et al.
Preoperative autologous blood donation before elective hysterectomy. JAMA 1996;276:
798-801.
Goodnough LT, Rudnick S, Price TH, et al. Increased preoperative collection of autologous
blood with recombinant human erythropoietin therapy. N Engl J Med 1989;321:1163-8.
Goodnough LT, Price TH, Rudnick S, Soegiarso
RW. Preoperative red blood cell production in
patients undergoing aggressive autologous
blood phlebotomy with and without erythropoietin therapy. Transfusion 1992;32:441-5.
Goodnough LT, Price TH, Friedman KD, et al.
A phase III trial of recombinant human erythropoietin therapy in non-anemic orthopedic
patients subjected to aggressive autologous
blood phlebotomy: Dose, response, toxicity,
efficacy. Transfusion 1994;34:66-71.
Goodnough LT, Monk TG, Andriole GL. Erythropoietin therapy. N Engl J Med 1997;336:
933-8.
Linden JV, Wagner K, Voytovich AE, Sheehan
J. Transfusion errors in New York State: An
analysis of 10 years’ experience. Transfusion
2000;40:1207-13.
Hébert PC, Wells G, Blajchman MA, et al. A
multicenter, randomized, controlled clinical
trial of transfusion requirements in critical
care. N Engl J Med 1999;340:409-17.

Chapter 5: Autologous Blood Donation and Transfusion

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

Kruskall MS, Yomtovian R, Dzik WH, et al. On
improving the cost effectiveness of autologous blood transfusion practices. Transfusion
1994;34:259-64.
Triulzi DJ, Gilmor GD, Ness PM, et al. Efficacy
of autologous fresh whole blood or plateletrich plasma in adult cardiac surgery. Transfusion 1995;35:627-34.
Kevy SV, Jacobson MS. Comparison of methods for point of care preparation of autologous platelet gel. J Extra Corpor Technol
2004;36:28-35.
Crovetti G, Martinelli G, Issi M, et al. Platelet
gel for healing cutaneous chronic wounds.
Transfus Apheresis Sci 2004;30:145-51.
Mazzucco L, Medici D, Serra M, et al. The use
of autologous platelet gel to treat difficult to
heal wounds: A pilot study. Transfusion 2004;
44:1013-8.
Goodnough LT, Brecher ME, Monk TG. Acute
normovolemic hemodilution in surgery. Hematology 1992;2:413-20.
Goodnough LT, Brecher ME, Kanter MH,
AuBuchon JP. Transfusion medicine. Second
of two parts. Blood conservation. N Engl J
Med 1999;340:525-33.
Messmer K, Kreimeier M, Intagliett A. Present
state of intentional hemodilution. Eur Surg
Res 1986;18:254-63.
Brecher ME, Rosenfeld M. Mathematical and
computer modeling of acute normovolemic
hemodilution. Transfusion 1994;34:176-9.
Goodnough LT, Bravo J, Hsueh Y, et al. Red
blood cell volume in autologous and homologous units: Implications for risk/benefit
assessment for autologous blood “crossover”
and directed blood transfusion. Transfusion
1989;29:821-2.
Goodnough LT, Grishaber JE, Monk TG,
Catalona WJ. Acute normovolemic hemodilution in patients undergoing radical suprapubic prostatectomy: A case study analysis.
Anesth Analg 1994;78:932-7.
Weiskopf RB. Mathematical analysis of isovolemic hemodilution indicates that it can decrease the need for allogeneic blood transfusion. Transfusion 1995;35:37-41.
Petry AF, Jost T, Sievers H. Reduction of homologous blood requirements by blood pooling at the onset of cardiopulmonary bypass. J
Thorac Cardiovasc Surg 1994;1097:1210-14.
Monk TG, Goodnough LT, Brecher ME, et al. A
prospective, randomized trial of three blood
conservation strategies for radical prostatectomy. Anesthesiology 1999;91:24-33.
Goodnough LT, Merkel K, Monk TG, Despotis
GJ. A randomized trial of acute normovolemic hemodilution compared to preopera-

60.

61.

62.

63.

64.

65.
66.

67.

68.

69.

70.

71.

72.

137

tive autologous blood donation in total knee
arthroplasty. Vox Sang 1999;77:11-16.
Goodnough LT, Despotis GJ, Merkel K, Monk
TG. A randomized trial of acute normovolemic hemodilution compared to preoperative autologous blood donation in total hip
arthroplasty. Transfusion 2000;40:1054-7.
Davies MJ, Cronin KD, Domanique C. Haemodilution for major vascular surgery using
3.5% polygeline (Haemaccel). Anaesth Intensive Care 1982;10:265-70.
Sejourne P, Poirier A, Meakins JL, et al. Effects
of haemodilution on transfusion requirements
in liver resection. Lancet 1989;ii:1380-2.
Rosenberg B, Wulff K. Regional lung function
following hip arthroplasty and preoperative
normovolemic hemodilution. Acta Anaesthesiol Scand 1979;23:242-7.
Kafer ER, Isley MR, Hansen T, et al. Automated acute normovolemic hemodilution reduces blood transfusion requirements for
spinal fusion (abstract). Anesth Analg 1986;
65(Suppl):S76.
Rose D, Coustoftides T. Intraoperative normovolemic hemodilution. J Surg Res 1981;31:375-81.
Ness PM, Bourke DL, Walsh PC. A randomized trial of perioperative hemodilution versus transfusion of preoperatively deposited
autologous blood in elective surgery. Transfusion 1991;31:226-30.
Monk TG, Goodnough LT, Birkmeyer JD, et al.
Acute normovolemic hemodilution is a costeffective alternative to preoperative autologous blood donation by patients undergoing
radical retropubic prostatectomy. Transfusion 1995;35:559-65.
Shander A. Acute normovolemic hemodilution. In: Spence RK, ed. Problems in general
surgery. Philadelphia: Lippincott Williams &
Wilkins, 1999;17:32-40.
Monk TG, Goodnough LT, Brecher ME, et al.
Acute normovolemic hemodilution can replace preoperative autologous blood donation as a standard of care for autologous
blood procurement in radical prostatectomy.
Anesth Analg 1997;85:953-8.
Goodnough LT, Monk TG, Brecher ME. Acute
normovolemic hemodilution should replace
preoperative autologous blood donation before elective surgery. Transfusion 1998;38:
473-7.
Segal JB, Blasco-Colmenares E, Norris EJ,
Guallar E. Preoperative acute normovolemic
hemodilution: A meta-analysis. Transfusion
2004;44:632-44.
Williamson KR, Taswell HF. Intraoperative
blood salvage: A review. Transfusion 1991;31:
662-75.

138

73.

74.

75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

AABB Technical Manual

Domen RE. Adverse reactions associated with
autologous blood transfusion: Evaluation
and incidence at a large academic hospital.
Transfusion 1998;38:296-300.
Gregoretti S. Suction-induced hemolysis at
various vacuum pressures: Implications for
intraoperative blood salvage. Transfusion
1996;36:57-60.
Bell K, Stott K, Sinclair CJ, et al. A controlled
trial of intra-operative autologous transfusion in cardiothoracic surgery measuring effect on transfusion requirements and clinical
outcome. Transfus Med 1992;2:295-300.
Tempe DK, Banjerjee A, Virmani S, et al.
Comparison of the effects of a cell saver and
low dose aprotinin on blood loss and homologous blood use in patients undergoing valve
surgery. J Cardiothorac Vasc Anesth 2001;15:
326-30.
Bovill DF, Moulton CW, Jackson WS, et al. The
efficacy of intraoperative autologous transfusion in major orthopaedic surgery: A regression analysis. Orthopedics 1986;9:1403-7.
Goodnough LT, Monk TG, Sicard G, et al.
Intraoperative salvage in patients undergoing
elective abdominal aortic aneurysm repair.
An analysis of costs and benefits. J Vasc Surg
1996;24:213-8.
Claggett GP, Valentine RJ, Jackson MR, et al. A
randomized trial of intraoperative transfusion during aortic surgery. J Vasc Surg 1999;
29:22-31.
Waters JH, Karafa MT. A mathematical model
of cell salvage efficiency. Anesth Analg 2002;
95:1312-7.
de Haan J, Boonstra P, Monnink S, et al. Retransfusion of suctioned blood during cardiopulmonary bypass impairs hemostasis. Ann
Thorac Surg 1995;59:901-7.
Ward HB, Smith RA, Candis KP, et al. A prospective, randomized trial of autotransfusion
after routine cardiac surgery. Ann Thorac
Surg 1993;56:137-41.
Thurer RL, Lytle BW, Cosgrove DM, Loop FD.
Autotransfusion following cardiac operations: A randomized, prospective study. Ann
Thorac Surg 1979;27:500-6.
Roberts SP, Early GL, Brown B, et al. Autotransfusion of unwashed mediastinal shed
blood fails to decrease banked blood requirements in patients undergoing aorta coronary
bypass surgery. Am J Surg 1991;162:477-80.
Schaff HV, Hauer JM, Bell WR, et al. Autotransfusion of shed mediastinal blood after

86.

87.

88.

89.

90.

91.

92.

93.

94.

95.

96.

97.

cardiac surgery. A prospective study. J Thorac
Cardiovasc Surg 1978;75:632-41.
Eng J, Kay PH, Murday AJ, et al. Post-operative autologous transfusion in cardiac surgery. A prospective, randomized study. Eur J
Cardiothorac Surg 1990;4:595-600.
Semkiw LB, Schurman OJ, Goodman SB,
Woolson ST. Postoperative blood salvage using the cell saver after total joint arthroplasty.
J Bone Joint Surg (Am) 1989;71A:823-7.
Faris PM, Ritter MA, Keating EM, Valeri CR.
Unwashed filtered shed blood collected after
knee and hip arthroplasties. J Bone Joint Surg
(Am) 1991;73A:1169-77.
Martin JW, Whiteside LA, Milliano MT, Reedy
ME. Postoperative blood retrieval and transfusion in cementless total knee arthroplasty. J
Arthroplasty 1992;7:205-10.
Umlas J, Jacobson MS, Kevy SV. Survival and
half-life of red cells salvaged after hip and knee
replacement surgery. Transfusion 1993;33:591-3.
Umlas J, Foster RR, Dalal SA, et al. Red cell
loss following orthopedic surgery: The case
against postoperative blood salvage. Transfusion 1994;34:402-6.
Ritter MA, Keating EM, Faris PM. Closed
wound drainage in total hip or knee replacement: A prospective, randomized study. J
Bone J Surg 1994;76:35-8.
Clements DH, Sculco TP, Burke SW, et al.
Salvage and reinfusion of postoperative
sanguineous wound drainage. J Bone Joint
Surg (Am)1992;74A:646-51.
Woda R, Tetzlaff JE. Upper airway oedema
following autologous blood transfusion from
a wound drainage system. Can J Anesth 1992;
39:290-2.
Blevins FT, Shaw B, Valeri RC, et al. Reinfusion of shed blood after orthopedic procedures in children and adolescents. J Bone
Joint Surg (Am) 1993;75A:363-71.
Goodnough LT, Verbrugge D, Marcus RE. The
relationship between hematocrit, blood lost,
and blood transfused in total knee replacement: Implications for postoperative blood
salvage and reinfusion. Am J Knee Surg 1995;
8:83-7.
Jackson BR, Umlas J, AuBuchon JP. The costeffectiveness of postoperative recovery of
RBCs in preventing transfusion-associated
virus transmission after joint arthroplasty.
Transfusion 2000;40:1063-6.

Chapter 6: Apheresis

Chapter 6

Apheresis

PHERESIS, FROM THE Greek
pheresis meaning “to take away,”
involves the selective removal of
blood constituents from blood donors or
patients.
The AABB provides standards1 for voluntary compliance for apheresis activities.
The Food and Drug Administration (FDA)
has established specific requirements that
are set forth in the Code of Federal Regula2
tions for apheresis activities. The American
Society for Apheresis (ASFA)3 has published
additional guidelines and recommendations.
In addition, hemapheresis practitioner (HP)
and apheresis technician (AT) certifications
are available through the American Society
of Clinical Pathology Board of Registry. All
personnel involved with apheresis activities
should be familiar with these sources and
should have documentation that they are
qualified by training and experience to perform apheresis.

Separation Techniques
Automated blood processing devices are
used for both component preparation and
therapeutic applications of apheresis.
Manual apheresis, in which whole blood
is collected in multiple bags and centrifuged offline, requires great care to ensure
that the bags are labeled correctly and are
returned to the correct donor. With the
currently available automated technology,
this process is seldom used.

Separation by Centrifugation
In most apheresis instruments, centrifugal force separates blood into components
on the basis of differences in density. A
measured amount of anticoagulant solution is added to the whole blood as it is
drawn from the donor or patient. The blood
is pumped into a rotating bowl, chamber,
or tubular rotor in which layering of com139

140

AABB Technical Manual

ponents occurs on the basis of their densities. The desired fraction is diverted and
the remaining elements are returned to the
donor (or patient) by intermittent or continuous flow.
All systems require prepackaged disposable sets of sterile bags, tubing, and centrifugal devices unique to the instrument. Each
system has a mechanism to allow the separation device to rotate without twisting the
attached tubing. In the intermittent flow
method, the centrifuge container is alternately
filled and emptied. Most instruments in use
today employ a method that involves the
continuous flow of blood through a separation chamber. Depending on the procedure
and device used, the apheresis procedure
time varies from 30 minutes to several hours.
Each manufacturer supplies detailed information and operational protocols. Each
facility must have, in a manual readily
available to nursing and technical personnel, detailed descriptions of each type of
procedure performed, specific for each type
of blood processor.4

Separation by Adsorption
Selective removal of a pathologic material
has theoretical advantages over the removal of all plasma constituents. Centrifugal devices can be adapted to protocols
that selectively remove specific soluble
plasma constituents by exploiting the
principles of affinity chromatography.5 Selective removal of low-density lipoproteins
(LDLs) in patients with familial hypercholesterolemia has been accomplished using
both immunoaffinity (anti-LDL) and che6
mical affinity (eg, dextran sulfate) columns.
Adsorbents such as staphylococcal protein
A (SPA), monoclonal antibodies, blood
group substances, DNA-collodion, and
polymers with aggregated IgG attached
can extract antibodies, protein antigens,
and immune complexes. Returning the

depleted plasma along with the cellular
components reduces or eliminates the
need for replacement fluids. Immunoadsorption can be performed online, or
the plasma can be separated from the cellular components, passed through an offline column, and then reinfused.

Component Collection
Whenever components intended for transfusion are collected by apheresis, the donor must give informed consent. Although
apheresis collection and preparation processes are different from those used for
whole-blood-derived components, storage conditions, transportation requirements, and some quality control steps are
the same. See Chapter 8 for more detailed
information. The facility must maintain
written protocols for all procedures used
and must keep records for each procedure
as required by AABB Standards for Blood
Banks and Transfusion Services.1(p2)

Platelets Pheresis
Plateletpheresis is used to obtain platelets
from random volunteer donors, from patients’ family members, or from donors with
matched HLA or platelet antigen phenotypes. Because large numbers of platelets
can be obtained from a single individual,
collection by apheresis reduces the number of donor exposures for patients. AABB
Standards requires the component to
11
contain at least 3 × 10 platelets in 90% of
1(p31)
sampled units.
When a high yield is
obtained, the original apheresis unit may
be divided into multiple units, each of which
must meet minimum standards independently. Some instruments are programmed to calculate the yield from the
donor’s hematocrit, platelet count, height,
and weight. For alloimmunized patients
who are refractory to random allogeneic

Chapter 6: Apheresis

platelets (see Chapters 16 and 21), platelets
from an apheresis donor selected on the
basis of a compatible platelet crossmatch
or matched for HLA antigens may be the
only way to achieve a satisfactory posttransfusion platelet increment. Within the
United States, the use of apheresis platelets has been steadily increasing over the
last 25 years. Currently, it is estimated that
77% of therapeutic platelet doses are
7
transfused as apheresis platelets.

Donor Selection and Monitoring
Plateletpheresis donors may donate more
frequently than whole blood donors but
must meet all other donor criteria. The interval between donations should be at
least 2 days, and donors should not undergo plateletpheresis more than twice
in a week or more than 24 times in a
year.1(pp19,20) If the donor donates a unit of
Whole Blood or if it becomes impossible
to return the donor’s red cells during plateletpheresis, at least 8 weeks should elapse
before a subsequent plateletpheresis procedure, unless the extracorporeal red cell
volume is less than 100 mL.1(p20) Platelets
may be collected from donors who do not
meet these requirements if the component is expected to be of particular value
to a specific intended recipient, and if a
physician certifies in writing that the donor’s health will not be compromised (eg,
an HLA-matched donor). Donors who
have taken aspirin-containing medications within 36 hours of donation are usually deferred because the platelets obtained by apheresis are often the single
source of platelets given to a patient.
Vasovagal and hypovolemic reactions are
rare in apheresis donors, but paresthesias
and other reactions to the citrate anticoagulant are common (see Complications,
later in this chapter). Serious reactions

141

occur less often among apheresis donors
than among whole blood donors.
Plateletpheresis donors should meet
usual donor requirements, including hemoglobin or hematocrit level. A platelet
count is not required before the first
apheresis collection or if 4 weeks or more
have elapsed since the last procedure. If the
donation interval is less than 4 weeks, the
donor’s platelet count should be above
150,000/µL before subsequent plateletpheresis occurs. AABB Standards permits
documentation of the platelet count from a
sample collected immediately before the
procedure or from a sample obtained either
before or after the previous procedure.1(p21)
Exceptions to these laboratory criteria should
be approved in writing by the apheresis physician. The FDA specifies that the total volume of plasma collected should be no more
than 500 mL (or 600 mL for donors weighing
more than 175 pounds).8 The platelet count
of each unit should be kept on record but
need not be written on the product label.8
Some plateletpheresis programs collect
plasma for use as Fresh Frozen Plasma
(FFP) in a separate bag during platelet collection. Apheresis can also be used to collect plasma for FFP without platelets, ie,
plasmapheresis. The FDA has provided
guidance with regard to the volume of
plasma that is allowed to be collected using
automated devices.9 A total serum or plasma
protein determination and a quantitative
determination of IgG and IgM (or a serum
protein electrophoresis) must be determined at 4-month intervals for donors undergoing large-volume plasma collection, if
the total annual volume of plasma collected
exceeds 12 liters (14.4 L for donors weighing more than 175 pounds) or if the donor
is a frequent (more often than every 4
weeks) plasma donor.10 AABB Standards requires that the donor’s intravascular volume
deficit must be less than 10.5 mL per kilogram of body weight at all times.1(p24)

142

AABB Technical Manual

Laboratory Testing

Plasma

Tests for ABO group and Rh type, unexpected alloantibodies, and markers for
transfusion-transmitted diseases must be
performed by the collecting facility in the
same manner as for other blood components. Each unit must be tested unless the
donor is undergoing repeated procedures
to support a single patient, in which case
testing for disease markers need be re1(p34)
peated only at 30-day intervals.
If red cells are visible in a product, the
hematocrit should be determined. FDA
guidelines require that if the component
contains more than 2 mL of red cells, a
sample of donor blood for compatibility
testing be attached to the container.8 In
some instances, it may be desirable for the
donor plasma to be ABO-compatible with
the recipient’s red cells—for example, if the
recipient is a child or an ABO-mismatched
allogeneic progenitor cell transplant recipient. In order to be considered leukocytereduced, apheresis platelets must contain
less than 5 × 106 leukocytes and must meet
the specifications of the apheresis device
manufacturer. Chapter 8 describes additional quality control measures that apply
to all platelet components.

Apheresis can be used to collect plasma as
FFP or for Source Plasma for subsequent
manufacturing. FDA requirements for
plasma collection are different from those
for whole blood or plateletpheresis; personnel who perform serial plasmapheresis
must be familiar with both AABB standards and FDA requirements. If plasma is
intended for transfusion, testing requirements are the same as those for red cell
components. Plasma collected for manufacture of plasma derivatives is subject to
different requirements for infectious disease testing.
A distinction is made between “occasional plasmapheresis,” in which the donor
undergoes plasmapheresis no more often
than once in 4 weeks, and “serial plasmapheresis,” in which donation is more frequent than every 4 weeks. For donors in an
occasional plasmapheresis program, donor
selection and monitoring are the same as
for whole blood donation. For serial plasmapheresis using either automated instruments or manual techniques, the following
principles apply:
1.
Donors must provide informed consent. They must be observed closely
during the procedure and emergency
medical care must be available.
2.
Red cell losses related to the procedure, including samples collected
for testing, must not exceed 25 mL
per week, so that no more than 200
mL of red cells are removed in 8
weeks. If the donor’s red cells cannot
be returned during an apheresis procedure, hemapheresis or whole blood
donation should be deferred for 8
weeks.
3.
In manual plasma collection systems,
there must be a mechanism to ensure safe reinfusion of the autologous
red cells. Before the blood container

Records
Complete records (see Chapter l) must be
kept for each procedure. All adverse reactions should be documented along with
the results of their investigation and follow-up. Records of all laboratory findings
and collection data must be periodically
reviewed by a knowledgeable physician
and found to be within acceptable limits.
FDA guidelines require review at least
once every 4 months.8 Facilities must have
policies and procedures in place to ensure
that donor red cell loss during each procedure does not exceed acceptable limits.

Chapter 6: Apheresis

is separated from the donor for processing, there should be two separate, independent means of identification, so that both the donor and
the phlebotomist can ascertain that
the contents are those of the donor.
Often, the donor’s signature is one
identifier, along with a unique identification number.
In manual procedures for donors
weighing 50 to 80 kg (110-176 lb), no
more than 500 mL of whole blood
should be removed at one time, or
1000 mL during the session or within
a 48-hour period. The limits for donors who weigh more than 80 kg are
600 mL and 1200 mL, respectively.
For automated procedures, the allowable volume has been determined
9
for each instrument by the FDA.
At least 48 hours should elapse between successive procedures; ordinarily, donors should not undergo
more than two procedures within a
7-day period. Exceptions are permissible when plasma is expected to
have special therapeutic value for a
single recipient.
At the time of initial plasmapheresis
and at 4-month intervals thereafter
for donors undergoing plasmapheresis more often than once every 4
weeks, serum or plasma must be
tested for total protein and serum
protein electrophoresis or quantitative immunoglobulins. Results must
2
be within normal limits.
A qualified, licensed physician, knowledgeable in all aspects of hemapheresis, must be responsible for the
program.

Red Cells
Both AABB standards and FDA-approved
protocols address the removal of two allo-

143

geneic or autologous Red Blood Cell units
every 16 weeks by an automated apheresis method. Saline infusion is used to minimize volume depletion, and the procedure is limited to persons who are larger
and have higher hematocrits than current
minimum standards for whole blood donors (for males: weight 130 lb, height 5′1″;
for females: weight 150 lb, height 5′5″;
hematocrit 40% for both genders).11

Granulocytes
The indications for granulocyte transfusion are controversial (see Chapter 21). A
meta-analysis of randomized controlled
trials of granulocyte transfusion indicates
that effectiveness depends on an adequate
dose (>1 × 10 10 granulocytes/day) and
crossmatch compatibility (no recipient
antibodies to granulocyte antigens). 1 2
There is renewed interest in granulocyte
transfusion therapy for adults because
much larger cell doses can be delivered
when cells are collected from donors who
receive colony-stimulating factors.13 Some
success with granulocyte transfusions has
been observed in the treatment of septic
infants,14 possibly because the usual dose
is relatively larger in these tiny recipients
and because HLA alloimmunization is absent.

Drugs Administered for Leukapheresis
A daily dose of at least 1 × 10 granulocytes is necessary to achieve a therapeutic
effect.15 Collection of this number of cells
requires administration of drugs or other
adjuvants to the donor. The donor’s consent should include specific permission
for any drugs or sedimenting agents to be
used.
Hydroxyethyl Starch. A common sedimenting agent, hydroxyethyl starch (HES),
causes red cells to aggregate and thereby
sediment more completely. Sedimenting

144

AABB Technical Manual

agents enhance granulocyte harvest and result in minimal red cell content. Because
HES can be detected in donors for as long
as a year after infusion, AABB Standards requires facilities performing granulocyte collections to have a process to control the
maximal cumulative dose of any sedimenting agent administered to the donor
1(p24)
Because HES is a
within a given interval.
colloid, it acts as a volume expander, and
donors who have received HES may experience headaches or peripheral edema because
of expanded circulatory volume.
Corticosteroids. Corticosteroids can
double the number of circulating granulocytes by mobilizing them from the marginal
pool. A protocol using 60 mg of oral prednisone as a single or divided dose before donation gives superior granulocyte harvests
16
with minimal systemic steroid activity. Alternatively, 8 mg of oral dexamethasone may
be used. Before administration of corticosteroids, donors should be questioned about
any history or symptoms of hypertension,
diabetes, cataracts,17 and peptic ulcer.
Growth Factors. Recombinant hematopoietic growth factors—specifically, granulocyte colony-stimulating factor (G-CSF)—
can effectively increase granulocyte yields.
Hematopoietic growth factors alone can result in collection of up to 4 to 8 × 1010 granulocytes per apheresis procedure.13 Typical
doses of G-CSF employed are 5 to 10 µg/kg
given 8 to 12 hours before collection.18 Preliminary evidence suggests that in-vivo recovery and survival of these granulocytes
are excellent and that growth factors are
13
well tolerated by donors.

should be ABO-compatible with the recipient’s plasma and, if more than 2 mL
are present, the component should be
crossmatched. Ideally, D-negative recipients should receive granulocyte concentrates
from D-negative donors. Leukocyte (HLA)
matching is recommended in alloimmunized patients.

Storage and Infusion
Because granulocyte function deteriorates
during storage, concentrates should be
transfused as soon as possible after preparation. AABB Standards prescribes a
storage temperature of 20 to 24 C, for no
longer than 24 hours.1(p57) Agitation during
storage is probably undesirable. Irradiation
is required before administration to immunodeficient recipients and will probably be indicated for nearly all recipients
because of their primary disease. Infusion
through a microaggregate or leukocyte reduction filter is contraindicated.

Hematopoietic Progenitor Cells
Cytapheresis for collection of hematopoietic
progenitor cells is useful for obtaining
progenitor cells for marrow reconstitution
in patients with cancer, leukemia in remission, and various lymphomas (see
Chapter 25). Cytapheresis procedures can
also be used to collect donor lymphocytes
for infusion as an immune therapy in these
patients (see Chapter 25). The AABB has
published Standards for Cellular Therapy
Product Services.19 Additional requirements
are reviewed in Chapter 25.

Laboratory Testing

Therapeutic Apheresis

Testing for ABO and Rh, alloantibodies,
and infectious disease markers on a sample drawn at the time of phlebotomy are
required. Red cell content in granulocyte
concentrates is inevitable; the red cells

Therapeutic apheresis has been used to
treat many different diseases. Cells, plasma,
or plasma constituents may be removed
from the circulation and replaced by normal plasma, crystalloid, or colloid solu-

Chapter 6: Apheresis

tions of starch or albumin. The term
“therapeutic apheresis” is used for the
general procedure and the term “therapeutic plasma exchange” (TPE) is used for
procedures in which the goal is the removal of plasma, regardless of the solution used as replacement.
The theoretical basis for therapeutic
apheresis is to reduce the patient’s load of a
pathologic substance to levels that will allow clinical improvement. In some conditions, replacement with normal plasma is
intended to supply an essential substance
that is absent. In the absence of the need to
replace plasma constituents, colloidal solutions and/or saline should be used as replacement fluids. Other possible outcomes
of therapeutic apheresis include alteration
of the antigen-to-antibody ratio, modification of mediators of inflammation or immunity, and clearance of immune complexes. Some perceived benefit may result
from a placebo effect. Despite difficulties in
documentation, there is general agreement
that therapeutic apheresis is effective treatment for the conditions listed in Table 6-1
as Category I or Category II.3,20-22

General Considerations
Appropriate use of therapeutic apheresis
requires considerable medical knowledge
and judgment. The patient should be
evaluated for treatment by his or her personal physician and by the apheresis physician. Close consultation between these
physicians is important, especially if the
patient is small or elderly, has poor vascular access or cardiovascular instability, or
has a condition for which apheresis is of
uncertain benefit. The apheresis physician should make the final determination
about appropriateness of the procedure
and eligibility of the patient (see Table
3,20-22
When therapeutic apheresis is
6-1).
anticipated, those involved with the pa-

145

tient’s care should establish a treatment
plan and the goal of therapy. The endpoint may be an agreed-upon objective
outcome or a predetermined duration for
the therapy, whichever is achieved first. It
is helpful to document these mutually acceptable goals in the patient’s medical record. The nature of the procedure, its expected benefits, its possible risks, and the
available alternatives should be explained
to the patient by a knowledgeable individual, and the patient’s consent should be
documented. The procedure should be
performed only in a setting where there is
ready access to care for untoward reactions, including equipment, medications,
and personnel trained in managing serious reactions.

Vascular Access
For most adults needing a limited number
of procedures, the antecubital veins are
suitable for removal and return of blood.
For critically ill adults and for children,
indwelling central or peripheral venous
catheters are typically used. Especially effective are rigid-wall, large-bore, doublelumen catheters placed in the subclavian,
femoral, or internal jugular vein. Catheters of the type used for temporary hemodialysis allow both removal and return of
blood at high flow rates. Central catheters
can be maintained for weeks if multiple
procedures are necessary. Tunnel catheters
can be used when long-term apheresis is
anticipated.

Removal of Pathologic Substances
During TPE, plasma that contains the
pathologic substance is removed and a replacement fluid is infused. The efficiency
with which material is removed can be
estimated by calculating the patient’s
plasma volume and using Fig 6-1. This estimate depends on the following assump-

146

AABB Technical Manual

Table 6-1. Indication Categories for Therapeutic Apheresis

Procedure

Indication
Category

Antiglomerular basement membrane antibody
disease

Plasma exchange

Rapidly progressive glomerulonephritis

Plasma exchange

Hemolytic uremic syndrome

Plasma exchange

III

Rejection

Plasma exchange

Sensitization

Plasma exchange

III

Recurrent focal glomerulosclerosis

Plasma exchange

III

Plasma exchange

III

Photopheresis

III

Acute hepatic failure

Plasma exchange

III

Familial hypercholesterolemia

Selective adsorption

Plasma exchange

Overdose or poisoning

Plasma exchange

III

Phytanic acid storage disease

Plasma exchange

Cryoglobulinemia

Plasma exchange

Idiopathic thrombocytopenic purpura

Immunoadsorption

Raynaud's phenomenon

Plasma exchange

III

Vasculitis

Plasma exchange

III

Autoimmune hemolytic anemia

Plasma exchange

III

Rheumatoid arthritis

Immunoadsorption

Lymphoplasmapheresis

Plasma exchange

Scleroderma or progressive systemic
sclerosis

Plasma exchange

III

Systemic lupus erythematosus

Plasma exchange

III

Lupus nephritis

Plasma exchange

Psoriasis

Plasma exchange

RBC removal (marrow)

Plasma exchange
(recipient)

Disease
Renal and metabolic diseases

Renal transplantation

Heart transplant rejection

Autoimmmune and rheumatic diseases

Hematolic diseases
ABO-mismatched marrow transplant

Chapter 6: Apheresis

147

Table 6-1. Indication Categories for Therapeutic Apheresis (cont'd)
Procedure

Indication
Category

Phlebotomy

Erythrocytapheresis

Leukocytosis and thrombocytosis

Cytapheresis

Thrombotic thrombocytopenia purpura

Plasma exchange

Posttransfusion purpura

Plasma exchange

Sickle cell diseases

RBC exchange

Myeloma, paraproteins, or hyperviscosity

Plasma exchange

Myeloma or acute renal failure

Plasma exchange

Coagulation factor inhibitors

Plasma exchange

Aplastic anemia or pure RBC aplasia

Plasma exchange

III

Cutaneous T-cell lymphoma

Photopheresis

Leukapheresis

III

Hemolytic disease of the fetus and newborn

Plasma exchange

III

Platelet alloimmunization and refractoriness

Plasma exchange

III

Immunoadsorption

III

Malaria or babesiosis

RBC exchange

III

AIDS

Plasma exchange

Chronic inflammatory demyelinating
polyradiculoneuropathy

Plasma exchange

Acute inflammatory demyelinating
polyradiculoneuropathy

Plasma exchange

Lambert-Eaton myasthenia syndrome

Plasma exchange

Relapsing

Plasma exchange

III

Progressive

Plasma exchange

III

Lymphocytapheresis

III

Myasthenia gravis

Plasma exchange

Acute central nervous system inflammatory
demyelinating disease

Plasma exchange

Paraneoplastic neurologic syndromes

Plasma exchange

III

Immunoadsorption

III

Disease
Erythrocytosis or polycythemia vera

Neurologic disorders

Multiple sclerosis

(cont'd)

148

AABB Technical Manual

Table 6-1. Indication Categories for Therapeutic Apheresis (cont'd)
Procedure

Indication
Category

Plasma exchange

Immunoadsorption

III

Sydenham's chorea

Plasma exchange

Polyneuropathy with IgM (with or without
Waldenstrom's)

Plasma exchange
Immunoadsorption

II
III

Cryoglobulinemia with polyneuropathy

Plasma exchange

Multiple myeloma with polyneuropathy

Plasma exchange

III

POEMS syndrome

Plasma exchange

III

Systemic (AL) amyloidosis

Plasma exchange

Polymyositis or dermatomyositis

Plasma exchange

III

Leukapheresis

Plasma exchange

III

Leukapheresis

Rasmussen's encephalitis

Plasma exchange

III

Stiff-person syndrome

Plasma exchange

III

PANDAS

Plasma exchange

Amyotrophic lateral sclerosis

Plasma exchange

Disease
Demyelinating polyneuropathy with IgG and
IgA

Inclusion-body myositis

POEMS = polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin lesions; PANDAS = pediatric autoimmune neuropsychiatric disorders; Category I = standard acceptable therapy; Category II = sufficient evidence
to suggest efficacy usually as adjunctive therapy; Category III = inconclusive evidence of efficacy or uncertain risk/benefit ratio; Category IV = lack of efficacy in controlled trials.

tions: 1) the patient’s blood volume does
not change; 2) mixing occurs immediately; and 3) there is relatively little production or mobilization of the pathologic
material to be removed during the procedure. As seen in Fig 6-1, removal is greatest early in the procedure and diminishes
progressively during the exchange. Exchange is usually limited to 1 or 1.5
plasma volumes, or approximately 40 to
60 mL plasma exchanged per kg of body
weight in patients with normal hematocrit and average body size. This maximizes the efficacy per procedure but may

make it necessary to repeat the process.
Rarely are two or more plasma volumes
exchanged in one procedure. Although
larger volume exchange causes greater initial diminution of the pathologic substance, overall it is less efficient and requires considerably more time. Larger
volume exchanges can increase the risk of
coagulopathy, citrate toxicity, or electrolyte imbalance, depending on the replacement fluid.
The rates at which a pathologic substance is synthesized and distributed between intravascular and extravascular com-

Chapter 6: Apheresis

149

Figure 6-1. The relationship between the volume of plasma exchange and the patient’s original plasma
remaining.

partments affect the outcome of TPE. For
example, the abnormal IgM of Waldenstrom’s macroglobulinemia is synthesized
slowly and remains almost entirely (about
75%) intravascular, making apheresis particularly effective in removing it.23 In addition, a relatively small change in intravascular protein concentration may result
in a large change in blood viscosity. In contrast, efforts to prevent hydrops fetalis with
intensive TPE to lower the mother’s level of
IgG anti-D have been less successful. This is
due in part to the fact that about 55% of IgG
is in the extravascular fluid. In addition,
rapid reduction of IgG may cause antibody
synthesis to increase rapidly and “rebound”
over pretreatment levels.24 Rebound synthesis may also complicate TPE treatment of
autoimmune diseases. Immuno-suppressive
agents such as cyclophosphamide, azathioprine, or prednisone may be administered
to blunt the autoantibody rebound response
to apheresis.

Plasma removed during TPE should be
handled carefully and disposed of properly.
Such plasma cannot be used for subsequent manufacture of transfusable plasma
derivatives.

Removal of Normal Plasma Constituents
When the quantity of plasma removed
during TPE exceeds 1.5 times the plasma
volume, different rates of removal and reconstitution are observed for different
25
constituents. In the case of fibrinogen,
the third component of complement (C3),
and immune complexes, 75% to 85% of the
original substance is removed after a 1.5
plasma-volume procedure. Pretreatment
levels are restored in 3 to 4 days. The concentrations of electrolytes, uric acid, Factor VIII, and other proteins are less affected by a plasma exchange. A 10% or
more decrease in platelet count generally
occurs, with 2 to 4 days needed for a re-

150

AABB Technical Manual

turn to pretreatment values. Coagulation
factors other than fibrinogen generally return to pretreatment values within 24
hours. Immunoglobulin removal occurs at
about the expected rate of 65% per plasma
volume, but recovery patterns vary for different immunoglobulin classes, depending on intravascular distribution and rates
of synthesis. (Table 11-3 describes immunoglobulin characteristics.) Plasma IgG
levels return to approximately 60% of the
pretreatment value within 48 hours because of reequilibration with protein in the
extravascular space. These issues are important in planning the frequency of therapeutic procedures. Weekly apheresis permits more complete recovery of normal
plasma constituents; daily procedures can
be expected to deplete many normal, as
well as abnormal, constituents. Additionally, intensive apheresis reduces the concentration of potentially diagnostic plasma
constituents, so blood for testing should be
drawn before TPE.

Replacement Fluids
Available replacement solutions include:
crystalloids, albumin solutions, plasma
(FFP, cryosupernatant plasma, or Plasma
Frozen within 24 Hours of Collection), and
HES.27 Table 6-2 presents advantages and
disadvantages of each. A combination is
often used, the relative proportions being
determined by the physician on the basis
of the patient’s disease and physical condition, the planned frequency of procedures, and cost. Acute treatment of
immediately life-threatening conditions
usually requires a series of daily plasma
exchange procedures, often producing a
significant reduction of coagulation factors.
Monitoring the platelet count, prothrombin time, activated partial thromboplastin
time, and fibrinogen level helps determine the need for supplemental platelets,

plasma, or cryoprecipitate. Because plasma
contains citrate, its use may increase the
risk of citrate toxicity.

Complications
With careful patient selection and attention to technical details, most therapeutic
apheresis procedures are completed without complications. Adverse effects of therapeutic apheresis were reported in only
4% of patients in one large study.28 However, therapeutic apheresis is often required
for patients who are critically ill and at risk
for a variety of complications.
Vascular Access. Patients requiring therapeutic apheresis have often been subjected
to multiple venipunctures and achieving
peripheral vascular access may be difficult.
Frequently, special venous access, such as
placement of an indwelling double-lumen
apheresis/dialysis catheter, is required. Venous access devices may cause further
vascular damage, sometimes resulting in
thrombosis. Infrequently, they may result in
severe complications such as pneumothorax or perforation of the heart or great
vessels. 28 Other complications include
arterial puncture, deep hematomas, and
arteriovenous fistula formation. Bacterial
colonization often complicates long-term
placement and may lead to catheter-associated sepsis, especially in patients who are
receiving steroids or other immunosuppressants. Inadvertent disconnection of
catheters may produce hemorrhage or air
embolism.
Alteration of Pharmacodynamics. TPE
can lower blood levels of drugs, especially
those that bind to albumin. Apheresis reduces plasma levels of antibiotics and anticonvulsants, but few clinical data exist to
suggest adverse patient outcomes due to
apheresis-associated lowering of drug levels. Nevertheless, the pharmacokinetics of
all drugs being given to a patient should be

Chapter 6: Apheresis

151

Table 6-2. Comparison of Replacement Fluids
Replacement
Solution

Advantages

Disadvantages

Crystalloids

Low cost
Hypoallergenic
No viral risk

2-3 volumes required
Hypo-oncotic
No coagulation factors
No immunoglobulins

Albumin

Iso-oncotic
No contaminating
“inflammatory mediators”
No viral risk

High cost
No coagulation factors

Hydroxyethyl starch

Moderate cost
Iso-oncotic
No contaminating “inflammatory
mediators”

No coagulation factors
Long-term residual levels of HES
Contraindicated with renal failure
Possible coagulopathy

Plasma

Maintains normal levels of:
immunoglobulins
complement
antithrombin
other proteins

Viral transmission risk
Citrate load
ABO incompatibility risk
Allergic reactions
Sensitization

considered before starting apheresis and
dosage schedules adjusted if necessary. It is
prudent to withhold the administration of
drugs scheduled to be given during or up to
an hour before apheresis until after the procedure has finished. Removal of plasma
cholinesterase may complicate the administration of paralyzing agents such as
succinylcholine in the immediate postexchange period.
Hypocalcemia. Most patients and donors
with normal parathyroid and liver function
maintain calcium homeostasis during apheresis. However, symptoms of hypocalcemia
related to citrate toxicity are the most common adverse effect reported in 3.0% of
therapeutic apheresis procedures in one
large study.28 Symptoms of reduced plasma
levels of ionized calcium (perioral paresthesias, tingling, a feeling of vibrations) reflect the rate at which citrate anticoagulant

No immunoglobulins

is returned, ionized and bound calcium are
removed, and ionized calcium is bound to
“calcium-stripped” albumin replacement.
Hyperventilation, hypothermia, hypomagnesemia, and the use of plasma as a replacement solution exacerbate citrate toxicity. Hypocalcemia can usually be controlled
by reducing the proportion of citrate or
slowing the reinfusion rate. If untreated,
symptoms may progress to muscle twitching, chills, pressure in the chest, nausea,
vomiting, and hypotension. Low ionized
calcium concentrations can induce severe
cardiac arrhythmias. Asking the patient to
report any vibrations or tingling sensations
can help determine the appropriate reinfusion rate. Extra precautions must be
taken in patients who are unable to communicate or who may metabolize citrate
poorly (eg, those with liver failure). Hypocalcemic toxicity can usually be managed

152

AABB Technical Manual

by administering oral calcium carbonate or
intravenous calcium.22,23,29
Circulatory Effects. Hypovolemia and
subsequent hypotension may occur during
apheresis, especially when the volume of
extracorporeal blood exceeds 15% of the total blood volume. Hypotension tends to occur in ill children, the elderly, neurology patients, anemic patients, and those treated
with intermittent-flow devices that have
large extracorporeal volumes. Continuousflow devices typically do not require large
extracorporeal volumes but can produce
hypovolemia if return flow is inadvertently
diverted to a waste collection bag, either
through operator oversight or mechanical
or software failures. Hypovolemia may also
be secondary to inadequate volume or protein replacement. During all procedures, it
is essential to maintain careful and continuous records of the volumes removed and
returned. The use of antihypertensive medications, especially angiotensin-converting
enzyme (ACE) inhibitors combined with albumin replacement, may also contribute to
hypotensive reactions (see Chapter 27). Patients taking agents that inhibit ACE have
experienced severe hypotensive episodes
when treated with SPA columns and with
other immunosorbents.30 Patients should not
receive these medications for 72 hours before undergoing immunoabsorption treatment. Because infusion of cold fluids
through a central venous catheter may induce arrhythmias, some apheresis programs
use blood warmers for selected patients.
Infections. Plasma is the only commonly
used replacement solution with the risk of
transmitting infectious viruses. Bacterial
colonization and infection related to repeated apheresis usually arise from within
the vascular catheter. Intensive apheresis
regimens decrease levels of immunoglobulins and the opsonic components of complement. In addition, immunosuppressive
drugs used to prevent rebound antibody

production may further compromise defense mechanisms.
Mechanical Hemolysis and Equipment
Failures. Collapsed or kinked tubing, malfunctioning pinch valves, or improper
threading of tubing may damage donor or
patient red cells in the extracorporeal circuit. Machine-related hemolysis was observed in 0.07% of over 195,000 apheresis
procedures performed in the United Kingdom.31 Similar rates of hemolysis, 0.06% and
0.01%, were reported in response to uniform
questionnaires regarding therapeutic and
donor apheresis procedures, respectively.32,33
Hemolysis can also occur with incompatible replacement fluids such as D5W
(eg, D5W used to dilute 25% albumin) or
ABO-discrepant plasma. The operator
should carefully observe plasma collection
lines for pink discoloration suggestive of
hemolysis. Other types of equipment failure, such as problems with the rotating
seal, leaks in the plastic, and roller pump
34
failure, are rare.
Allergic Reactions and Respiratory Distress. Respiratory difficulty during or immediately following apheresis can have
many causes: pulmonary edema, massive
pulmonary embolism, air embolism, obstruction of the pulmonary microvasculature, anaphylactic reactions, and transfusionrelated acute lung injury.35 Hemothorax or
hemopericardium due to vascular erosion
by a central venous catheter is typically un36,37
Pulmonary
suspected yet may be fatal.
edema that results from volume overload or
cardiac failure is usually associated with
dyspnea, an increase in the diastolic blood
pressure, and characteristic chest X-ray
findings. Acute pulmonary edema can also
arise from damage to alveolar capillary
membranes secondary to an immune reaction or to vasoactive substances in FFP or
colloid solutions prepared from human
plasma. The use of FFP as a replacement
fluid has been associated with complement

Chapter 6: Apheresis

activation and with allergic reactions that
produce urticaria, swelling of oral mucosa,
and bronchospasm; these usually respond
to antihistamines and corticosteroids. Hypotension and flushing associated with the
rapid infusion of albumin in patients taking
ACE inhibitors are discussed in Chapter 27.
Predominantly ocular (periorbital edema,
conjunctival swelling, and tearing) reactions have occurred in donors sensitized to
the ethylene oxide gas used to sterilize disposable plastic apheresis kits.38
Fatalities During Apheresis. Despite the
fact that patients undergoing therapeutic
apheresis are often critically ill, fatalities
during apheresis are comparatively rare.
Estimates of fatality rates range from 3 in
10,00039 to 1 in 50040 procedures. Most deaths
were due to cardiac arrhythmias or arrest
during or shortly after the procedure or to
acute pulmonary edema or adult respiratory distress syndrome occurring during a
procedure. Rare fatalities resulted from
anaphylaxis, vascular perforation, hepatitis,
sepsis, thrombosis, and hemorrhage.

Indications for Therapeutic Apheresis
Although therapeutic apheresis has been
used in the treatment of many diseases,
most published studies are case reports or
small uncontrolled series, often providing
insufficient evidence of efficacy. Publication bias tends to favor positive results,
and physicians should avoid subjecting
patients to the risks and high costs of
apheresis procedures based on marginal
clinical studies. Controlled, randomized,
blinded studies of therapeutic apheresis
are difficult to conduct, especially because using sham treatments as a control
is expensive and carries some risk. However, the complicated apheresis instruments and associated attention from
nursing and medical personnel may cre-

153

ate or amplify a placebo effect and bias
the evaluation of clinical improvement.
For many of the diseases being treated,
the etiology, pathogenesis, and natural history are incompletely understood, and reductions in such measured variables as
complement components, rheumatoid factor, or immune complexes cannot be correlated reliably with changes in disease activity. An example is the use of the erythrocyte
sedimentation rate (ESR) as an index of disease activity in rheumatoid arthritis. The
ESR invariably decreases during intensive
TPE, but this reflects removal of fibrinogen
and not necessarily a decrease in disease
activity. For the same reasons, the optimal
volume and frequency of exchange are often not established. For severe imminently
life-threatening disease, when albumin/saline is the replacement fluid, TPE is initially
performed daily. After a few days, the
fibrinogen or platelet count may be low
enough to significantly increase the risk of
bleeding. Clinical judgment must then be
exercised to decide whether to proceed
with TPE using clotting factor/platelet
transfusions or to withhold TPE until these
parameters normalize. The conditions discussed below are established indications
for therapeutic apheresis.3,20,21,41,42

Hematologic Conditions
Serum Hyperviscosity Syndrome. Serum
hyperviscosity resulting from multiple
myeloma or Waldenstrom’s macroglobulinemia can cause congestive heart failure;
reduced blood flow to the cerebral, cardiac, or pulmonary circulation; or symptoms of headache, vertigo, somnolence, or
obtundation. Paraproteins may interfere
with hemostasis, leading to hemorrhagic
symptoms.
The presence of hyperviscosity correlates
only in very general terms with the concen-

154

AABB Technical Manual

tration of paraprotein. Measurement of serum viscosity relative to water is a simple
procedure that provides more objective information. For some pathologic proteins,
serum viscosity is highly temperature dependent, so serum viscosity should be
measured at physiologically relevant temperatures. Normal serum viscosity ranges
from 1.4 to 1.8 relative to water. Because
most patients are not symptomatic until
their relative serum viscosity is more than
4.0 or 5.0, patients with mild elevations may
not require treatment. For symptomatic
hyperviscosity, a single apheresis procedure
is usually highly effective.23
Hyperleukocytosis. Leukapheresis is often used to treat the dramatically elevated
white cell count that can occur in acute leukemia. Several different thresholds have
been used: fractional volume of leukocytes
(leukocrit) above 10%; total circulating leukocytes above 100,000/µL; and circulating
blasts above 50,000/µL.43 However, the use
of a single laboratory value as an indication
for treatment is an oversimplification. Such
factors as erythrocyte concentration, leukemic cell type, rate at which the count is rising, potential obstructions to cerebral or
pulmonary blood flow, and the patient’s coagulation status and general condition must
be considered. Most leukemic patients with
extreme leukocytosis have significant anemia. Reduced red cell mass reduces blood
viscosity, so unless there is an acute need to
increase oxygen-carrying capacity, red cells
should not be transfused until the hyperviscosity crisis has been resolved.43
In some patients with acute blast crisis
or in unusual types of leukemia, both the
hematocrit and leukocrit are elevated. If
there is evidence of cerebral or pulmonary
symptoms, rapid reduction of leukocyte
concentration should be considered, although the efficacy of such leukocyte reduction is unproved. More commonly,
however, the white cell count rises over

weeks or longer, and leukocyte reduction
can be effected with chemotherapy, with or
without leukapheresis. Leukapheresis is
sometimes used to reduce the white cell
count to <100,000/µL before the start of
chemotherapy, to reduce the likelihood of
tumor lysis syndrome. However, there have
been no controlled clinical trials to substantiate this approach, and it must be recognized that more malignant cells are
present outside the circulation than within
the bloodstream.
Thrombocythemia. Therapeutic plateletpheresis is usually undertaken for symptomatic patients with platelet counts above
1,000,000/µL. The measured count, by itself,
should not determine whether platelet
reduction is indicated. In patients with
evidence of thrombosis secondary to
thrombocythemia, plateletpheresis can be
beneficial. The rationale for platelet reduction in bleeding patients with thrombocythemia is less clear. There are no accepted
indications for prophylactic plateletpheresis in asymptomatic patients, although the
risk of placental infarction and fetal death
may justify the procedure in a pregnant
woman with severe thrombocythemia.43
Thrombotic Thrombocytopenic Purpura/
Hemolytic-Uremic Syndrome (TTP/HUS).
The conditions described as TTP/HUS are
multisystem disorders, in which platelet/fibrin thrombi occlude the microcirculation.
They are characterized by varying degrees
of thrombocytopenia, microangiopathic
hemolytic anemia, renal dysfunction, neurologic abnormalities, and fever. Patients
presenting with fulminant TTP usually have
platelet counts below 50,000/µL and lactic
dehydrogenase (LDH) levels above 1000
IU/mL, resulting from systemic ischemia
and hemolysis. 44 The peripheral blood
smear characteristically shows increased
numbers of schistocytes. Evidence for disseminated intravascular coagulation is generally absent.

Chapter 6: Apheresis

TTP usually develops without obvious
cause, although episodes may occur after
infections, pregnancy, or use of some common drugs such as ticlopidine, or clopidogrel. Recent reports suggest that it is caused
by a transient antibody to a protease
(ADAMTS13) that normally cleaves large
von Willebrand factor (vWF) multimers.
The unusually large vWF multimers avidly
aggregate circulating platelets, triggering
the syndrome.45,46 Increasingly, cases of recurrent or relapsing TTP are being recognized.
HUS is a similar condition that occurs
more commonly in children than adults.
HUS may follow diarrheal infections with
verotoxin-secreting strains of Escherichia
coli (strain 0157:H7) or Shigella. Compared
with patients who have classic TTP, those
with HUS have more renal dysfunction and
less prominent neurologic and hematologic
findings. Most patients with HUS do not
have antibody to the vWF protease and
have normal concentrations of vWF protease. TTP/HUS can occur after treatment with
certain cytotoxic drugs, including mitomycin C. A microangiopathic hemolysis
similar to TTP/HUS can occur in organ or
stem cell transplant recipients receiving
cyclosporine and tacrolimus.47,48 Transplantassociated microangiopathic hemolysis appears to be less responsive to therapy and
probably represents a different disease process.48
TPE with plasma or Plasma Cryoprecipitate Reduced replacement has become
49,50
the treatment of choice for TTP/HUS.
Protease levels have been shown to be stable for at least 2 weeks in citrated plasma
stored at 37 C.51 TPE is now thought to remove both antibody to the protease and
vWF and to replace deficient protease.
Other largely unproved treatments include
prednisone, antiplatelet agents, splenectomy, vincristine, rituximab, and intravenous immunoglobulin. Because platelet
transfusions have anecdotally been associ-

155

ated with disease exacerbation and death,
they are usually contraindicated (except in
the presence of life-threatening hemorrhage).
TPE is typically performed daily for l to 2
weeks, but the intensity and duration of
treatment should be guided by the individual patient’s course. Occasionally, prolonged courses of treatment are required.
Therapeutic plasma exchange has impressively improved the survival rate in TTP,
from being almost universally fatal before
1964 to 80% survival in a recent series.49
Signs of response to therapy include a rising platelet count and reduction of LDH
between procedures. As patients recover to
near normal LDH and platelet count (100150,000/µL), TPE is discontinued. Some
programs switch from intensive TPE to intermittent plasma exchange or simple
plasma infusion, but the efficacy of this approach has not been established.52 Despite
the success of TPE, TTP/HUS remains a
serious condition. Treatment failures continue to occur and to cause major organ
damage or death.
Complications of Sickle Cell Disease.
Several complications of sickle cell disease
are syndromes that can be treated by red
cell exchange. These conditions include
stroke or impending stroke, acute chest
syndrome, and multiorgan failure. Either
manual or automated techniques can be
used for red cell exchange, but automated
techniques are faster and better controlled.
The goal is to replace red cells containing
hemoglobin S with a sufficient number of
red cells containing hemoglobin A so that
the overall proportion of hemoglobin A in
the blood is 60% to 80%. Some centers provide partially phenotypically matched red
cells (eg, C, E, and K1) to avoid alloimmunizing long-term transfusion recipients to
these antigens. At the end of the procedure,
the patient’s hematocrit should be no higher
than 30% to 35% to avoid increased blood
viscosity. Chronic erythrocytapheresis can be

156

AABB Technical Manual

used to manage iron overload in patients
requiring long-term transfusion therapy.53

Cryoglobulinemia
Significant elevations of cryoglobulins may
cause cold-induced vascular occlusion,
abnormalities of coagulation, renal insufficiency, or peripheral nerve damage. Removal of cryoglobulins by apheresis can
be used to treat acute symptomatic episodes, but definitive therapy depends on
identifying and treating the underlying
causative condition.

Neurologic Conditions
Myasthenia Gravis. Myasthenia gravis results from autoantibody-mediated blockade of the acetylcholine receptor located
on the postsynaptic motor endplate of
muscles. Standard treatment includes steroids and acetylcholinesterase inhibitors.
TPE is used as adjunctive treatment for
patients experiencing exacerbations not
controlled by medications and for patients being prepared for thymectomy. A
typical treatment protocol is five or six
TPE procedures over l to 2 weeks. Concurrent immunosuppression to prevent antibody rebound is recommended. Chronic
TPE has been used with some success in a
small number of patients.
Acute Guillain-Barré Syndrome.
Guillain-Barré syndrome is an acute autoimmune demyelinating polyneuropathy
that can produce dramatic paralysis in otherwise healthy individuals. The cause is unknown; many cases appear to follow benign
viral infections or Campylobacter jejuni infection. Most patients recover spontaneously, but as many as one in six may become unable to walk or may develop
respiratory failure requiring ventilatory
support. Early treatment is beneficial for
patients with rapidly progressive disease.
The response to therapy is inferior in pa-

tients who remain untreated for several
weeks. Recent controlled studies suggest
that intravenous immunoglobulin gives results equivalent to five TPE procedures over
a 2-week period.54 A cost-effectiveness analysis has suggested that TPE is less costly
than a course of intravenous immune glob55
ulin. Multicenter trials have suggested that
TPE, if initiated early, can decrease the period of minimal sensorimotor function.56
Patients whose illness is not acute in onset,
is not characteristic of Guillain-Barré syndrome, or in whom nerve conduction studies show complete axonal block may have a
poorer prognosis and less response to
apheresis therapy.
Chronic Inflammatory Demyelinating
Polyneuropathy. Chronic inflammatory
demyelinating polyneuropathy (CIDP), often seen in HIV patients, is a group of disorders with slow onset and progressive or
intermittent course, characterized by elevated spinal fluid protein, marked slowing
of nerve conduction velocity, and segmental demyelination of peripheral nerves. Various sensorimotor abnormalities result.
Polyneuropathy may also occur in the
POEMS syndrome, characterized by polyneuropathy, organomegaly, endocrinopathy, MGUS (monoclonal gammopathy of
unknown significance), and skin changes.
Corticosteroids are the first-line treatment
for CIDP. TPE and intravenous immunoglobulin have equivalent efficacy in patients
unresponsive to corticosteroids.57
Polyneuropathy Associated with Monoclonal Gammopathy of Undetermined Significance. When polyneuropathy is associated with monoclonal paraproteins of uncertain significance, TPE has been shown to
58,59
be effective for all variants.

Renal Diseases
Rapidly progressive glomerulonephritis
(RPGN) associated with antibodies to

Chapter 6: Apheresis

basement membranes of glomeruli and alveoli, which may result in pulmonary
hemorrhage (Goodpasture’s disease), usually responds to TPE as an adjunct to immunosuppressive drugs.41 TPE accelerates
the disappearance of antibodies to basement membranes and improves renal
function. TPE is particularly effective in
halting pulmonary hemorrhage in these
patients, even if renal function does not
completely normalize following treatment. Therapeutic apheresis has been
used in treating the vasculitis associated
with RPGN and the presence of antineutrophil cytoplasmic antibody (ANCApositive RPGN).60,61 TPE is most effective
41
in the more severe cases.
Myeloma light chains may be toxic to renal tubular epithelium and cause renal failure in up to 10% of cases. TPE is useful as
adjunctive therapy in some myeloma patients with cast nephropathy but is not associated with improved survival.41,62

Other Conditions
TPE has been used as adjunctive treatment
for a variety of multisystem diseases. A
combination of steroid, cytotoxic agents,
and TPE has been used for severely ill patients with polyarteritis nodosa, 6 3 although most rheumatologic conditions
respond poorly to TPE. Clinical trials using standard TPE have not shown benefit
in the treatment of systemic lupus erythematosus, polymyositis, dermatomyositis, or
scleroderma.21 Immunoadsorption columns are of benefit in patients with rheumatoid arthritis refractory to medical
management.64
Homozygous Type II Familial Hypercholesterolemia. Homozygous hypercholesterolemia, a rare disorder of the receptor
for low-density lipoproteins, results in severe premature atherosclerosis and early
death from coronary artery disease. Pro-

157

longed reduction in circulating lipids can
be achieved with repeated TPE, often with
selective adsorption or filtration techniques.43 Heterozygous hypercholesterolemia results from several gene defects in
the LDL receptor. Some patients with heterozygous hypercholesterolemia also develop high levels of cholesterol and are at
increased risk for developing premature
atherosclerotic heart disease.
Two apheresis systems for selective LDL
removal in patients with homozygous or
heterozygous hypercholesterolemia have
been cleared by the FDA. The Liposorber
LA-150 (Kaneka Pharma America, New
York, NY) is based on a dextran sulfate adsorption system. The H.E.L.P. LDL system
(B. Braun, Melsugen, Germany) is a heparin-induced LDL cholesterol precipitation
system. The LDL apheresis procedure selectively removes apolipoprotein-B-containing cholesterols such as LDL and very
LDL, sparing high-density lipoprotein
(HDL) cholesterol. This provides an advantage over standard apheresis, which removes all plasma proteins, including the
“protective” HDL. The procedure acutely
lowers levels of targeted cholesterols by
60% to 70%. Treatment is usually performed every 1 to 2 weeks, and cholesterollowering drugs are generally employed simultaneously. Several studies have shown
that use of LDL apheresis can achieve significant lowering of lipids in nearly all patients with severe hypercholesterolemia.65,66
Promising results have also been reported
from a system capable of direct adsorption
of LDL and Lp(a) (DALI) from whole
blood.67
Refsum’s Disease (Phytanic Acid Disease). Refsum’s disease is a rare inborn error of metabolism resulting in toxic levels of
phytanic acid, causing neurologic, cardiac,
skeletal, and skin abnormalities.21 TPE is
useful in conjunction with a phytanicacid-deficient diet and should be started as

158

AABB Technical Manual

soon as possible, before permanent damage occurs.

Staphylococcal Protein A
Immunoadsorption

References
1.

SPA immunoadsorption is approved by the
FDA for treatment of acute and chronic
immune thrombocytopenic purpura. The
device is also FDA-approved to treat
adults with rheumatoid arthritis unresponsive to disease-modifying antirheu64
matic drugs. Although not FDA-approved, this technique has been used,
with limited success, to treat other autoimmune thrombocytopenias.68 Many of
these protocols are still experimental and
randomized trials have not been conducted. Anecdotal cases of TTP/HUS refractory to TPE that have responded to SPA
immunoadsorption have also been reported.69

Photopheresis
Photopheresis is a technique that involves
the treatment of patients with psoralens,
the separation of lymphocytes by apheresis, and treatment of the cells with ultraviolet radiation. This renders the lymphocytes and other nucleated cells incapable
of division. The treated cells are then reinfused. This procedure, also known as
extracorporeal photochemotherapy, has
been approved by the FDA for the treatment of cutaneous T-cell lymphoma and
is considered the first line of treatment for
the erythrodermic phase of this disease.70
Clinical trials are under way to determine
the efficacy of photopheresis in the following conditions: cellular-mediated rejection of heart and lung allografts, and
acute and chronic graft-vs-host disease
following allogeneic marrow transplant.
This technology is not available in most
apheresis centers.

10.

11.

12.

13.

Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.
Code of federal regulations. Title 21 CFR Part
640. Washington, DC: US Government Printing Office, 2004 (revised annually).
McLeod BC. Introduction to the third special
issue: Clinical applications of therapeutic
apheresis. J Clin Apheresis 2000;15(½):2-3.
Burgstaler EA. Current instrumentation for
apheresis. In: McLeod BC, Price TH, Weinstein
R, eds. Apheresis: Principles and practice.
2nd ed. Bethesda, MD: AABB Press, 2003:95130.
Vamvakas EC, Pineda AA. Selective extraction
of plasma constituents. In: McLeod BC, Price
TH, Weinstein R, eds. Apheresis: Principles and
practice. 2nd ed. Bethesda, MD: AABB Press,
2003:437-76.
Berger GM, Firth JC, Jacobs P, et al. Three different schedules of low-density lipoprotein
apheresis compared with plasmapheresis in
patients with homozygous familial hypercholesterolemia. Am J Med 1990;88:94-100.
Silva MA, Brecher ME. Summary of the AABB
interorganizational taskforce on bacterial
contamination of platelets, fall 2004 impact
survey. Transfusion 2005 (in press).
Food and Drug Administration. Memorandum: Revised guideline for the collection of
Platelets, Pheresis. (October 7, 1988) Rockville,
MD: CBER Office of Communication, Training, and Manufacturers Assistance, 1988.
Food and Drug Administration. Memorandum: Volume limits for automated collection
of source plasma. (November 4, 1992) Rockville,
MD: CBER Office of Communication, Training, and Manufacturers Assistance, 1992.
Food and Drug Administration. Memorandum: Requirements for infrequent plasmapheresis donors. (March 10, 1995) Rockville,
MD: CBER Office of Communication, Training, and Manufacturers Assistance, 1995.
Food and Drug Administration. Guidance for
industry: Recommendations for collecting
red blood cells by automated apheresis methods. (January 30, 2001; technical correction
February 13, 2001) Rockville, MD: CBER Office of Communication, Training, and Manufacturers Assistance, 2001.
Vamvakas EC, Pineda AA. Determinants of the
efficacy of prophylactic granulocyte transfusions: A meta-analysis. J Clin Apheresis 1997;
12:74-81.
Stroncek DF, Yau YY, Oblitas J, Leitman SF.
Administration of G-CSF plus dexamethasone produces greater granulocyte concen-

Chapter 6: Apheresis

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

trate yields while causing no more donor toxicity than G-CSF alone. Transfusion 2001;41:
1037- 44.
Strauss RG. Neutrophil collection and transfusions. In: Simon TL, Dzik WH, Snyder EL, et
al, eds. Rossi’s principles of transfusion medicine. 3rd ed. Baltimore, MD: Lippincott Williams and Wilkins, 2002:258-67.
McCullough J. Granulocyte transfusion. In:
Petz LD, Swisher SN, Kleinman S, et al, eds.
Clinical practice of transfusion medicine. 2nd
ed. New York: Churchill-Livingstone, 1996:
413-32.
Barnes A, DeRoos A. Increased granulocyte
yields obtained with an oral three-dose prednisone premedication schedule (abstract).
Am J Clin Pathol 1982;78:267.
Strauss RG, Ghodsi Z. Cataracts in neutrophil
donors stimulated with adrenal corticosteroids. Transfusion 2001;41:1464-8.
Stroncek DF, Matthews CL, Follman D,
Leitman SF. Kinetics of G-CSF induced
granulocyte mobilization in healthy subjects:
Effects of route of administration and addition of dexamethasone. Transfusion 2002;42:
597-602.
Szczepiorkowski ZM, ed. Standards for cellular therapy product services. 1st ed. Bethesda,
MD: AABB, 2004.
Smith JW, Weinstein R, Hillyer KL for the
AABB Hemapheresis Committee. Therapeutic
apheresis: A summary of current indications
categories endorsed by the AABB and the
American Society for Apheresis. Transfusion
2003;43:820-2.
Strauss RG, Ciavarella D, Gilcher RO, et al. An
overview of current management. J Clin
Apheresis 1993;8:189-272.
Jones HG, Bandarenko N. Management of the
therapeutic apheresis patient. In: McLeod
BC, Price TH, Weinstein R, eds. Apheresis:
Principles and practice. 2nd ed. Bethesda,
MD: AABB Press, 2003:253-82.
McLeod BC. Therapeutic plasma exchange.
In: Simon TL, Dzik WH, Snyder EL, et al, eds.
Rossi’s principles of transfusion medicine.
3rd ed. Baltimore, MD: Lippincott Williams
and Wilkins, 2002:662-82.
Williams WJ, Katz VL, Bowes WA. Plasmapheresis during pregnancy. Obstet Gynecol
1990;76:451-7.
Orlin JB, Berkman EM. Partial plasma replacement: Removal and recovery of normal
plasma constituents. Blood 1980;56:1055-9.
Weinstein R. Basic principles of therapeutic
blood exchange. In: McLeod BC, Price TH,
Weinstein R, eds. Apheresis: Principles and
practice. 2nd ed. Bethesda, MD: AABB Press,
2003:295-320.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

159

Brecher ME, Owen HG, Bandarenko N. Alternatives to albumin: Starch replacement for
plasma exchange. J Clin Apheresis 1997;12:
146-53.
Kiprov DD, Golden P, Rohe R, et al. Adverse
reactions associated with mobile therapeutic
apheresis; analysis of 17,940 procedures. J
Clin Apheresis 2001;16:130-3.
Weinstein R. Prevention of citrate reactions
during therapeutic plasma exchange by constant infusion of calcium gluconate with the
return fluid. J Clin Apheresis 1996;11:204-10.
Olbricht CJ, Schaumann D, Fischer D. Anaphylactoid reactions, LDL apheresis with
dextran sulphate, and ACE inhibitors. Lancet
1993;341:60-1.
Robinson A. Untoward reactions and incidents in machine donor apheresis. Transfus
Today 1990;7:7-8.
McLeod BC, Sniecinski I, Ciavarella D, et al.
Frequency of immediate adverse effects associated with therapeutic apheresis. Transfusion 1999;39:282-8.
McLeod BC, Price TH, Owen H, et al. Frequency
of immediate adverse effects associated with
apheresis donation. Transfusion 1998;38:
938-43.
Westphal RG. Complications of hemapheresis.
In: Westphal RG, Kasprisin DO, eds. Current
status of hemapheresis: Indications, technology and complications. Arlington, VA: AABB,
1987:87-104.
Askari S, Nollet K, Debol SM, et al. Transfusion-related acute lung injury during plasma
exchange: Suspecting the unsuspected. J Clin
Apheresis 2002;17:93-6.
Duntley P, Siever J, Korwes ML, et al. Vascular
erosion by central venous catheters. Clinical
features and outcome. Chest 1992;101:16338.
Quillen K, Magarace L, Flanagan J, Berkman
EM. Vascular erosion caused by a double-lumen central venous catheter during therapeutic plasma exchange. Transfusion 1995;
35:510-2.
Leitman SF, Boltansky H, Alter HJ, et al. Allergic reactions in healthy plateletpheresis donors caused by sensitization to ethylene oxide gas. N Engl J Med 1986;315:1192-6.
Gilcher RO. Apheresis: Principles and technology of hemapheresis. In: Simon TL, Dzik
WH, Snyder EL, et al, eds. Rossi’s principles of
transfusion medicine. 3rd ed. Baltimore, MD:
Lippincott Williams and Wilkins, 2002:64858.
Schmitt E, Kundt G, Klinkmann H. Three
years with a national apheresis registry. J Clin
Apheresis 1992;7:58-72.

160

41.

42.
43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

AABB Technical Manual

Madore F, Lazarus JM, Brady HR. Therapeutic
plasma exchange in renal diseases. J Am Soc
Nephrol 1996;7:367-86.
McLeod BC. Apheresis principles and practice. 2nd ed. Bethesda, MD: AABB Press, 2003.
Klein HG. Principles of apheresis. In: Anderson KC, Ness PM, eds. Scientific basis of
transfusion medicine. 2nd ed. Philadelphia:
WB Saunders, 2000:553-68.
Cohen JA, Brecher ME, Bandarenko N. Cellular source of serum lactate dehydrogenase elevation in patients with thrombotic thrombocytopenic purpura. J Clin Apheresis 1998;13:
16-9.
Furlan M, Robles R, Galbusera M, et al. von
Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura and the
hemolytic-uremic syndrome. N Engl J Med
1998;339:1578-84.
Tsai H-M, Chun-Yet Lian E. Antibodies to von
Willebrand factor-cleaving protease in acute
thrombotic thrombocytopenic purpura. N Engl
J Med 1998;339:1585-94.
McLeod BD. Thrombotic microangiopathies
in bone marrow and organ transplant patients. J Clin Apheresis 2002;17:118-23.
George JN, Li X, McMinn JR, Terrell DR, et al.
Thrombotic thrombocytopenic purpurahemolytic uremic syndrome following allogeneic HPC transplantation: A diagnostic dilemma. Transfusion 2004;44:294-304.
George JN, Rizui MA. Thrombocytopenia. In:
Beutler E, Lichtman MA, Coller BS, et al, eds.
Williams’ hematology. 6th ed. New York:
McGraw-Hill, 2001:1495-539.
Rock G, Shumak KH, Sutton DM, et al. Cryosupernatant as replacement fluid for plasma
exchange in thrombotic thrombocytopenic
purpura. Br J Haematol 1996;94:383-6.
Gerritsen HE, Robles R, Lammle B, Furlan M.
Partial amino acid sequence of purified von
Willebrand factor-cleaving protease. Blood 2001;
98;1654-61.
Bandarenko N, Brecher ME, and members of
the US TTP Apheresis Study Group. United
States Thrombotic Thrombocytopenic Purpura Apheresis Study Group (US TTP ASG):
Multicenter survey and retrospective analysis
of current efficacy of therapeutic plasma exchange. J Clin Apheresis 1998;13:133-41.
Adans DM, Schultz WH, Ware RE, Kinney TR.
Erythrocytapheresis can reduce iron overload
and prevent the need for chelation therapy in
chronically transfused pediatric patients. J
Pediatr Hematol Oncol 1996;18:46-50.
Plasma Exchange/Sandoglobulin GuillainBarré Syndrome Trial Group. Randomized
trial of plasma exchange, intravenous immunoglobulin, and combined treatments in

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

Guillain-Barré syndrome. Lancet 1997;349:
225-30.
Nagpal S, Benstead T, Shumak K, et al. Treatment of Guillain-Barré syndrome: A cost-effectiveness analysis. J Clin Apheresis 1999;14:
107-13.
Guillain-Barré Syndrome Study Group.
Plasmapheresis and acute Guillain-Barré syndrome. Neurology 1985;35:1096-104.
vanDoorn PA, Vermeulen M, Brand A. Intravenous immunoglobulin treatment in patients with chronic inflammatory demyelinating polyneuropathy. Arch Neurol 1991;
48:217-20.
Dyck PJ, Low PA, Windebank AJ, et al. Plasma
exchange in polyneuropathy associated with
monoclonal gammopathy of undetermined
significance. N Engl J Med 1991;325:1482-6.
Simovic D, Gorson KC, Popper AH. Comparison of IgM-MGUS and IgG-MGUS polyneuropathy. Acta Neurol Scand 1998;97:194-200.
Frasca GM, Zoumparidis NG, Borgnino LC, et
al. Plasma exchange treatment in rapidly progressive glomerulonephritis associated with
anti-neutrophil cytoplasmic autoantibodies.
Int J Artif Organs 1992;3:181-4.
Pusey CD, Rees AJ, Evans JJ, et al. A randomized controlled trial of plasma exchange in
rapidly progressive glomerulonephritis without anti-GBM antibodies. Kidney Int 1991;40:
757-63.
Johnson WJ, Kyle RA, Pineda AA, et al. Treatment of renal failure associated with multiple
myeloma. Arch Intern Med 1990;150:863-9.
Guillevin L, Lhote F, Leon A, et al. Treatment
of polyarteritis nodosa related to hepatitis B
virus with short-term steroid therapy associated with antiviral agents and plasma exchanges: A prospective trial in 33 patients J
Rheumatol 1993;20:289-98.
Felson DT, LaValley MP, Baldassare AR, et al.
The Prosorba column for treatment of refractory rheumatoid arthritis: A randomized,
double-blind, sham-controlled trial. Arthritis
Rheum 1999;42:2153-9.
Kroon AA, Aengevaeren WRM, van der Werf T,
et al. LDL-apheresis atherosclerosis regression study (LAARS): Effect of aggressive versus conventional lipid lowering treatment on
coronary atherosclerosis. Circulation 1996;
93:1826-35.
Thompson GR, Maher VMG, Matthews S, et
al. Familial hypercholesterolemia regression
study: A Randomized trial of low-density-lipoprotein apheresis. Lancet 1995;345:811-16.
Bosch T, Lennertz A, Schenzle D, et al. Direct
adsorption of low density lipoprotein and lipoprotein (a) from whole blood: Results of
the first clinical long-term multicenter study

Chapter 6: Apheresis

68.

using DALI apheresis. J Clin Apheresis 2002;
17:161-9.
Handelsman H. Office of health technology
assessment report, No. 7. Protein A columns
for the treatment of patients with idiopathic
thrombocytopenic purpura and other indications. Rockville, MD: DHHS, PHS, Agency for
Health Care Policy and Research, 1991:1-8.

69.

70.

161

Gaddis TG, Guthrie TH, Drew MJ, et al. Treatment of refractory thrombotic thrombocytopenic purpura with protein A immunoadsorption. Am J Hematol 1997;55:55-8.
Lim HW, Edelson RL. Photopheresis for treatment of cutaneous T-cell lymphoma. Hematol Oncol Clin North Am 1995;9:1117-26.

Chapter 7: Blood Component Testing and Labeling

Chapter 7

Blood Component Testing
and Labeling

ACH DONOR UNIT must be tested
and properly labeled before its release for transfusion. Although the
scope and characteristics of donor tests
changed with the release of new tests and
the advent of new regulatory requirements, the intent of donor testing remains
constant: to enhance the safety of the
blood supply. This chapter presents the
general principles that apply to testing
and labeling donor blood, and it provides
a description of the specific tests that are
required or done voluntarily at most
blood banks on each donation. Discussion of the infectious complications of
blood transfusion is found in Chapter 28.
Other aspects of component preparation
are covered in Chapter 8.

Testing
General Requirements
Each laboratory needs to develop standard
operating procedures for the performance

of blood component testing strictly in
compliance with current instructions provided by the test manufacturers. Testing
must be performed in a planned, orderly
manner under a quality plan and a written set of procedures that instruct the
staff how to perform testing, under what
circumstances additional testing needs to
be done, and what to do if things go
wrong. The facilities and equipment must
be adequate for the activity being conducted. Access to the area must be limited. The environment must be controlled
so that temperature specifications for the
tests will be met, and the test will not be
adversely affected by the environment.
The test materials and equipment in use
must be those previously approved and
validated by the facility. If a facility uses
reagents or equipment from several different manufacturers, the facility is responsible for documentation that validation of
the equipment or reagent combination
for each test in use has occurred and that
staff have been trained on the most cur163

164

AABB Technical Manual

rent applicable instructions. For tests required by the Food and Drug Administration (FDA)1 and/or AABB Standards for
Blood Banks and Transfusion Services, all
reagents used must meet or exceed the re2(p9)
quirements of the FDA. If the manufacturer of a licensed test supplies controls,
they must be used for that test. However,
if these controls are used for calibration,
different controls must be used to verify
test performance. These controls may need
to be purchased separately. The manufacturer defines acceptable sample (specimen) requirements and considerations
that usually include the presence and nature of anticoagulant, the age of suitable
samples, and permissible storage intervals and conditions. Tests must be performed on a properly identified sample
from the current donation. Testing must
be completed for each blood donation before release. Each test result must be recorded concurrently with its observation;
interpretation is to be recorded only when
testing has been completed. Testing results must be recorded and records maintained so that any results can be traced for
a specific unit and/or component.
The facility should have a policy for notifying donors of positive infectious disease
test results. Test results are confidential and
must not be released to anyone (other than
the donor) without the donor’s written consent. At the time of donation, the donor
must be told if the policy is to release positive test results to state or local public
health agencies, and the donor must agree
to those conditions before phlebotomy. Donors must sign a consent form before they
donate blood acknowledging that the facility maintains a registry of donors who gave
disqualifying donor histories or have positive infectious disease results. The donor
must also be informed if the sample will
undergo research testing, including investigational new drug (IND) protocols. The

most problematic notifications are those in
which the donor has a false-positive test result. For some analytes [eg, antibodies to
hepatitis C virus (anti-HCV) or human immunodeficiency virus, types ½ (antiHIV-1/2)], confirmatory or supplemental
testing is routinely performed for donor
counseling purposes and possible donor
reentry, if applicable. In the case of a minor,
state and local laws apply.3

Required Tests
ABO group and Rh type must be determined
at each donation. A sample from each donation intended for allogeneic use must
be tested for the following1,2(p33):
Syphilis
■
Hepatitis B surface antigen (HbsAg)
■
HIV nucleic acid (individual or com■
bined HIV/HCV assay)
HCV nucleic acid (individual or
■
combined HIV/HCV assay)
■
Anti-HIV-1
■
Anti-HIV-2
■
Antibodies to hepatitis B core antigen (anti-HBc)
■
Anti-HCV
■
Antibodies to human T-cell lymphotropic viruses, types I/II (anti-HTLVI/II)
A combination test for anti-HIV-1/2 may
be used. A test for alanine aminotransferase
(ALT) is not required by the FDA or the
AABB. Recommendations for labeling units
associated with an elevated ALT have been
released by the FDA.4

Equipment Requirements
All equipment used for testing must be
properly calibrated and validated upon
installation, after repairs, and periodically.
There must be a schedule for planned
maintenance. All calibration, maintenance, and repair activities must be documented for each instrument. Software

Chapter 7: Blood Component Testing and Labeling

used to control the instrument or to interface with the institution’s computer system must also be properly validated.5

Records Requirements
Records must show each production step
associated with each blood component
from its source to its final disposition.6,7
Records must be kept in a manner that
protects the identity and personal information of the donor from discovery by
anyone other than the facility doing the
donor recruitment, qualification, and
blood collection, with the exception of
government agencies that require certain
test-positive results to be reported by law
for public health purposes. Testing records
on donor units must be kept in a manner
that makes it possible to investigate adverse consequences to a recipient. In addition, donor testing records must be suitable for look-back to previously donated
components when a donor’s blood gives
positive results on a new or improved infectious disease test.
Previous records of a donor’s ABO and D
typing results must be reviewed and compared with the ABO and D test findings on
the current donation. This is a very valuable
quality check on both the sample identity
correctness and the operation of the laboratory. If a discrepancy is found between
any current or historic test required, the
unit must not be released until there is unequivocal resolution of the discrepancy.2(p39)

ABO and D Testing
Every unit of blood intended for transfu2(p32),8
sion must be tested for ABO and D.
The ABO group must be determined by
testing donor red cells with reagent anti-A
and anti-B, and donor serum or plasma
with A1 and B red cells. The Rh type must
be determined by testing donor red cells
with anti-D serum. Red cells that are

165

nonreactive with anti-D in direct agglutination tests must be tested by a method
designed to detect weak D. Red cells that
react with anti-D either by direct agglutination or by the weak D test must be
labeled Rh positive. Red cells that are
nonreactive with anti-D by direct agglutination and the weak D test must be labeled Rh negative. Some of the automated
techniques have sufficient D sensitivity to
obviate the need for a weak D test. These
instruments add reagents, incubate reagent and sample appropriately, read the
reaction, and provide a result ready for interpretation. In addition, the automated
devices incorporate positive sample identification with the use of barcode readers
and use anticoagulated blood so that only
one tube is needed for both red cell and
plasma sampling. See Chapter 13 and
Chapter 14 for a more complete discussion of the principles of ABO and D testing.

Antibody Screening
Blood from donors with a history of transfusion or pregnancy must be tested for
unexpected antibodies. Because it is usually impractical to segregate blood that
should be tested from units that need not
be tested, most blood centers test all donor units for unexpected red cell antibodies. Donor serum or plasma may be tested
against individual or pooled reagent red
cells of known phenotypes. Methods must
be those that demonstrate clinically significant red cell antibodies. See Methods
Section 3 for antibody detection techniques and Chapter 19 for a discussion of
antibody detection.

Serologic Test for Syphilis
Serologic testing for syphilis (STS) has been
carried out on donor samples for over 50
years. Although experimental studies in

166

AABB Technical Manual

the 1980s showed that survival of spirochetes at 4 C is dependent on the concentration, it is not known how long the spirochete (Treponema pallidum) survives at
refrigerated temperatures in a naturally
9
infected blood component. The last reported transfusion transmitted case of
syphilis was reported in fresh blood com10
ponents in 1969.
The majority of screening tests for syphilis in US blood collection centers are microhemagglutinin or cardiolipin-based tests
that are typically automated. Donor units
testing positive for syphilis (STS) may not
be used for allogeneic transfusion. Results
can be confirmed before a donor is notified. Volunteer donors are much more
likely to have a false-positive test result
than a true-positive one.

Viral Marker Testing
Two screening methods are widely used
to detect viral antigens and/or antibodies.
The first is the enzyme-linked immunosorbent assay (EIA). The EIA tests for the
viral antigen HBsAg employ a solid support (eg, a bead or microplate) coated
with an unlabeled antiserum against the
antigen. The indicator material is the
same or another antibody, labeled with an
enzyme whose presence can be detected
by a color change in the substrate. If the
specimen contains antigen, it will bind to
the solid-phase antibody and will, in turn,
be bound by the enzyme-labeled indicator antibody. To screen for viral antibodies, ie, anti-HIV-1, anti-HIV-2, anti-HBc,
anti-HCV, or anti-HTLV-I/II, the solid
phase (a bead or microtiter well) is coated
with antigens prepared from the appropriate viral recombinant proteins or synthetic peptides. The second technology
for virus detection is based on nucleic
acid amplification and detection of viral
11
nuclear material. The RNA viruses being

tested for routinely are HIV and HCV. Two
experimental tests for West Nile virus
( WNV ) are undergoing study nationwide.12,13 Nucleic acid testing (NAT) for
HBsAg is undergoing clinical trials in
some centers.
In the capture approach frequently used
in assays, serum or plasma is incubated
with fixed antigen. If present, antibody
binds firmly to the solid phase and remains
fixed after excess fluid is washed away. An
enzyme-conjugated preparation of antigen
or antiglobulin is added; if fixed antibody is
present, it binds the labeled antigen or
antiglobulin, and the antigen-antibody-antigen (or antiglobulin) complex can be
quantified by measuring enzyme activity.
One assay for anti-HBc uses an indirect
capture method (competitive assay), in
which an enzyme-antibody conjugate is
added to the solid-phase antigen along
with the unknown specimen. Any antibody
present in the unknown specimen will
compete with the enzyme-conjugated antibody and significantly reduce the level of
enzyme fixed, compared with results seen
when nonreactive material is present. Antigens used in the viral antibody screening
tests may be made synthetically by recombinant technology or extracted from viral
particles.
NAT is a powerful but expensive technology that reduces the exposure window for
HIV and HCV by detecting very low numbers of viral copies after they appear in the
bloodstream. Primers for HCV and HIV viruses are placed in microplate wells, either
separately or in combination according to
the specific test design. In the use of pooled
sera, 16 to 24 donor samples are mixed and
tested. If viral RNA matching the fragments
already in the well is present in the donor
samples, heat-cycling nucleic acid amplification using a heat-cycling technique will
cause the viral fragments to multiply and
be easily detectable. The microplate with

Chapter 7: Blood Component Testing and Labeling

the aliquot of pooled sera is placed in the
well with the viral primers and substrate so
that if the primers and viral material in the
donor samples are the same, the primer
and viral particles will increase geometrically with each cycle. Viral presence can
then be detected reliably. When a pool is
found to be positive, all the individual samples making up the pool are tested separately for the individual viruses HIV and
HCV to find the positive donor sample. The
second major advantage of this test is the
relative lack of false-positive results as long
as sample and laboratory cross-contamination are controlled.12
For most of the assays, samples giving
nonreactive results on the initial screening
test, as defined by the manufacturer’s package insert, are considered negative and
need not be tested further. Samples that are
reactive on the initial screening test must
be repeated in duplicate. Reactivity in one
or both of the repeated tests constitutes a
positive result and is considered repeatedly
reactive. All components must be discarded
in this case. If both the duplicate repeat
tests are nonreactive, the test is interpreted
as having a negative result.
Before a donor is designated as antigenor antibody-positive, a status that may have
significant clinical and social consequences
and cause permanent exclusion from blood
donation, it is important to determine
whether the screening result is a true- or
false-positive result.

Invalidation of Test Results
In the course of viral marker testing, it
may be necessary to invalidate test results
if the test performance did not meet the
requirements of the manufacturer’s package insert (eg, faulty equipment, improper procedure, compromised reagents),
or if the control results do not meet the
acceptance criteria defined in the pack-

167

age insert. All results, both the reactive
and the nonreactive, obtained in the run
must be declared invalid; all specimens
involved must be tested in a new run,
which becomes the initial test of record.13
However, if the batch controls are acceptable and no error is recognized in test
performance, the reactive and nonreactive
results from the initial run remain as the
initial test of record for the specimens involved. Specimens with reactive results
must be retested in duplicate, as required
by the manufacturer’s instructions.
Before a test run is invalidated, the problems observed should be reviewed by a supervisor or equivalent, causes should be
analyzed, and corrective action should be
taken, if applicable. A record of departure
from normal standard operating procedures should be prepared, with a complete
description of the reason for invalidation
and the nature of corrective actions.13

Use of External Controls
Other considerations may need to be addressed before the invalidation of test results when external controls are used.13
Internal controls are the validation materials provided with the licensed assay kit;
they are used to demonstrate that the test
performs as expected. External controls
generally consist of at least one positive
control and one negative control. If the
negative control from the kit is used to
calculate the assay cutoff, it cannot be
used as an assay control reagent for testing. An external negative control should
be used in its place (see Table 7-1). External controls are frequently used to demonstrate the ability of the test to identify
weakly reactive specimens. External controls are surrogate specimens, either purchased commercially or developed by the
institution, that are not a component of
the test kit; they are used for surveillance

168

AABB Technical Manual

Table 7-1. Use of External Controls
Used to Calculate
Assay Cutoff?

Test Kit Reagents
Negative control only
Positive control only
Both positive and negative
controls

Yes
No
Yes
No
Yes

of test performance. External controls are
tested in the same manner as donor samples to augment blood safety efforts and
to alert the testing facility to the possibility of an increasing risk of error.
Before being entered into routine use,
external controls must be qualified, lot by
lot, because each control lot may vary with
the test kit. One way to qualify an external
control is:
1.
Run the external control for 2 days,
four replicates per day, using two to
three different test kit lots. The performance of the external controls
must meet specified requirements
before use.
2.
If the external controls do meet the
specifications, the acceptable sample-to-cutoff ratio for the external
control (eg, within three standard
deviations of the mean) must be
determined.
Additional qualification testing must be
performed whenever a new lot of test kits
or external controls is introduced.
When a change of test kit lot occurs, replicates (eg, 20 replicates) of the external
control should be run with the current kit
lot and the new lot. The new sample-tocutoff ratio and limits should be determined.
Users should verify special requirements
for external control handling in pertinent

External Controls Required?
Yes–negative control
No
Yes–positive control
No
Yes–positive and negative
controls

state, federal, and international regulations,
as applicable.
A facility may invalidate nonreactive test
results on the basis of external controls, but
if the assay was performed in accordance
with manufacturer’s specifications and the
internal controls performed as expected,
external controls cannot be used to invalidate reactive test results. The use of external
controls may be more stringent than, but
must be consistent with, the package insert’s criteria for rejection of test results.
Observation of donor population data,
such as an unexpectedly increased reactive
rate within a test run, may cause nonreactive results to be considered invalid.
The next assay, performed on a single
aliquot from affected specimens, becomes
the initial test of record for those samples
nonreactive in the invalidated run. However, reactive results obtained in a run with
an unexpectedly increased reactive rate
may not be invalidated unless the entire
run fails to meet the performance criteria
specified in the package insert. Such reactive results remain the initial test of record.
The samples must be tested in duplicate as
the repeat test.
External controls may also be used to invalidate a duplicate repeat test run when an
assay run is valid by test kit acceptance criteria and both the repeated duplicate tests
are nonreactive. The duplicate samples

Chapter 7: Blood Component Testing and Labeling

may be repeated in duplicate; the second
duplicate test becomes the test of record. If
either of the original duplicate repeat tests
is reactive, the donor(s) must be classified
as repeatedly reactive and no further repeat
screening tests should be performed.
When samples are reactive on the initial
screening test, allogeneic donor units must
be quarantined until the results of duplicate repeat testing are available. Components associated with repeatedly reactive
test results must not be used for allogeneic
transfusion. Supplemental or confirmatory
testing may be performed on samples that
are repeatedly reactive to obtain additional
information for donor counseling and possible reentry, depending on the viral marker
and availability of approved assays.

Supplemental Tests: Neutralization
In confirmatory antigen neutralization
tests (eg, HBsAg), the reactive specimen is
incubated with human serum known to
contain antibody specific for the antigen
in question. If incubation causes the positive reaction to disappear or to diminish
by at least 50% (or the percentage specified in the package insert) and all controls
behave as expected, the presence of antigen is confirmed and the original result is
considered a true positive. If incubation
with a known antibody does not affect
subsequent reactivity, the original reactivity is considered a false-positive result.
Known positive and negative control samples must be tested in parallel with donor
or patient samples. Parallel incubations
must be performed with a preparation
known to contain antibody specific for
the antigen in question and with a preparation known to be free of both antigen
and antibody. If the positive and negative
control values do not fall within limits
stated in the package insert, the test must
be repeated.

169

Supplemental Test for EIA-Reactive
Anti-HIV-1/2, -HTLV I/II, and -HCV Tests
Western blot is the method most frequently used for the confirmation of repeatedly reactive anti-HIV EIA tests. The
technique separates antigenic viral material into bands according to molecular
weight. The material is transferred to
nitrocellulose membranes. Antibody in
the test serum reacts with the individual
bands, depending on the specificity.
Most persons infected with HIV, whether
asymptomatic or exhibiting AIDS, show
multiple bands, representing antibodies
to virtually all of the various gene products. A fully reactive test serum should react with the p17, p24, and p55 gag proteins; the p31, p51, and p66 pol proteins;
and the gp41, gp120, and gp160 env
glycoproteins. Western blot results in
EIA-reactive blood donor samples are
classified as positive, negative, or indeterminate. Positive results are those with reactivity to at least two of the following HIV
proteins: p24, core protein; gp41, transmembrane protein; and gp120/160, external protein and external precursor protein. Indeterminate results are those with
other patterns of reactivity.
An immunofluorescence assay (IFA) is
used in some blood centers as an alternative to Western blot testing. Cells infected
with virus are fixed on a slide. The sample is
incubated with the fixed cells. Antibody in
the sample will bind to the antigen sites on
the viral particles. The reaction mixture is
incubated with fluorescent-labeled antihuman IgG. Following incubation and
washing, binding of the labeled antihuman
IgG is read using a fluorescence microscope, with subsequent interpretation of
the fluorescence pattern.
Although there are FDA-approved Western blot confirmatory tests for anti-HIV, the
Western blot test using recombinant DNA

170

AABB Technical Manual

and viral lysate antigens for anti-HTLV-I/II
has not been approved by the FDA. An appropriate supplemental test to confirm a
reactive anti-HTLV test result is to repeat
the test using another manufacturer’s EIA
test. If that test is repeatedly reactive, the
result is considered confirmed. If the test is
negative, the anti-HTLV test result is
considered a false-positive result.
The FDA has approved a recombinant
immunoblot assay (RIBA) system to further
differentiate anti-HCV EIA repeatedly reactive samples. The RIBA system is based on
the fusion of HCV antigens to human
superoxide dismutase in the screening test
and to a recombinant superoxide dismutase in the confirmatory test to detect nonspecific reactions. A positive result requires
reactivity to two HCV antigens and no reactivity to superoxide dismutase. Reactivity to
only one HCV antigen or to one HCV antigen and superoxide dismutase is classified
as an indeterminate reaction. Results are
usually presented as positive, negative, or
indeterminate. As with all procedures, it is
essential to follow the manufacturer’s instructions for classification of test results.
The use of nucleic acid amplification testing is discussed in Chapter 28.

Cytomegalovirus Testing
Optional tests may be performed on units
intended for recipients with special needs.
For example, cytomegalovirus (CMV )
testing is a commonly performed optional
test. CMV can persist in the tissues and
leukocytes of asymptomatic individuals
for years after initial infection. Blood from
persons lacking antibodies to the virus
has reduced risk of transmitting infection
compared with untested (nonleukocytereduced) units. Only a small minority of
donor units with positive test results for
anti-CMV will transmit infection. However,
there is presently no way to distinguish

infective antibody-positive units from
noninfective units containing anti-CMV.
Routine testing for anti-CMV is not required by AABB Standards,2(p43) but, if it is
performed, the usual quality assurance
considerations apply. The most common
CMV antibody detection methods in use
are EIA and latex agglutination. Other
methods, such as indirect hemagglutination, complement fixation, and immunofluorescence, are also available.

Labeling, Records, and
Quarantine
Labeling
Labeling is a process that includes a final
review of records of donor history, collection, testing, blood component modification, quality control functions, and any
additional information obtained after donation. This also includes a review of labels attached to the components and checks
to ensure that all labels meet regulatory
requirements and are an accurate reflection of the contents of the blood or blood
components.1,8
All labeling of blood components must
be performed in compliance with AABB
Standards2(p12) and FDA regulations. Blood
centers and transfusion services must ensure that labeling is specific and controlled.
Before the labeling process begins, there
should be a mechanism or procedure in
place that ensures the use of appropriate
labels. This process should include assurance of acceptable label composition, inspection on receipt, secure storage and distribution of labels, archiving of superseded
labels, and availability of a master set of labels in use. In addition, procedures should
address generation of labels, changes in labels, and modification of labels to reflect la-

Chapter 7: Blood Component Testing and Labeling

bel control of altered or new components.
Labels should be checked for the proper
product code for the component being labeled, which is based on collection method,
anticoagulant, and modifications to the
component during processing. All aspects
of labeling (the bag label as well as the Circular of Information for the Use of Human
Blood and Blood Components,14 including
the label size, type size, wording, spacing,
and the base label adhesive) are strictly
controlled.

ISBT 128
The ISBT 128 labeling system is an internationally defined system based on barcode symbology called Code 128. It standardizes the labeling of blood so that
bar-coded labels can be read by blood
centers and transfusion services around
the world. The system allows for each
number assigned to a unit of blood (blood
identification number) to be unique. The
unique number will allow tracking of a
unit of blood from donor to recipient, regardless of where the unit was drawn or
transfused. As outlined in the United States
Industry Consensus Standard for the Uniform Labeling of Blood and Blood Components Using ISBT 128,15 the information
appearing on the label, the location of the
label, and the exact wording on the label
are standardized.
ISBT 128 differs from its predecessor,
which used CODABAR symbology, by including more specific information on the
label. One advantage of the standardized
system is that additional information on
the label allows for better definition of
product codes. Other changes include an
expanded donation identification number
to include the collection facility’s identification; bar-coded manufacturer’s lot number,
bag type, etc; bar-coded expiration date;
and special testing barcode. A benefit will

171

be that the use of standardized computergenerated barcode labels (with better differentiation between components, preparation methods, and expiration dates) enhances efficiency, accuracy, and ultimately
safety of labeled components. For example,
the ISBT 128 label shows conspicuously
that an autologous, biohazard unit is not a
standard unit by making the blood type a
different, smaller size and filling the space
usually reserved for blood type with a biohazard symbol. Adherence to the guidelines
ensures compliance with AABB standards2(p12)
and FDA regulations. The United States
blood banking community has recently
been prompted by a general directive from
the Secretary of Health and Human Services and a subsequent proposal from the
FDA for uniform acceptance of this more
comprehensive labeling system. Until the
new international guidelines are implemented, the 1985 FDA Uniform Labeling
Guideline remains in effect. More information on ISBT 128 is available from the International Council on Commonality in Blood
Banking Automation at www.iccbba.com.

Label Requirements
The following pieces of information are
required2(p12),16,17 in clear readable letters on
a label firmly attached to the container of
all blood and component units:
■
The proper name of the component,
in a prominent position.
■
A unique numeric or alphanumeric
identification that relates the original unit to the donor and each component to the original unit.
■
For components, the name, address,
and FDA license number or registration number (whichever is appropriate) of the facility that collected the
blood and/or the component. The label must include the name and location of all facilities performing any

172

■

■
■

■

■
■
■

■

AABB Technical Manual

part of component manufacturing.
This includes facilities that wash, irradiate, and reduce leukocytes by filtration. If a process is performed
under contract, and the process is
performed under processes controlled by the contracting facility,
only that facility’s name is required
in this case. There should not be
more than two alphanumeric identifiers on the unit.
The expiration dates, including the
date and year; if the shelf life is 72
hours or less, the hour of expiration
must be stated.
The amount of blood collected.
The kind and quantity of anticoagulant (not required for frozen, deglycerolized, rejuvenated, or washed red
cells).
For all blood and blood components,
including pooled components, the
approximate volume of the component must appear on the container.
Recommended storage temperature.
ABO group and Rh type.
Interpretation of unexpected red cell
antibody tests for plasma-containing
components when positive (not required for cryoprecipitate or frozen,
deglycerolized, rejuvenated, or washed
RBCs).
Results of unusual tests or procedures
performed when necessary for safe
and effective use. Routine tests performed to ensure the safety of the
unit need not be on the label if they
are listed in the Circular of Information for the Use of Human Blood and
Blood Components.14
Reference to the Circular of Information for the Use of Human Blood and
Blood Components,14 which must be
available for distribution, and contains information about actions, indications, contraindications, dosage,

■

administration, side effects, and
hazards.
Essential instructions or precautions
for use, including the warning that
the component may transmit infectious agents, and the statements: “Rx
only” and “Properly Identify Intended Recipient.”
The appropriate donor classification
statement—“autologous donor,” “paid
donor,” or “volunteer donor”—in
type no less prominent than that
used for the proper name of the
component.
Any additives, sedimenting agents,
or cryoprotective agents that might
still be present in the component.

Special Labeling
■

■

Cellular blood components issued as
“Leukocytes Reduced” must be labeled as such.
The name and final volume of the
component and a unique identifier
for a pool must appear on all pooled
components.
The number of units in a pool and
their ABO group and Rh type must
be on the label or an attached tie tag.
Identification numbers of the individual units in a pool should not be
on the label but must be in the records of the facility preparing the
pool.
Cellular blood components issued as
“CMV negative” must be labeled as
such.
Irradiated blood components must
carry the appropriate irradiated label.

Records
Current good manufacturing practice regulations, as defined by Title 21 CFR Parts
200 and 600,6,7,16,18 state that master production and control records must be a

Chapter 7: Blood Component Testing and Labeling

part of the labeling process. These records
must be described in the facility’s procedures. Before labeling, these records must
be reviewed for accuracy and completeness. Appropriate signatures and dates
(either electronic or manual) must document the review process.

■

173

Transfer of those results must be
performed by a system that properly
identifies test results to all appropriate blood and blood components.
Quarantine: that any nonconforming
unit is appropriately isolated.

Production Records
Control Records
Control records include but may not be
limited to:
Donation process: that all questions
■
are answered on the donor card,
consent is signed, all prequalifying
tests are acceptable (eg, hemoglobin, blood pressure), and a final review is documented by qualified
supervisory personnel.
Infectious disease testing: if per■
formed at the collecting facility, that
tests have acceptable quality control
and performance; that daily equipment maintenance was performed
and was acceptable; and that final
results are reviewed to identify the
date and person performing the
review.
■
Donor deferral registry: That the list
of deferred donors has been checked
to ensure that the donor is eligible.
■
Component preparation: that all
blood and blood components were
processed and/or modified under
controlled conditions of temperature and other physical requirements of each component.
■
Transfer of records: if testing is performed at an outside facility, that all
records of that facility are up to date
and that the appropriate licensure is
indicated. Records, either electronic
or manual, must transfer data appropriately. All electronically transferred test records must be transmitted
by a previously validated system.

Master production records must be traceable back to:
Dates of all processing or modifica■
tion.
Identification of the person and equip■
ment used in the process steps.
Identification of batches and in-pro■
cess materials used.
Weights and measures used in the
■
course of processing.
In-laboratory control results (tem■
peratures, refrigerator, etc).
Inspection of labeling area before
■
and after use.
■
Results of component yield when
applicable.
■
Labeling control.
■
Secondary bag and containers used
in processing.
■
Any sampling performed.
■
Identification of person performing
and checking each step.
■
Any investigation made on nonconforming components.
■
Results of examinations of all review
processes.

Quarantine
Before final labeling, there must be a process to remove nonconforming blood and
blood components from the labeling process until further investigation has occurred. This process must be validated to
capture and isolate all blood and blood
components that do not conform to requirements in any of the critical areas of
collecting, testing, and processing. This

174

AABB Technical Manual

process must also capture verbal (eg, telephone calls) information submitted to the
collection facility after the collection process. All nonconforming units must remain in quarantine until they are investigated and all issues are resolved. The units
may then be discarded, labeled as nonconforming units (eg, autologous units),
or labeled appropriately for transfusion if
the investigation resolved the problems. If
the nonconformance cannot be resolved
and the units are from an allogeneic donation, they must be discarded.

10.

11.

12.

13.

References
1.

Code of federal regulations. Title 21 CFR
610.40. Washington, DC: US Government
Printing Office, 2004 (revised annually).
Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.
Dodd RY, Stramer SL. Indeterminate results
in blood donor testing: What you don’t know
can hurt you. Transfus Med Rev 2000;14:151-9.
Food and Drug Administration. Memorandum: Recommendations for labeling and use
of units of whole blood, blood components,
source plasma, recovered plasma or source
leukocytes obtained from donors with elevated levels of alanine aminotransferase
(ALT). (August 8, 1995) Rockville, MD: CBER
Office of Communication, Training, and
Manufacturers Assistance, 1995.
Code of federal regulations. Title 21 CFR
606.60. Washington, DC: US Government
Printing Office, 2004 (revised annually).
Code of federal regulations. Title 21 CFR
606.160. Washington, DC: US Government
Printing Office, 2004 (revised annually).
Code of federal regulations. Title 21 CFR
606.165. Washington, DC: US Government
Printing Office, 2004 (revised annually).
Code of federal regulations. Title 21 CFR
640.5. Washington, DC: US Government
Printing Office, 2004 (revised annually).
van der Sluis JJ, ten Kate FJ, Vuzevski VD, et
al. Transfusion syphilis, survival of Treponema pallidum in donor blood. II. Dose
dependence of experimentally determined
survival times. Vox Sang 1985;49:390-9.

14.

15.

16.

17.

18.

Chambers RW, Foley HT, Schmidt PJ. Transmission of syphilis by fresh blood components. Transfusion 1969;9:32-4.
Busch MP, Stramer SL, Kleinman SH. Evolving
applications of nucleic acid amplification assays for prevention of virus transmission by
blood components and derivatives. In:
Garratty G, ed. Applications of molecular biology to blood transfusion medicine. Bethesda,
MD: AABB, 1997:123-76.
Vargo K, Smith K, Knott C, et al. Clinical specificity and sensitivity of a blood screening assay for detection of HIV-1 and HCV RNA.
Transfusion 2002;42:876-85.
Food and Drug Administration. Guidance for
industry. Revised recommendations regarding invalidation of test results of licensed and
510(k)-cleared blood-borne pathogen assays
used to test donors. (July 11, 2001) Rockville,
MD: CBER Office of Communication, Training, and Manufacturers Assistance, 2001.
AABB, American Red Cross, and America’s
Blood Centers. Circular of information for the
use of human blood and blood components.
Bethesda, MD: AABB, 2002.
Food and Drug Administration. Guidance for
Industry: United States industry consensus
standard for the uniform labeling of blood
and blood components using ISBT 128, Version 1.2.0. (November 28, 1999) Rockville,
MD: CBER Office of Communication, Training, and Manufacturers Assistance, 1999.
Code of federal regulations. Title 21 CFR
606.210, and 606.211. Washington, DC: US
Government Printing Office, 2004 (revised
annually).
Food and Drug Administration. Guidelines
for the uniform labeling of blood and blood
components. (August 1985) Rockville, MD:
CBER Office of Communication, Training and
Manufacturers Assistance, 1985.
Code of federal regulations. Title 21 CFR Part
210, 211 and 606. Washington, DC: US Government Printing Office, 2004 (revised annually).

Suggested Reading
AABB, American Red Cross, and America’s Blood
Centers. Circular of information for the use of human blood and blood components. Bethesda, MD:
AABB, 2002.

Chapter 8: Components from Whole Blood Donations

Chapter 8

Collection, Preparation,
Storage, and Distribution of
Components from Whole
Blood Donations

ONOR CENTERS AND transfusion
services share a common goal in
blood component production: to
provide a safe and efficacious component
that benefits the intended recipient. To this
end and in keeping with Food and Drug
Administration (FDA) current good manufacturing practice regulations, all processes involved in the collection, testing,
preparation, storage, and transport of blood
and components are monitored for quality, including procedures, personnel, reagents, equipment, and the contents of the
components themselves. Processes should
ensure the potency and purity of the final
product, minimize microbial contamination and proliferation, and prevent or delay the detrimental physical and chemical
changes that occur when blood is stored.

Blood Component
Descriptions
Readers should refer to Chapters 21, 23, and
24 and the current Circular of Information
for the Use of Human Blood and Blood
Components 1 for more detailed indications and contraindications for transfusion.

Whole Blood
Fresh Whole Blood contains all blood elements plus the anticoagulant-preservative
in the collecting bag. It is used commonly
as a source for component production.
After 24-hour storage, it essentially becomes red cells suspended in a protein
solution equivalent to liquid plasma, with
a minimum hematocrit of approximately
33%.
175

176

AABB Technical Manual

Red Blood Cells
Red Blood Cells (RBCs) are units of Whole
Blood with most of the plasma removed
(see Method 6.4). If prepared from whole
blood collected into citrate-phosphatedextrose (CPD), citrate-phosphate-dextrosedextrose (CP2D), or citrate-phosphatedextrose-adenine (CPDA-1), the final
hematocrit must be ≤80%. Additive red
cell preservative systems consist of a primary collection bag containing an anticoagulant-preservative with at least two satellite bags integrally attached; one is
empty and one contains an additive solution (AS). AS contains sodium chloride,
dextrose, adenine, and other substances that
support red cell survival and function up
2
to 42 days (see Table 8-1). The volume of
the AS in a 450-mL collection set is 100
mL and the volume in 500-mL sets is 110
mL. AS is added to the red cells remaining
in the primary bag after most of the
plasma has been removed. This allows
blood centers to use or recover a maximum amount of plasma, yet still prepare
a red cell component with a final hematocrit between 55% and 65%, a level that facilitates excellent flow rates and allows
easy administration.

RBCs can be prepared at any time during
their shelf life, but AS must be added within
the time frame specified by the manufacturer, generally within the first 72 hours of
storage. Shelf life at 1 to 6 C storage depends on the anticoagulant-preservative
used and the method of preparation.

Platelets
Platelet concentrates (Platelets) are prepared
from units of whole blood that have not
been allowed to cool below 20 C. Platelet-rich plasma (PRP) is separated within
4 hours after completion of the phlebotomy or within the time frame specified in
the directions for the use of the blood collecting, processing, and storage system—
typically 8 hours.3 The platelets are concentrated by an additional centrifugation step
and the removal of most of the supernatant plasma. A procedure for preparation
of platelets from single units of whole
blood appears in Method 6.13. The final
component should contain resuspended
platelets in an amount of plasma adequate to maintain an acceptable pH; generally, 40 to 70 mL is used. Although not
approved in the United States, platelet
concentrates are commonly manufac-

Table 8-1. Content of Additive Solutions (mg/100 mL)
AS-1
(Adsol)

AS-3
(Nutricel)

AS-5
(Optisol)

2200

1100

900

276

Mannitol

750

525

Sodium chloride

900

410

877

Sodium citrate

588

Citric acid

Dextrose
Adenine
Monobasic sodium phosphate

Chapter 8: Components from Whole Blood Donations

tured in Europe using buffy coat as an intermediate product. In this schema, the
initial centrifugation is a “hard-spin” in
which the platelets are concentrated in the
buffy coat. The supernatant platelet-poor
plasma and the red cells can be expressed
using a top-and-bottom device. The buffy
coats can then be centrifuged in a “softspin” to remove the white cells or, more
commonly, pooled before storage (not
currently allowed by the FDA), then softspun as a pooled concentrate, with ex4-6
pression of the PRP.

Plasma
Plasma in a unit of Whole Blood can be
separated at any time during storage, up
to 5 days after the expiration date of the
Whole Blood. When stored frozen at –18 C
or colder, this component is known as
Plasma and can be used up to 5 years after the date of collection. If not frozen, it
is called Liquid Plasma, which is stored at
1 to 6 C and transfused up to 5 days after
the expiration date of the Whole Blood
from which it was prepared.
Fresh Frozen Plasma (FFP) is plasma
prepared from whole blood, either from the
primary centrifugation of whole blood into
red cells and plasma or from a secondary
centrifugation of PRP. The plasma must be
3
frozen within 8 hours of collection. See
Methods 6.10 and 6.13.
Blood centers often convert plasma and
liquid plasma to an unlicensed component,
“Recovered Plasma (plasma for manufacture),” which is usually shipped to a fractionator and processed into derivatives
such as albumin and/or immune globulins.
To ship recovered plasma, the collecting facility must have a “short supply agreement”
with the manufacturer.7 Because recovered
plasma has no expiration date, records for
this component must be retained indefinitely.

177

See further discussion of additional
plasma products later in the chapter.

Cryoprecipitated AHF
Cryoprecipitated antihemophilic factor
(AHF) is the cold-insoluble portion of
plasma that precipitates when FFP is
thawed between 1 to 6 C. It is essentially a
concentrate of high-molecular-weight
glycoproteins also known as CRYO. This
component is prepared from a single
Whole Blood unit collected into CPDA-1,
CPD, or CP2D and suspended in less than
15 mL of plasma. It contains ≥80 IU Factor
VIII (AHF), >150 mg of fibrinogen, and
most of the Factor XIII originally present
in the fresh plasma. CRYO contains both
the procoagulant activity (Factor VIII) and
the von Willebrand factor of the Factor
VIII von Willebrand complex.
Once separated, CRYO is refrozen within
1 hour of preparation and stored at –18 C or
colder for up to 1 year after the date of phlebotomy. See Method 6.11 for a preparation
procedure.

Plasma Cryoprecipitate Reduced
If cryoprecipitate has been removed from
plasma, this must be stated on the label.
When stored at –18 C or colder, this component has a 12-month expiration date
from the date of collection.8(p59) This component is used primarily in the treatment
of thrombotic thrombocytopenic purpura.9

Granulocytes
Granulocytes are usually collected by apheresis techniques; however, buffy coats harvested from fresh Whole Blood units can
9
provide a source (<1 × 10 ) of granulocytes
in urgent neonatal situations. Their effectiveness is controversial, however.10 Granulocytes should be transfused as soon as
possible after collection but may be
stored at 20 to 24 C without agitation for

178

AABB Technical Manual

up to 24 hours. Arrangements for precollection testing are often useful. This
product is becoming obsolete.

Collection
Blood component quality begins with a
healthy donor and a clean venipuncture
site to minimize bacterial contamination.
To prevent activation of the coagulation
system during collections, blood should
be collected rapidly and with minimal
trauma to tissues. Although the target collection time is usually 4 to 10 minutes,
one study has shown platelets and fresh
frozen plasma to be satisfactory after collection times of up to 15 minutes.11 The facility’s written procedures should be followed regarding these collection times.
There should be frequent, gentle mixing
of the blood with the anticoagulant. If prestorage filtration is not intended after collection, the tubing to the donor arm may be
stripped into the primary collection bag, allowed to fill, and segmented, so that it will
represent the contents of the donor bag for
compatibility testing. Blood is then cooled

toward 1 to 6 C unless it is to be used for
room temperature component production,
in which case it should be cooled toward,
but not below, 20 C. Chapter 4 discusses
blood collection in detail.

Anticoagulants and Preservatives
Whole blood is collected into a bag that
contains an FDA-approved anticoagulantpreservative solution designed to prevent
clotting and to maintain cell viability and
function during storage. Table 8-2 compares some common solutions. Citrate
prevents coagulation by chelating calcium, thereby inhibiting the several calcium-dependent steps of the coagulation
2
cascade.
The FDA approves 21-day storage at 1 to
6 C for red cells from whole blood collected
in CPD and CP2D and 35-day storage for
red cells collected in CPDA-1.12 Most blood
centers now collect up to 500 mL ± 50 mL
(450-550 mL) whole blood in bags specifically designed for this larger volume. Blood
bags intended for a collection volume of
450 mL ± 45 mL of whole blood (ie, 405-495
mL) contain 63 mL of anticoagulant-pre-

Table 8-2. Anticoagulant-Preservative Solutions (mg in 63 mL) for 450 mL
Collections
CPD
Ratio (mL solution to blood)
FDA-approved shelf life (days)

1.4:10

CP2D
1.4:10

CPDA-1
1.4:10

1660

Citric acid

206

Dextrose

1610

3220

2010

140

Content
Sodium citrate

Monobasic sodium phosphate
Adenine

With 500 mL collections, the volume is 70 mL and the content 10% to 11% higher.

17.3

Chapter 8: Components from Whole Blood Donations

servative. The volume of anticoagulant-preservative in 500 mL ±50 mL bags is 70 mL.
The allowable range of whole blood collected is dependent upon the collection bag
selected and can vary with manufacturer,
but the total amount collected including
testing samples must not exceed 10.5
mL/kg donor weight per donation.
If only 300 to 404 mL of blood is collected into a blood bag designed for a
450-mL8(p28) collection, the red cells may be
used for transfusion provided the unit is labeled “Red Blood Cells Low Volume.” However, other components should not be prepared from these low-volume units. If a
whole blood collection of less than 300 mL
is planned, the volume of anticoagulantpreservative solution in that bag should be
reduced proportionately (see Chapter 4 for
calculations), to ensure that the correct
amount of anticoagulant is used (ratio of
anticoagulant: whole blood).

Transportation from a Collection Site
Whole blood should be transported from
the collection site to the component preparation laboratory as soon as possible.
Units should be cooled toward 1 to 6 C
unless platelets are to be harvested, in
which case, units should be cooled toward,
but not below, 20 C. The time between
collection and the separation of components must not exceed the time frame
specified in the directions for use of the
blood collection,3 processing, and storage
system.

Prestorage Processing
Differential Centrifugation
To simplify the separation of whole blood
into its component parts, blood is collected
into primary bags to which up to three
satellite bags are attached.5 Set design is

179

based on intended use: RBCs, platelets,
FFP, Cryoprecipitated AHF, or neonatal
aliquots. Refer to Methods Section 6 for
specific component preparation procedures. Whole blood must be separated
and prepared components placed into their
required storage temperatures within the
anticoagulant-preservative manufacturer’s
3
recommended times of collection. Records of component preparation should
identify each individual performing a significant step in processing.
Because red cells, platelets, and plasma
have different specific gravities, they are
separated using differential centrifugation.13
Rotor size, speed, and duration of spin are
critical variables in centrifugation. Method
7.4 describes how to calibrate a centrifuge
for platelet separation, but each centrifuge
must be calibrated for optimal speeds and
spin times for each combination of components prepared in like fashion and for each
different type of collection bag. Times include the time of acceleration and “at
speed,” not deceleration time. Once the operating variables are identified for component production, timer accuracy, rpm, and
temperature (if appropriate), centrifuges
should be monitored periodically to verify
equipment performance.
Another practical way to assess centrifugation is to monitor quality control data on
components prepared in each centrifuge. If
component quality does not meet defined
standards, eg, if platelet concentrate yields
are inconsistent, the entire process should
be evaluated. Factors affecting the quality
assessment are the calibration of the centrifuge, the initial platelet count on the whole
blood donations, storage time, conditions
between blood collection and platelet preparation, sampling technique, and counting
methods.
Large centrifuges rotate at high speeds,
exerting gravitational forces of thousands of
pounds on blood bags. Weaknesses in these

180

AABB Technical Manual

blood bags or the seals between tubing segments can cause rupture and leakage. The
addition of filters for blood sets presents
different challenges for cup insertion.
Blood bags may be overwrapped with plastic bags to contain any leaks. Bags should
be positioned so that a broad surface faces
the outside wall of the centrifuge to reduce
the centrifugal force on blood bag seams.
Contents in opposing cups of the centrifuge must be equal in weight to improve
centrifuge efficiency and prevent damage to
the rotor. Dry balancing materials are preferable to liquid material. Weighted rubber
discs and large rubber bands are excellent
and come in several thicknesses to provide
flexibility in balancing. Swinging centrifuge
cups provide better separation between cells
and plasma than fixed-angle cups.

the specific filter in use. This method may
be preferable if special units are to be selected for leukocyte reduction.
During inline filtration of red cells, platelets and/or plasma are first removed from
the whole blood donation and the additive
solution is transferred to the red cells. The
red cells are then filtered through an inline
filter into a secondary storage container.
This filtration step should occur as early in
the shelf life as possible and within the allowed time frame for the specific filter in
use.
Leukocyte-reduced platelets may be prepared from PRP using inline leukocyte reduction filtration.14 FFP manufactured using
this intermediate step typically will have a
leukocyte content of <5 × 106.

Freezing
Testing and Labeling of Donor Units
Chapter 7 contains detailed information
on testing and labeling blood components.

Filtration
During inline filtration of whole blood, the
anticoagulated whole blood is filtered by
gravity through an inline leukocyte reduction filter contained in the collection system. The filtered whole blood may be
manufactured into leukocyte-reduced
RBCs. Whole blood leukocyte reduction
filters retain the platelets to a variable degree, so platelet concentrates cannot be
routinely prepared. However, newly designed platelet sparing filters are under
investigation. This filtration should occur
within the time specified by the filter
manufacturer.
A leukocyte reduction filter can be attached to a unit of Whole Blood or RBCs
using a sterile connection device. Ideally,
such filtration should occur as early as possible after collection but must conform to
the manufacturer’s recommendations for

Acellular Components
When stored at –18 C or colder, FFP contains maximum levels of labile and nonlabile clotting factors (about 1 IU per mL)
and has a shelf life of 12 months from the
date of collection. FFP frozen and maintained at –65 C may be stored up to 7 years.
See Method 6.10 for preparation details.
Plasma can be rapidly frozen by placing the
bag 1) in a dry ice-ethanol or dry ice-antifreeze bath, 2) between layers of dry ice, 3)
in a blast freezer, or 4) in a mechanical
freezer maintained at –65 C or colder.
Plasma frozen in a liquid bath should be
overwrapped with a plastic bag to protect
the container from chemical alteration.
When a mechanical freezer is used, care
must be taken to avoid slowing the freezing
process by introducing too many units at
one time.
It is prudent practice to use a method to
facilitate detection of inadvertent thawing
of plasma during storage, such as:
1.
Pressing a tube into the bag during
freezing to leave an indentation that

Chapter 8: Components from Whole Blood Donations

disappears if the unit thaws. Remove
tube(s).
2.
Freezing the plasma bag in a flat,
horizontal position but storing it upright. Air bubbles trapped along the
bag’s uppermost broad surface during freezing will move to the top if
the unit is thawed in a vertical position.
3.
Placing a rubber band around the
liquid plasma bag and removing it
after freezing to create an indentation that disappears with thawing.
Plasma separated and frozen at –18 C
between 8 and 24 hours (eg, plasma that
does not meet the stricter time requirements of FFP) may be labeled as “Plasma
Frozen Within 24 Hours after Phlebotomy.”
It contains all the stable proteins found in
FFP (see Table 21-3). FFP thawing guidelines apply.

Cellular Components
Frozen storage can significantly extend the
shelf life of red cell components. Unfortunately, the process can also cause cell damage and add considerable expense.
Effects of Freezing and Thawing. When
unprotected cells are frozen, damage may
result from cellular dehydration and from
mechanical trauma caused by intracellular
ice crystals. At rates of freezing slower than
10 C/minute, extracellular water freezes before intracellular water, producing an osmotic
gradient that causes water to diffuse from
inside the cell to outside the cell. This leads
to intracellular dehydration. Controlling the
freezing rate, however, is not sufficient by
itself to prevent cellular damage, so cryoprotective agents must be used.
Cryoprotective agents are classified as
penetrating and nonpenetrating. Penetrating agents such as glycerol are small molecules that freely cross the cell membrane

181

into the cytoplasm. The intracellular cryoprotectant provides an osmotic force that
prevents water from migrating outward as
extracellular ice is formed. A high concentration of cryoprotectant prevents formation of ice crystals and consequent membrane damage.15 Glycerol, a trihydric alcohol,
is a colorless, sweet-tasting, syrup-like fluid
that is miscible with water. Pharmacologically, glycerol is relatively inert.
Nonpenetrating cryoprotective agents
(eg, hydroxyethyl starch) are large molecules that do not enter the cell. The molecules protect the cells by a process called
“vitrification” because they form a noncrystalline “glassy” shell around the cell.
This prevents loss of water and dehydration
injury and alters the temperature at which
the solution undergoes transition from
liquid to solid.
Freezing of RBCs. Frozen preservation of
RBCs with glycerol is primarily used for
storing units with rare blood types and
autologous units. Frozen cells can be effectively stockpiled for military mobilization
or civilian disasters, but the high cost and
the 24-hour shelf life after deglycerolization
make them less useful for routine inventory
management. Recently, an effectively closed
system was approved with a 2-week postthaw shelf life when the blood is collected
in CPDA-1, frozen within 6 days, and stored
at –80 C.
Two concentrations of glycerol have been
used to cryopreserve red cells, as shown in
Table 8-3. This chapter and Methods 6.7
and 6.8 discuss only the high-concentration
glycerol technique used by most blood
banks. Modifications have been developed
for glycerolizing, freezing, storing, thawing,
and deglycerolizing red cells and are discussed elsewhere.16 Several instruments are
available that partially automate glycerolization and deglycerolization of red cells.
The manufacturer of each instrument provides detailed instructions for its use.

182

AABB Technical Manual

Table 8-3. Comparison of Two Methods of Red Blood Cell Cryopreservation
Consideration

High-Concentration
Glycerol

Low-Concentration
Glycerol

Final glycerol concentration (wt/vol)

Approx. 40%

Approx. 20%

Initial freezing temperature

–80 C

–196 C

Freezing rate

Slow

Rapid

Freezing rate controlled

Yes

Type of freezer

Mechanical

Liquid nitrogen

Storage temperature (maximum)

–65 C

–120 C

Change in storage temperature

Can be thawed and
refrozen

Critical

Type of storage

Polyvinyl chloride;
polyolefin

Polyolefin

Shipping

Dry ice

Liquid nitrogen

Special deglycerolizing equipment
required

Yes

Deglycerolizing time

20-40 minutes

30 minutes

Hematocrit

55-70%

50-70%

Blood intended for freezing can be collected into CPD, CP2D, or CPDA-1 and stored
as Whole Blood or RBCs (including ASRBCs). Ordinarily, red cells are glycerolized
and frozen within 6 days of collection or rejuvenated and frozen up to 3 days after they
expire, but RBCs preserved in AS have been
frozen up to 42 days after collection, with
adequate recovery.17,18
Some glycerolization procedures require
removal of most of the plasma or additive
from the RBCs; others do not. The concentration of glycerol used for freezing is hypertonic to blood. Its rapid introduction can
cause osmotic damage to red cells, which
manifests as hemolysis only after thawing.
Therefore, when initiating the cryopreservation process, glycerol should be introduced slowly to allow equilibration within
the red cells.
The US Department of Defense has
adopted a method for high-concentration

glycerolization that uses an 800-mL primary collection container suitable for
freezing (see Method 6.8). Because the cytoprotective agent for freezing is transferred
directly into the primary collection containers, there is less chance of contamination and/or identification error. In addition,
the amount of extracellular glycerol is
smaller and it is more efficient to store and
ship units prepared by this method.
Storage Bags. Storage bag composition can
affect the freezing process; less hemolysis
may occur in polyolefin than in some polyvinyl chloride (PVC) bags. Contact between
red cells and the PVC bag surface may cause
an injury that slightly increases hemolysis
upon deglycerolization. In addition, polyolefin bags are less brittle at –80 C and less
likely to break during shipment and handling than PVC bags.
Freezing Process. Red cells frozen within
6 days of collection with a final glycerol

Chapter 8: Components from Whole Blood Donations

concentration of 40% (wt/vol) must be
stored at –65 C or colder. Red cells are usually placed in canisters to protect the plastic
bag during freezing, storage, and thawing.
Although up to 18 hours at room temperature may elapse between glycerolizing
and freezing without increased postthaw
hemolysis, an interval not exceeding 4
16
hours is recommended. With current 40%
(wt/vol) glycerol methods, controlled rate
freezing is unnecessary; freezing is accomplished by placing the RBC container into a
–80 C freezer.
Refreezing Deglycerolized RBCs. It may
occasionally be desirable to refreeze thawed
RBC units that have not been used as expected or have been unintentionally thawed.
Units that were deglycerolized, stored 20
hours at refrigerator temperature, and then
reglycerolized and refrozen showed no loss
of adenosine triphosphate (ATP), 2,3diphosphoglycerate (2,3-DPG), or in-vivo
survival,19 and RBCs subjected three times
to glycerolizing, freezing, and thawing exhibited a 27% loss of total hemoglobin.20
AABB Standards for Blood Banks and Transfusion Services does not address refreezing
thawed units because this should not be
considered a routine practice. If thawed
units are refrozen, the records should document the valuable nature of such units and
the reasons for refreezing them.
Freezing of Platelets. Perhaps because of
their greater complexity, platelets appear to
sustain greater injury during cryopreservation than red cells, although several protocols have successfully used dimethyl sulfoxide as a cryoprotectant. 21,22 Because
postthaw platelet recovery and function are
significantly reduced when compared with
those of liquid-stored platelets, the clinical
use of cryopreserved platelets is not widespread. The primary use of this procedure
is to freeze autologous platelets for future
use. Platelet cryopreservation is essentially
a research technique.

183

Irradiation
Cellular blood components may be irradiated before storage to prevent transfusionassociated graft-vs-host disease (GVHD).
This does not shorten the shelf life of platelets or granulocytes, but red cells expire
28 days after irradiation or at the end of
the storage period, whichever comes first.

Pooling
Sterile connection devices are used to attach additional bags and compatible tubing to a blood bag without breaking the
sterile integrity of the system. The shelf
life of components thus prepared is the
same as those prepared in a closed system
except for Pooled Platelets, which expire 4
hours after pooling. All sterile connection
device welds must be inspected for completeness, integrity, leakage, and air bubbles; procedures must address the action
to take if the weld is not satisfactory. Record-keeping should include documentation of the products welded, weld quality
control, and lot numbers of disposables.23

Cryoprecipitated AHF
Units of CRYO may be pooled before labeling, freezing, and storage. If pooled
promptly after preparation using aseptic
technique and refrozen immediately, the
resulting component is labeled “Cryoprecipitated AHF Pooled,” with the number
of units pooled stated on the label. The
volume of saline, if added to facilitate
pooling, must also appear on the label.
The statement may appear in the Circular
of Information for the Use of Human Blood
and Blood Components1 instead of on the
container label.
The facility preparing the pool must
maintain a record of each individual donor
traceable to the unique identifier used for
the pooled component.8(p26)

184

AABB Technical Manual

If an open-system pool or component is
to be stored frozen, it should be placed in
the freezer within 6 hours after the seal has
been broken. The AABB and the FDA require transfusion within 4 hours for pooled
thawed components stored at 20 to 24
C.8(p58)

Platelets
Prestorage pooling of PRP whole-bloodderived platelets is possible using a sterile
connection device and a storage container suitable for storage of a high-yield
platelet concentrate. Platelet concentrates
can be leukocyte-reduced using inline filtration 14 or they can be pooled into a
pooling container, then subsequently leukocyte-reduced by filtration and stored in
a storage container. Although current FDA
requirements limit the dating of these pools
to 4 hours, studies of such prestorage leukocyte-reduced pooled platelet concentrates show good preservation of platelet
function without any evidence of a mixed
lymphocyte reaction, with up to 7 days of
storage.24 However, the AABB and FDA require transfusion of pooled platelets within
4 hours of pooling.
As indicated earlier, buffy-coat-derived
platelets are commonly pooled before storage with filtration of the PRP after centrifugation.4-6

Storage
Refrigerated Storage
Blood must be stored only in refrigerators
that, by design and capacity, maintain the
required blood storage temperatures of 1
to 6 C throughout their interior space.
There must be a system to monitor temperatures continuously and record them
at least every 4 hours, and an alarm system with an audible signal that activates

before blood reaches unacceptable storage
temperatures.
Interiors should be clean, adequately
lighted, and well organized. Clearly designated and segregated areas are needed for:
1) unprocessed blood; 2) labeled blood
suitable for allogeneic transfusion; 3) rejected, outdated, or quarantined blood; 4)
autologous blood; and 5) biohazardous
autologous blood. Refrigerators used for
the storage of blood and blood components
may also be used for blood derivatives, tissues, patient and donor specimens, and
blood bank reagents.
Refrigerators for blood storage outside
the blood bank, as may be found in surgical
suites or emergency rooms, must meet
these same standards. Temperature records
are required at all times when blood is present. It is usually most practical to make
blood bank personnel responsible for monitoring these refrigerators.

Frozen Storage
The FDA licenses Red Blood Cells Frozen
for storage up to 10 years when prepared
with high glycerol (40% wt/vol) methods.
Units stored for up to 21 years have been
transfused successfully. A facility’s medical director may wish to extend the storage period; however, storage beyond 10
years requires exceptional circumstances.
The distinctive nature of such units and
the reason for retaining them past the
10-year storage period should be documented. As units are put into long-term
storage, many consider it prudent to freeze
samples of serum or plasma for subsequent testing should new donor screening
tests be introduced in the future. The type
of any specimen saved, date of collection,
date of freezing, and specimen location, if
necessary, should be included in records
of frozen blood.

Chapter 8: Components from Whole Blood Donations

Not all such specimens may meet the
sample requirements of new tests. If stored
samples are not available or inappropriate
for testing, blood centers may attempt to
call the donor back for subsequent testing.
Frozen rare RBCs that have not been tested
for all required disease markers should be
transfused only after weighing the risks and
benefits to the patient. The label should indicate that the unit has not been completely tested and should identify the missing test(s) results.
Blood freezers have the same temperature monitoring and alarm requirements as
blood refrigerators and must be kept clean
and well organized. Freezers designated for
plasma storage must maintain temperatures colder than –18 C (many function at
–30 C or colder); RBC freezers must maintain temperatures colder than –65 C (many
maintain temperatures colder than –80 C).
Self-defrosting freezers must maintain acceptable temperatures throughout their
defrost cycle.
Freezer alarm sensors should be accessible and located near the door, although
older units may have sensors located between the inner and outer freezer walls
where they are neither apparent nor accessible. In such cases, the location of the sensor can be obtained from the manufacturer
and a permanent mark placed on the wall
at that location. Clinical engineers may be
able to relocate the sensor thermocouple
for easier use.
Liquid nitrogen tanks used for blood
storage also have alarm system requirements. The level of liquid nitrogen should
be measured and the sensor placed somewhere above the minimum height needed.

Room Temperature Storage
Platelets require gentle, continuous agitation during storage because stationary
platelets display increased lactate pro-

185

duction and a decrease in pH. Elliptical,
circular, and flat-bed agitators are available for tabletop or chamber use. Elliptical rotators are not recommended for use
with storage bags made of polyolefin
without plasticizer (PL-732).25
Other components that require 20 to 24
C temperatures, eg, cryoprecipitate, can be
stored on a tabletop in any room with an
appropriate ambient temperature, provided the temperature is recorded every 4
hours during storage. Because room temperatures fluctuate, “environmental” or
“platelet chambers” have been developed to
provide consistent, controlled room temperatures. These chambers are equipped with
circulating fans, temperature recorders, and
alarm systems.

Liquid Storage

Red Blood Cells
Biochemical changes occur when red cells
are stored at 1 to 6 C; these changes, some
of which are reversible, contribute to the
“storage lesion” of red cells and to a reduction in viability and levels of 2,3-DPG
affecting oxygen delivery to tissues.26 The
most striking biochemical changes that
affect stored red cells are listed in Table
8-4, but some of these changes rarely
have clinical significance, even in massively transfused recipients. Hemoglobin
becomes fully saturated with oxygen in
the lungs but characteristically releases
only some of its oxygen at the lower oxygen pressure (pO2) of normal tissues. The
relationship between pO2 and oxygen saturation of hemoglobin is shown by the oxygen dissociation curve (see Fig 8-1). Release of oxygen from hemoglobin at a
given pO 2 is affected by ambient pH,
intracellular red cell levels of 2,3-DPG, and
other variables. High levels of 2,3-DPG in
the red cells cause greater oxygen release
at a given pO2, which occurs as an adap-

186

CPD

Variable
Days of Storage

CPDA-1

Whole Blood

Whole
Blood

Red Blood
Cells

Whole
Blood

Red Blood
Cells
35

AS-1

AS-3

AS-5

Red Blood
Cells

(24 hours
posttransfusion)

100

76 (64-85)

pH (measure at 37 C)

7.20

6.84

7.60

7.55

6.98

6.71

6.6

6.5

ATP (% of initial
value)

100

56 (± 16)

45 (± 12)

68.5

2,3-DPG (% of initial
value)

100

<10

Plasma K+ (mmol/L)

3.9

4.20

5.10

27.30

78.50*

45.6

Plasma hemoglobin

191

461

658.0*

N/A

386

N/A

% Hemolysis

N/A

0.5

0.9

0.6

% Viable cells

*Values for plasma hemoglobin and potassium concentrations may appear somewhat high in 35-day stored RBC units; the total plasma in these units is only about 70 mL.

AABB Technical Manual

Table 8-4. Biochemical Changes in Stored Non-Leukocyte-Reduced Red Blood Cells

Chapter 8: Components from Whole Blood Donations

187

Figure 8-1. Oxygen dissociation of hemoglobin under normal circumstances and in Red Blood Cells
(RBCs) stored in excess of 14 days. At tissue pO 2 (40 mm Hg), 25% to 30% of the oxygen is normally released. In stored RBCs, this will decrease to 10% to 15%.

tive change in anemia; lower red cell levels of 2,3-DPG increase the affinity of hemoglobin for oxygen, causing less oxygen
release at the same pO2. In red cells stored
in CPDA-1 or in current additive systems,
2,3-DPG levels fall at a linear rate to zero
after approximately 2 weeks of storage.
This is caused by a decrease in intracellular pH caused by lactic acid, which
26
increases the activity of a diphosphatase.
This causes the dissociation curve to shift
to the left, resulting in less oxygen release
(Fig 8-1).
Upon entering the recipient’s circulation,
stored red cells regenerate ATP and 2,3-DPG,
resuming normal energy metabolism and
hemoglobin function as they circulate in
the recipient. It takes approximately 12
hours for severely depleted red cells to regenerate half their 2,3-DPG levels, and
about 24 hours for complete restoration of
2,3-DPG and normal hemoglobin function.26,27

Red cells lose potassium and gain sodium during the first 2 to 3 weeks of storage
at 1 to 6 C because sodium/potassium
adenosine triphosphatase, which pumps
sodium out of red cells and replaces it with
potassium, has a very high temperature coefficient and functions poorly in the cold.
Supernatant levels of potassium in a unit of
CPDA-1 RBCs have been reported to increase from 5.1 mmol/L on the day of collection to 23 mmol/L on day 7 and 75
mmol/L on day 35. Intracellular levels of
potassium will be replenished after transfusion.
Supernatant potassium levels in red cell
components seem high when compared
with levels in units of Whole Blood of
equivalent age. However, the smaller
supernatant fluid volumes must be considered when determining total potassium
load. Blood stored at 1 to 6 C for more than
24 hours has few functional platelets, but
levels of coagulation Factors II, VII, IX, X,

188

AABB Technical Manual

and fibrinogen are well maintained. Labile
factors (Factors V and VIII) decrease with
time and are not considered sufficient to
correct specific deficiencies in bleeding patients, although levels of 30% for Factor V
and 15% to 20% for Factor VIII have been
reported in Whole Blood stored for 21 days,
and platelets stored at room temperature
have been shown to have Factor V levels of
47% (see Chapter 21) and Factor VIII levels
of 68% after 72 hours.13 For better preservation of Factors V and VIII and platelets,
whole blood is separated into its component parts and the plasma is stored as FFP.

Platelets
Platelets stored in the liquid state at 20 to
24 C are suspended in either autologous
anticoagulated plasma (United States and
Europe) or in platelet additive solutions
(Europe). Under these conditions, the
current shelf life of the platelets in most
countries is 5 days (Table 8-5). This time
limitation is partly related to concerns
about storage-related deterioration in
product potency28 and partly to the potential for bacteria to grow rapidly in this
temperature range.29-30 With regard to potency, liquid-stored platelets undergo
in-vitro changes, which are related to the
duration of storage and are collectively
known as the platelet storage lesion.31,32
This is characterized by a change in platelet shape from discoid to spherical; the
generation of lactic acid from glycolysis,
with an associated decrease in pH; the release of cytoplasmic and granule contents; a decrease in various in-vitro measures of platelet function, particularly
osmotic challenge; shape changes induced by adenosine diphosphate; and reduction in in-vivo recovery and survival.
The in-vitro measures are useful measures
of qualitative potency, but controversy
still exists regarding their utility as practi-

cal surrogates for predicting in-vivo via33
bility and function. Attempts to date to
define the biochemical nature of the
platelet storage lesion have not been conclusive. These observed changes may represent a normal aging process, which is
attenuated by the lower temperature of
storage (20-24 C), rather than the in-vivo
temperature of 37 C. However, a role for
mitochondrial injury as a contributing
cause of these changes is plausible. Resting platelets derive substantial energy
34
from β-oxidation of fatty acids. Alteration in mitochondrial integrity would result in a reduction in carbon flux through
the tricarboxylic acid cycle and require
energy metabolism through glycolysis,
with increased lactate production. Such a
reduction would compromise the generation of efficient ATP and result in a decrease in the metabolic pool of ATP and,
therefore, the energy charge of the pla35
telet. This decrease in the energy charge
would be expected to affect membrane
integrity, resulting in a leakage of cytoplasmic contents, a diminished response
to physiologic stimuli, and an inability to
repair oxidized membrane lipids, with
subsequent distortions in platelet mor36
phology.

Shelf Life
The maximum allowable storage time for
a blood component held under acceptable
temperatures and conditions is called its
“shelf life.” For red cells, the criteria for
determining shelf life for an approved anticoagulant-preservative require that at
least 75% of the original red cells (from a
normal allogeneic donor) be in the recipient’s circulation 24 hours after transfusion.
For other components, their shelf life is
based on functional considerations. Storage times are listed in Table 8-5.

Chapter 8: Components from Whole Blood Donations

Table 8-5. Expiration Dates for Selected Blood Components
Category

Expiration

Whole Blood

ACD/CPD/CP2D – 21 days

189

8(pp53-60)

CPDA-1 – 35 days
Whole Blood Irradiated

Original outdate (see outdates above per anticoagulant) or
28 days form date of irradiation, whichever is sooner

Red Blood Cells (RBCs)

ACD/CPD/CP2D – 21 days
CPDA-1 – 35 days
Open system – 24 hours
Additive solutions – 42 days

RBCs Washed

24 hours

RBCs Leukocytes Reduced

ACD/CPD/CP2D – 21 days
CPDA-1 – 35 days
Open system – 24 hours
Additive solutions – 42 days

RBCs Rejuvenated

24 hours

RBCs Rejuvenated Washed

24 hours

RBCs Irradiated

Original outdate above or 28 days from date of irradiation,
whichever is sooner

RBCs Frozen 40% Glycerol

10 years

RBCs Frozen 20% Glycerol

10 years

RBCs Deglycerolized

24 hours – or 14 days depending on method

RBCs Open System

24 hours

RBCs Open System – Frozen

10 years, 24 hours after thaw

RBCs Closed System – Frozen

10 years, 2 weeks after thaw as approved by the FDA

RBCs Frozen – Liquid Nitrogen

10 years

Platelets

24 hours to 5 days, depending on collection system*

Platelets Pooled or in Open
System

4 hours, unless otherwise specified

Platelets Leukocytes Reduced

Open system – 4 hours
Closed system – no change from original expiration date*

Platelets Irradiated

Open system – 4 hours
Closed system – no change from original expiration date

Granulocytes

24 hours

FFP

12 months (–18 C)
7 years (–65 C), as approved by the FDA
(cont'd)

190

AABB Technical Manual

Table 8-5. Expiration Dates for Selected Blood Components
Category

Expiration

FFP Thawed

24 hours

FFP Thawed – Open System

24 hours

Plasma Frozen within 24 hours
after Phlebotomy

12 months

Plasma Frozen within 24 hours
after Phlebotomy Thawed

24 hours

Thawed Plasma

<5 days if whole blood derived

8(pp53-60)

(cont'd)

24 hours if apheresis
Plasma Liquid

5 days after expiration of RBCs

Plasma Cryoprecipitate
Reduced Frozen

12 months

Plasma Cryoprecipitate
Reduced Frozen

24 hours to 5 days

Cryoprecipitated AHF

12 months

Cryoprecipitated AHF Thawed

4 hours if open system or pooled, 6 hours if single unit

*Maximum time without agitation is 24 hours.

Poststorage Processing
Additional discussion of some of the following topics can be found in Chapters 21
and 22.

Filtration for Leukocyte Reduction
Only special leukocyte reduction filters
reliably provide the ≥99.9% (log 3) removal needed to meet the 5 × 106 specification.37 Red cell leukocyte reduction filters contain multiple layers of synthetic
nonwoven fibers that retain white cells and
platelets, allowing red cells to flow through.
Leukocyte reduction filters are commercially available in a number of set configurations to facilitate filtration during the
separation process at the bedside or in the
laboratory before issue.38 Intact leukocyte
removal efficiency is best when per-

formed soon after collection; therefore, pre39
storage leukocyte reduction is preferred.
There were concerns that early removal
of leukocytes would allow bacteria, present
at the time of collection, to proliferate.
However, studies suggest that early removal
(within 24 hours) in the case of RBCs may
reduce the likelihood of significant bacterial contamination.40 Bedside filtration, particularly of platelets, may not be as effective
in preventing reactions in multitransfused
patients and is less desirable for this reason
41
than prestorage leukocyte reduction. Bedside filtration has also caused hypotensive
reactions.42 Cytokines and other substances
that accumulate during storage (particularly in platelet components) may account
for some failures of bedside filtration to
prevent febrile reactions43 (see Chapter 27).
Furthermore, quality control is difficult to
attain at the bedside.44

Chapter 8: Components from Whole Blood Donations

Thawing

Thawing FFP
FFP is thawed either at temperatures between 30 and 37 C or in an FDA-cleared
device. 8 ( p 5 9 ) It is then known as “FFP
Thawed” and should be transfused immediately or stored between 1 and 6 C for no
more than 24 hours. The expiration date
and time must be indicated on the label.
FFP thawed in a waterbath should be
protected so that entry ports are not contaminated with water. This can be accomplished by wrapping the container in a
plastic overwrap, or by positioning the container upright with entry ports above the
water level. Microwave devices should be
shown not to exceed temperature limits
and not to damage the plasma proteins,
and there should be a warning device to indicate if the temperature rises unacceptably. As with any device, there should be a
procedure for the quality control of indicated functions.
When whole-blood-derived FFP prepared in a closed system is thawed but not
transfused within 24 hours, the label must
be modified. This product should be labeled “Thawed Plasma” and can be stored
at 1 to 6 C and transfused up to 5 days after
thawing. It is similar to FFP except for a reduction in both Factor V and Factor VIII,
particularly Factor VIII.

Thawing CRYO
CRYO is thawed at temperatures between
30 to 37 C for no more than 15 minutes
[CFR 606.122(n) (4)]. Bags should not remain at 30 to 37 C once thawed, so that
degradation of Factor VIII is minimized.
As with FFP, entry ports should be protected from water contamination if the
unit is thawed in a waterbath. Single-unit
thawed CRYO must be transfused immediately or can be stored at room temperature (20 to 24 C) for no more than 6 hours.

191

All pooled CRYO, whether prepared in an
open or a closed system, must be transfused within 6 hours after thawing, or 4
hours after pooling, whichever comes
first. CRYO may be pooled into one bag
after thawing to simplify transfusion to a
patient requiring multiple units. The
pooled product is assigned a unique pool
number, but records must document the
individual units included. See Method 6.12
for guidelines on how to thaw and pool
CRYO for transfusion.

Thawing and Deglycerolizing RBCs
The protective canister and enclosed frozen
cells may be placed directly in a 37 C dry
warmer or can be overwrapped and immersed in a 37 C waterbath. Units frozen
in the primary collection bag system
16
should be thawed at 42 C. The thawing
process takes at least 20 to 25 minutes and
should not exceed 40 minutes. Gentle agitation may be used to speed thawing.
Thawed cells contain a high concentration of glycerol that must be reduced gradually to avoid in-vivo or in-vitro hemolysis.
Deglycerolization is achieved by washing
the red cells with solutions of decreasing
osmolarity. In one procedure (see Method
6.9), glycerolized cells are diluted with 150
mL of 12% saline, then washed with 1 L of
1.6% saline, followed by 1 L of 0.9% saline
with 0.2% dextrose. The progressive decrease in osmolarity of the washing solutions causes osmotic swelling of the cells,
so each solution must be added slowly, with
adequate time allowed for mixing and osmotic equilibration. Any of the commercially available instruments for batch or
continuous-flow washing can be used to
deglycerolize red cells frozen in a high concentration of glycerol. Because there are
many potentially important variations in
deglycerolization protocols for each instru-

192

AABB Technical Manual

ment, personnel in each facility should not
only follow the manufacturer’s instructions,
but also validate the local process. The process selected must ensure adequate removal of cryoprotectant agents, minimal
free hemoglobin, and recovery of greater
than 80% of the original red cell volume after the deglycerolization process.8(p27)
When deglycerolization is complete, the
integrally connected tubing should be filled
with an aliquot of red cells and sealed in
such a manner that it can be detached for
subsequent compatibility testing. The label
must identify both the collecting facility
and the facility that prepares the deglycerolized unit if it is different from the collection facility.
When glycerolized frozen red cells from
persons with sickle cell trait are suspended
in hypertonic wash solutions during deglycerolization and centrifuged, they form a
jelly-like mass and hemolyze.16 Modified
wash procedures using only 0.9% saline
with 0.2% dextrose after the addition of
12% saline can eliminate this problem.45 In
some cryopreservation programs, donations are screened for the presence of hemoglobin S before being frozen.
When glycerolization or deglycerolization involves entering the blood bag, the
system is considered “open” and the resulting suspension of deglycerolized cells can
be stored for only 24 hours at 1 to 6 C. A
method for glycerolization and deglycerolization in an effectively closed system
allows for the resulting suspension of
deglycerolized red cells to be stored for 2
weeks at 1 to 6 C. This method allows more
effective inventory management of the
deglycerolized RBC units.
When deglycerolized RBCs are stored at
1 to 6 C for periods up to 14 days, the major
changes observed are increased concentrations of potassium and hemoglobin in the
supernatant fluid. Red cells that have undergone gamma irradiation and subse-

quent storage at 1 to 6 C tolerate freezing
with no more detectable damage than
unirradiated cells.46,47

Irradiation
Blood components that contain viable
lymphocytes (including red cell, platelet,
and granulocyte components) should be
irradiated to prevent proliferation of transfused T lymphocytes in recipients at risk
of acquiring, or from donors at risk of causing, GVHD. The AABB and FDA recommend a minimum 25 Gy dose of gamma
radiation to the central portion of the
container, with no less than 15 Gy deliv8(p26)
ered to any part of the bag.
Irradiation is accomplished using cesium-137 or cobalt-60, in self-contained
blood irradiators or hospital radiation therapy machines. More recently, an x-ray device has been developed that is capable of
adequate dose delivery. Measurement of
dose distribution; verification of exposure
time, proper mechanical function, and
turntable rotation; and adjustment of exposure time as the radioactive source decays
should be addressed in the facility’s procedures.48 Records must be maintained, and
all steps, supplies, and equipment used in
the irradiation process must be documented.
To confirm the irradiation of individual
units, radiochromic film labels (available
commercially) may be affixed to bags before irradiation. When exposed to an adequate amount of radiation, the film portion
of the label darkens, indicating that the
component has been exposed to an adequate radiation dosage. Because irradiation
damages red cells and reduces the overall
viability (24-hour recovery),49 red cell components that have been irradiated expire on
their originally assigned outdate or 28 days
from the date of irradiation, whichever comes first. Platelets sustain minimal damage
from irradiation, so their expiration date

Chapter 8: Components from Whole Blood Donations

does not change. Irradiated blood is essential for patients at risk from transfusion-associated GVHD, including fetuses
receiving intrauterine transfusion, select
immunocompetent or immunocompromised recipients, recipients who are undergoing hematopoietic transplantation, recipients of platelets selected for HLA or platelet
compatibility, and recipients of donor units
from blood relatives.

193

with any visible red cells present in the
pool. Only one unique number is affixed
to the final component, but records must
reflect the pooling process and all units
included in the pool. This pooled product
has an expiration time of 4 hours.
Single CRYO units may be pooled after
thawing and labeled appropriately. The
AABB and FDA require transfusion of CRYO
within 4 hours of pooling and subsequent
storage at 20 to 24 C.

Washing
Washing a unit of RBCs with 1 to 2 L of
sterile normal saline removes about 99%
of plasma proteins, electrolytes, and antibodies. Automated and manual washing
methods remove some of the leukocytes
in the RBCs, but not enough to prevent
alloimmunization. Up to 20% of the red
cell mass may be lost depending on the
protocol used. Washed red cells must be
used within 24 hours because preparation
is usually accomplished in an open system, and removal of the anticoagulantpreservative solution compromises longterm preservation of cell viability and
function.
Platelets can be washed with normal saline or saline-buffered with ACD-A or citrate,
using manual or automated methods. The
procedures may result in a reduction in
radiolabeled recovery (about 33% less), but
not in survival of the washed platelets51;
white cell content is not significantly
changed. Washed platelets must be used
within 4 hours of preparation.

Pooling
When a patient requires multiple units of
Platelets, pooling them into a single bag
simplifies issue and transfusion. This product should be labeled “Platelets Pooled.”
If Platelets contain a significant number
of red cells and ABO groups are mixed,
plasma antibodies should be compatible

Volume Reduction
Platelets may be volume-reduced in order
to decrease the total volume of the component transfused or partially remove
supernatant substances, such as ABO alloantibodies. The former need may arise in
patients with small intravascular volumes
or those with fluid overload (eg, resulting
from renal or cardiac failure). The latter
need may be better addressed by washing
(see below). If sterility is broken, the expiration of the product becomes 4 hours. If
sterility is not broken (eg, a sterile connection device is used23), removal of the
supernatant still reduces glucose availability and buffering capacity, and the
subsequent storage of the platelets is in a
suboptimal environment. Transfusion as
soon as possible is generally advocated.

Aliquoting
Blood components may be aliquoted in
smaller volumes into other containers in
order to meet the needs of very-low-volume transfusion recipients or to provide a
component to meet the needs of patients
with fluid overload. The composition of
red cell and plasma components is not altered by aliquoting, unlike volume reduction. Therefore, the expiration date is not
altered if a sterile connection device is
used to perform the aliquoting.23 The shelf
life and viability of platelets, however, are

194

AABB Technical Manual

dependent on the storage bag, plasma
volume, and storage environment. Removing aliquots of platelets from the
“mother” bag changes the storage environment of the platelets remaining in the
“mother” bag. If the altered storage environment does not meet the storage bag
manufacturer’s requirements, the expiration period of the remaining component
should also be modified.

Rejuvenation
It is possible to restore levels of 2,3-DPG
and ATP in red cells stored in CPD or
CPDA-1 solutions by adding an FDA-licensed solution containing pyruvate,
inosine, phosphate, and adenine (see
Method 6.6). RBCs may be rejuvenated after 1 to 6 C storage up to 3 days after expiration; then, they may be glycerolized and
frozen in the same manner as fresh red
cells. If rejuvenated RBC units are to be
transfused within 24 hours, they may be
stored at 1 to 6 C; however, they must be
washed before use to remove the inosine,
which might be toxic to the recipient. The
blood label and component records must
indicate the use of rejuvenating solutions.

Inspection, Shipping,
Disposition, and Issue
Inspection
Stored blood components are inspected
immediately before issue for transfusion
or shipment to other facilities.8(pp14,15) These
inspections must be documented; records
should include the date, donor number,
description of any abnormal units, the action taken, and the identity of personnel
involved. Visual inspections cannot always detect contamination or other deleterious conditions; nonetheless, blood

components that look abnormal must not
be shipped or transfused.
Deleterious conditions should be suspected if 52 : 1) segments appear much
lighter in color than what is in the bag (for
AS-RBCs), 2) the red cell mass looks purple,
3) a zone of hemolysis is observed just
above the cell mass, 4) clots are visible, 5)
blood or plasma is observed in the ports or
at sealing sites in the tubing, or (6) the
plasma or supernatant fluid is murky, purple, brown, or red. A green hue from
light-induced changes in bilirubin pigments need not cause the unit to be rejected. Mild lipemia, characterized by a
milky appearance, does not render a donation unsuitable provided that all infectious
disease testing can be performed. Grossly
lipemic specimens are unsuitable.
A component that is questioned for any
reason should be quarantined until a responsible person determines its disposition. Evaluation might include inverting the
unit gently a few times to mix the cells with
the supernatant fluid because considerable
undetected hemolysis, clots, or other alterations may be present in the undisturbed
red cells. If, after resuspension, resettling,
and careful examination, the blood no longer appears abnormal, it may be returned
to inventory. Appropriate records should be
maintained documenting the actions
taken, when, and by whom. Units of FFP
and CRYO should be inspected when they
are removed from frozen storage for evidence of thawing and refreezing and for evidence of cracks in the tubing or plastic
bag. Unusual turbidity in thawed components may be cause for discard.
All platelet components should be inspected before release and issue. Units with
macroscopically visible platelet aggregates
should not be used for transfusion. Some
facilities assess the “swirling” appearance
of platelets by holding platelet bags up to a
light source and gently tapping them. This

Chapter 8: Components from Whole Blood Donations

swirl phenomenon correlates well with pH
values associated with adequate platelet
in-vivo viability.53 Some platelet components have been noted to contain small
amounts of particulate matter. These components are suitable for use.
Bacterial contamination of transfusion
components is rare because of the use of
aseptic technique and screening for bacteria in platelets, the availability of closed systems for collection and preparation, and
careful control of storage conditions. Sterility testing of blood components plays a role
in validating initial production processes. If
a transfusion component has an abnormal
appearance, or if an adverse clinical reaction appears to be related to contaminated
donor blood, culturing should be performed
and a Gram’s stain should be evaluated.
Microbiologists can best advise blood center staff on sample requirements and appropriate test methods for detecting potential
blood contaminants, including cryophilic microorganisms. Making cultures directly from
the contents of the bag and from the recipient can provide useful diagnostic information.
Any facility that collected a reportedly
contaminated component should be notified so that donor bacteremia, potentially
inadequate donor arm preparation, or improper handling or pooling technique can
be investigated. The donor’s health should
be reviewed, and other components prepared
from that collection should be withdrawn.

Shipping
Shipment to areas outside the facility requires additional packaging. Transport
containers or coolers and packaging procedures must be validated before use to
verify that they are able to maintain blood
components at required temperatures for
the intended time and conditions. Containers must also be able to withstand
leakage, pressure, and other conditions

195

incidental to routine handling. Refer to
Chapter 2 for more information on shipping regulations and guidelines.
Simple exposure to temperatures outside
the acceptable range does not necessarily
render blood unsuitable for transfusion. Exceptions may be made under unusual circumstances such as for autologous units or
cells of a rare phenotype, but the records
must document the reasons for preserving
the unit, the evaluation of its continued
suitability for transfusion, and the identity
of the person responsible for the decision.
Other factors to consider when assessing
component acceptability after transport include the length of time in shipment, mode
of transportation, magnitude of variance
above or below the acceptable range, presence of residual ice in the shipping box, appearance of the unit(s), age of the unit(s),
and likelihood of additional storage before
transfusion. The shipping facility should be
notified when a receiving facility observes
that acceptable transport temperatures
have been exceeded.

Whole Blood and RBCs
Liquid Whole Blood and RBCs shipped
from the collection facility to another facility must be transported in a manner
that ensures a temperature of 1 to 10 C.
The upper limit of 10 C can be reached in
30 minutes if a unit of blood taken from 5
C storage is left at an ambient temperature of 25 C. Smaller units, as are commonly used for pediatric patients, can
warm even more quickly.
Wet ice, securely bagged to prevent leakage, is the coolant of choice to maintain required temperatures during transport and
shipping. An appropriate volume is placed
on top of the units within the cardboard
box or insulated container. Clinical coolant
packs or specially designed containers may
also be used to maintain acceptable trans-

196

AABB Technical Manual

port temperatures. Super-cooled ice (eg,
large blocks of ice stored at –18 C) in contact with Whole Blood or RBCs may result
in temperatures below 1 C, with resultant
hemolysis of the red cells.

Platelets and Granulocytes
Every reasonable effort must be made to
ensure that platelets and granulocytes are
maintained at 20 to 24 C during shipment. A well-insulated container without
added ice, or with a commercial coolant
designed to keep the temperature at 20 to
24 C, is recommended. Fiber-filled envelopes or newspaper are excellent insulators. For very long distances or travel
times in excess of 24 hours, double-insulated containers may be needed.

Frozen Components
Frozen components must be packaged for
transport in a manner designed to keep
them frozen. This may be achieved by using a suitable quantity of dry ice in wellinsulated containers or in standard shipping cartons lined with insulation material, such as plastic air bubble packaging
or dry packaging fragments. The dry ice,
obtained as sheets, may be layered at the
bottom of the container, between each
layer of frozen components, and on top.
Shipping facilities should determine optimal conditions for shipping frozen components, which depend on the temperature
requirements of the component, the distance to be shipped, the shipping container
used, and ambient temperatures to be encountered. Procedures and shipping containers should be validated and periodically
monitored. The receiving facility should always observe the shipment temperature
and report unacceptable findings to the
shipping facility.
Red cells cryopreserved with high-concentration glycerol (40% wt/vol) tolerate

fluctuations in temperatures between –85 C
and –20 C with no significant change in
in-vitro recovery or 24-hour posttransfusion survival, so transport with dry ice is
acceptable. Blood shipments containing
dry ice as a coolant are considered “dangerous goods,” and special packaging and labeling requirements apply (see Chapter 2).
Shipping boxes containing dry ice must not
be completely sealed so that the carbon dioxide gas released as the dry ice sublimes
can escape without risk of explosion.

Disposition
Both blood collection sites and transfusion services must maintain records on all
blood components handled so that units
can be tracked from collection to final
disposition. Units of blood that cannot be
released for transfusion should be returned to the provider or discarded as
biohazardous material. The nature of the
problem disqualifying the unit should be
investigated and the results reported to
the blood supplier. Findings may indicate
a need to improve phlebotomy techniques, donor screening methods, or the
handling of units during processing, storage, or transport.
Disposal procedures must conform to the
local public health code for biohazardous
waste. Autoclaving or incineration is recommended. If disposal is carried out offsite, a contract with the waste disposal firm
must be available and should specify that
appropriate Environmental Protection Agency,
state, and local regulations are followed
(see Chapter 2 for the disposal of biohazardous waste).

Issuing from a Transfusion Service
Blood transported short distances within
a facility, eg, to the patient care area for
transfusion, requires no special packaging
other than that dictated by perceived safety

Chapter 8: Components from Whole Blood Donations

concerns and institutional preferences.
However, blood should never unnecessarily be allowed to reach temperatures outside its accepted range. Specific transport
guidelines may be warranted if transport
time is prolonged.
Units that have left the control of the
transfusion service or donor center and
then have been returned must not be reissued for transfusion unless the following
conditions have been met:
1.
The container closure has not been
penetrated, entered, or modified in
any manner.
2.
Red cell components have been maintained continuously between 1 and
10 C, preferably 1 to 6 C. Blood centers and transfusion services usually
do not reissue RBC units that have
remained out of a monitored refrigerator or a validated cooler for longer than 30 minutes because, beyond
that time, the temperature of the
component may have risen above 10 C.
3.
At least one sealed segment of integral donor tubing remains attached
to the red cell component, if the
blood has left the premises of the issuing facility.
4.
Records indicate that the blood has
been reissued and has been inspected
before reissue.

Blood Component Quality
Control
Ensuring safe and efficacious blood components requires applying the principles
of quality assurance to all aspects of component collection, preparation, testing,
storage, and transport. All procedures and
equipment in use must be validated before their implementation and periodically monitored thereafter. Staff must be
appropriately trained and their compe-

197

tency evaluated. The contents of final
products should be periodically assessed
to make sure that they meet expectations.
How much quality control is performed is
best determined by the institution, with
input from the compliance officer and consideration of AABB and FDA requirements.
See Appendix 8-1.

Quality Control of Equipment

Continuous Temperature Monitoring
Systems
Most blood refrigerators, freezers, and
chambers have built-in temperature monitoring sensors connected to recording
charts or digital readout systems for easy
surveillance. Digital recording devices
measure the differences in potential generated by a thermocouple; this difference
is converted to temperature. Because warm
air rises, temperature-recording sensors
should be placed on a high shelf and immersed in a volume of liquid not greater
than the volume of the smallest component stored. Either a glass container or a
plastic blood bag may be used. Recording
charts and monitoring systems must be inspected daily to ensure proper function.
When recording charts or tapes are
changed, they should be dated inclusively
(ie, start and stop dates), and labeled to
identify the facility, the specific refrigerator
or freezer, and the person changing the
charts. Any departure from normal temperature should be explained in writing on the
chart beside the tracing or on another document and should include how the stored
components were managed. A chart with a
perfect circle tracing may indicate that the
recorder is not functioning properly or is
not sensitive enough to record the expected
variations in temperature that occur in any
actively used refrigerator.
Blood banks with many refrigerators and
freezers may find it easier to use a central

198

AABB Technical Manual

alarm monitoring system that monitors all
equipment continuously and simultaneously and prepares a hard copy tape of
temperatures at least once every 4 hours.
These systems have an audible alarm that
sounds as soon as any connected equipment reaches its predetermined temperature alarm point and indicates the equipment in question. Blood storage equipment
so monitored does not require a separate
independent recording chart.

Thermometers
Visual thermometers in blood storage equipment provide ongoing verification of temperature accuracy. One should be immersed
in the container with the continuous monitoring sensor. The temperature of the
thermometer should be compared periodically with the temperature on the recording chart. If the two do not agree
within 2 C, both should be checked
against a thermometer certified by the
National Institute of Standards and Technology (NIST) and suitable corrective action should be taken (see Method 7.2). (A
2 C variation between calibrated thermometers allows for the variation that
may occur between thermometers calibrated against the NIST thermometers.)
Thermometers also help verify that the
temperature is appropriately maintained
throughout the storage space. Large refrigerators or freezers may require several thermometers to assess temperature fluctuations. In addition to the one immersed with
the continuous monitoring sensor (usually
located on a high shelf), at least one other
in a similar container is placed on the lowest shelf on which blood is stored. The temperature in both areas must be within the
required range at all times.
Either liquid-in-glass (analog) thermometers or electronic and thermocouple (digital) devices can be used for assessing stor-

age temperatures, as long as their accuracy
is calibrated against a NIST-certified thermometer or a thermometer with a NISTtraceable calibration certificate (see Method
7.2). Of equal importance is that they be
used as intended, according to the manufacturer’s recommendations.

Alarm Systems
To ensure that alarm signals will activate
at a temperature that allows personnel to
take proper action before blood reaches
undesirable temperatures, both temperature of activation and power source are
tested periodically. The electrical source
for the alarm system must be separate
from that of the refrigerator or freezer; either a continuously rechargeable battery
or an independent electrical circuit served
by an emergency generator is acceptable.
Method 7.3.1 provides a detailed procedure to test the temperatures of activation
for refrigerator alarms. Suggestions for
freezer alarms are in Method 7.3.2 Thermocouple devices that function at freezer
temperatures are especially useful for determining the temperature of activation
with accuracy when sensors are accessible.
When they are not, approximate activation
temperatures can be determined by checking a freezer’s thermometer and recording
chart when the alarm sounds after it is shut
down for periodic cleaning or maintenance. It can also be assessed by placing a
water bottle filled with cold tap water
against the inner freezer wall where the
sensor is located.
When the alarm goes off, usually in a
short time, the recording chart can be
checked immediately for the temperature
of activation. There must be written instructions for personnel to follow when the
alarm sounds. These instructions should
include steps to determine the immediate
cause of the temperature change and ways

Chapter 8: Components from Whole Blood Donations

to handle temporary malfunctions, as well
as steps to take in the event of prolonged
failure. It is important to list the names of
key people to be notified and what steps
should be taken to ensure that proper storage temperature is maintained for all blood,
components, and reagents.

Quality Control of Red Blood Cells
RBCs prepared without additive solutions
must have a hematocrit ≤80%. This
should be established during process validation and periodically confirmed by
quality control procedures. RBCs Leukocytes Reduced should contain <5 × 106residual leukocytes and retain 85% of the
original red cells.54 Quality control should
demonstrate that at least 95% of units
sampled meet this specification. Units
tested must meet the leukocyte reduction
specification.6,8(p28) (See Appendix 8-1.)

8(p30)

mg of fibrinogen.
Each pool must have
a Factor VIII content of at least 80 mg
times the number of donor units in the
pool; for fibrinogen, the content should be
150 mg times the number of donor units.8(p30)
(See Appendix 8-1.)

References
1.

Quality Control of Platelets
The quality of every method of platelet
preparation must be assessed periodically. Data must show that at least 90% of
components tested contain an acceptable
number of platelets (5.5 × 1010 for wholeblood-derived platelets) and have a plasma
pH of 6.2 or higher at the end of the allowable storage period.8(p36)
Prestorage leukocyte-reduced Platelets
derived from filtration of platelet-rich
plasma must contain less than 8.3 × 105 residual leukocytes per unit to be labeled as
leukocyte reduced. Validation and quality
control should demonstrate that at least
95% of units sampled meet this require8(p31)
ment.

Quality Control of Cryoprecipitated AHF
AABB Standards for Blood Banks and
Transfusion Services requires that all
tested individual units of CRYO contain a
minimum of 80 IU of Factor VIII and 150

199

10.

AABB, American National Red Cross, and
America’s Blood Centers. Circular of information for the use of human blood and blood
components. Bethesda, MD: AABB, 2002.
Buetler E. Liquid preservation of red blood
cells. In: Simon TL, Dzik WH, Snyder EL, et al,
eds. Rossi’s principles of transfusion medicine. 3rd ed. Philadelphia: Lippincott Williams & Wilkins, 2002:50-61.
Code of federal regulations. Title 21 CFR
640.24(b) and 640.34(b). Washington, DC: US
Government Printing Office, 2004 (revised
annually).
Pietersz RN, van der Meer PF, Steneker I, et al.
Preparation of leukodepleted platelet concentrates from pooled buffy-coats: Prestorage
filtration with Autostop BC. Vox Sang 1999;76:
231-6.
Bo o m g a a rd M N , Jo u s t ra - Di j k h u i s A M ,
Gouwerok CW, et al. In vitro evaluation of
platelets, prepared from pooled buffy-coats,
stored for 8 days after filtration. Transfusion
1994;34:311-6.
van der Meer PF, Pietersz RN, Tiekstra MJ, et
al. WBC-reduced platelet concentrates from
pooled buffy-coats in additive solution: An
evaluation of in vitro and in vivo measures.
Transfusion 2001;41:917-22.
Code of federal regulations. Title 21 CFR
601.22. Washington, DC: US Government
Printing Office, 2004 (revised annually).
Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.
Owens MR, Sweeney JD, Tahhan RH, et al. Influence of type of exchange fluid on survival
in therapeutic apheresis for thrombotic
thrombocytopenic purpura. J Clin Apheresis
1995;10:178-82.
Rosenthal J, Mitchell SC. Neonatal myelopoiesis and immunomodulation of host defenses. In: Petz LD, Swisher SN, Kleinman S,
et al, eds. Clinical practice of transfusion
medicine. 3rd ed. New York: Churchill Livingstone, 1996:685-703.

200

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

AABB Technical Manual

Huh YO, Lichtiger B, Giacco GG, et al. Effect
of donation time on platelet concentrates and
fresh-frozen plasma. Vox Sang 1989;56:21-4.
Code of federal regulations. Title 21 CFR
610.53 (c). Washington, DC: US Government
Printing Office, 2004 (revised annually).
Calhoun L. Blood product preparation and
administration. In: Petz LD, Swisher SN,
Kleinman S, et al, eds. Clinical practice of
transfusion medicine. 3rd ed. New York:
Churchill Livingstone, 1996:305-33.
Sweeney JD, Holme S, Heaton WAL, Nelson E.
White cell-reduced platelet concentrates prepared by in-line filtration of platelet-rich
plasma. Transfusion 1995;35:131-6.
Valeri CR. Frozen preservation of red blood
cells. In: Simon TL, Dzik WH, Snyder EL, et al,
eds. Rossi’s principles of transfusion medicine. 3rd ed. Philadelphia: Lippincott Williams & Wilkins, 2002:62-8.
Meryman HT, Hornblower M. A method for
freezing and washing RBCs using a high glycerol concentration. Transfusion 1972;12:14556.
Lovric VA, Klarkowski DB. Donor blood
frozen and stored between –20 C and –25 C
with 35-day liquid post-thaw shelf-life. Lancet 1989;i:71-3.
Rathbun EJ, Nelson EJ, Davey RJ. Posttransfusion survival of red cells frozen for 8
weeks after 42-day liquid storage in AS-3.
Transfusion 1989;29:213-7.
Kahn RA, Auster MJ, Miller WV. The effect of
refreezing previously frozen deglycerolized
red blood cells. Transfusion 1978;18:204-5.
Myhre BA, Nakasako YUY, Schott R. Studies
on 4 C stored frozen reconstituted red blood
cells. III. Changes occurring in units which
have been repeatedly frozen and thawed.
Transfusion 1978;18:199-203.
Angelini A, Dragani A, Berardi A, et al. Evaluation of four different methods for platelet
freezing: In vitro and in vivo studies. Vox Sang
1992;62:146-51.
Borzini P, Assali G, Riva MR, et al. Platelet cryopreservation using dimethylsulfoxide/polyethylene glycol/sugar mixture as cytopreserving solution. Vox Sang 1993;64:248-9.
Food and Drug Administration. Memorandum: Use of an FDA cleared or approved sterile connection device (STCD) in blood bank
practice. (July 29, 1994) Rockville, MD: CBER
Office of Communication, Training, and
Manufacturers Assistance, 1994.
Sweeney JD, Kouttab NM, Holme SH, et al.
Prestorage pooled whole blood derived
leukoreduced platelets stored for seven days
preserve acceptable quality and do not show

25.

26.

27.

28.

29.

30.

31.
32.

33.

34.

35.

36.

37.

38.
39.
40.

evidence of a mixed lymphocyte reaction.
Transfusion 2004;44:1212-9.
Moroff G, Holme S. Concepts about current
conditions for the preparation and storage of
platelets. Transfus Med Rev 1991;5:48-59.
Högman CF, Meryman HT. Storage parameters affecting red blood cell survival and
functions after transfusion. Transfus Med Rev
1999;13:275-6.
Heaton A, Keegan T, Holme S. In vivo regeneration of red cell 2,3-diphosphoglycerate following transfusion of DPG-depleted AS-1,
AS-3 and CPDA-1 red cells. Br J Haematol
1989;71:131-6.
Sweeney JD, Holme S, Heaton WAL. Quality of
platelet concentrates. In: Van Oss CJ, ed.
Transfusion immunology and medicine. New
York: Marcel Dekker, 1995:353-70.
Heal JM, Singl S, Sardisco E, et al. Bacterial
proliferation in platelet concentrates. Transfusion 1996;26:388-9.
Punsalang A, Heal JM, Murphy PJ. Growth of
gram-positive and gram-negative bacteria in
platelet concentrates. Transfusion 1989;29:
596-9.
Murphy S. Platelet storage for transfusion.
Semin Hematol 1985;22:165-77.
Murphy S, Rebulla P, Bertolini F, et al. In vitro
assessment of the quality of stored platelet
concentrates. The BEST (Biomedical Excellence of Safer Transfusion) Task Force of the
International Society of Blood Transfusion.
Transfus Med Rev 1994;8:29-36.
Rinder HM, Smith BR. In vitro evaluation of
stored platelets: Is there hope for predicting
post-transfusion platelet survival and function? Transfusion 2003;43:2-6.
Murphy S. The oxidation of exogenously
added organic anions by platelets facilitates
maintenance of pH during their storage for
transfusion at 22 C. Blood 1995;85:1929-35.
Holme S, Heaton WA, Courtright M. Platelet
storage lesion in second-generation containers: correlation with platelet ATP levels. Vox
Sang 1987;53:214-20.
Holme S, Sawyer S, Heaton A, Sweeney JD.
Studies on platelets exposed or stored at temperatures below 20 C or above 24 C. Transfusion 1997;37:5-11.
Dzik S. Leukodepletion blood filters: Filter
design and mechanisms of leukocyte removal. Transfus Med Rev 1993;7:65-77.
Leukocyte reduction. Association Bulletin
99-7. Bethesda, MD: AABB, 1999.
Heaton A. Timing of leukodepletion of blood
products. Semin Hematol 1991;28:1-2.
Buchholz DH, AuBuchon JP, Snyder EL, et al.
Effects of white cell reduction on the resis-

Chapter 8: Components from Whole Blood Donations

41.

42.

43.

44.

45.

46.

47.

48.

tance of blood components to bacterial multiplication. Transfusion 1994;34:852-7.
Sweeney JD, Kouttab N, Penn LC, et al. A
comparison of prestorage leukoreduced
whole blood derived platelets with bedside
filtered whole blood derived platelets in
autologous stem cell transplant. Transfusion
2000;40:794-800.
Cyr M, Hume H, Sweeney JD, et al. Anomaly
of the des-Arg9-bradykinin metabolism associated with severe hypotensive reaction during blood transfusions: A preliminary report.
Transfusion 1999;39:1084-8.
Heddle NM, Klama L, Singer J, et al. The role
of the plasma from platelet concentrates in
transfusion reactions. N Engl J Med 1994;331:
625-8.
Sweeney JD. Quality assurance and standards
for red cells and platelets. Vox Sang 1998;74:
201-5.
Meryman HT, Hornblower M. Freezing and
deglycerolizing sickle-trait red blood cells.
Transfusion 1976;16:627-32.
Suda BA, Leitman SF, Davey RJ. Characteristics of red cells irradiated and subsequently
frozen for long-term storage. Transfusion
1993;33:389-92.
Miraglia CC, Anderson G, Mintz PD. Effect of
freezing on the in vivo recovery of irradiated
cells. Transfusion 1994;34:775-8.
Food and Drug Administration. Guidance for
industry: Gamma irradiation of blood and

49.

50.

51.

52.

53.

54.

201

blood components: A pilot program for licensing. (March 15, 2000) Rockville, MD:
CBER Office of Communication, Training,
and Manufacturers Assistance, 2000.
Moroff G, Holme S, AuBuchon JP, et al. Viability and in vitro properties of gamma irradiated AS-1 red blood cells. Transfusion 1999;
39:128-34.
Sweeney JD, Holme S, Moroff G. Storage of
apheresis platelets after gamma radiation.
Transfusion 1994;34:779-83.
Pineda AA, Zylstra VW, Clare DE, et al. Viability and functional integrity of washed platelets. Transfusion 1989;29:524-7.
Kim DM, Brecher ME, Bland LA, et al. Visual
identification of bacterially contaminated red
cells. Transfusion 1992;32:221-5.
Bertoloni F, Murphy S. A multicenter inspection of the swirling phenomenon in platelet
concentrates prepared in routine practice.
Transfusion 1996;36:128-32.
Dumont LJ, Dzik WH, Rebulla P, Brandwein
H, and the Members of the BEST Working
Party of the ISBT. Practical guidelines for process validation and process control of white
cell-reduced blood components: Report of
the Biomedical Excellence for Safer Transfusion (BEST) Working Party of the International Society of Blood Transfusion (ISBT).
Transfusion 1996;36:11-20.

202

AABB Technical Manual

Appendix 8-1. Component Quality Control
Component

Specifications
and Standards*

AABB
Standards†

Red Blood Cells

Hematocrit ≤80% (in all)

5.7.5.1

Red Blood Cells
Leukocytes
Reduced

Retain 85% of original red cells, 95% of tested
units <5 × 106 leukocytes in the final container

5.7.5.6

Cryoprecipitated AHF

Factor VIII: ≥80 IU/bag (100%)
Fibrinogen ≥150 mg/bag (100%)

5.7.5.14

Platelets

≥5.5 × 1010 platelets per unit and pH ≥6.2 in
90% of units tested

5.7.5.16

Platelets Leukocytes
Reduced

≥5.5 × 1010 platelets in 75% of units tested,
≥6.2 pH in 90% of units tested, and
<8.3 × 105 leukocytes in 95% of units tested

5.7.5.17

Platelets Pheresis

≥3.0 × 1011 platelets in final container of components tested; and pH ≥6.2 in 90% of units
tested

5.7.5.19

Platelets Pheresis
Leukocytes
Reduced

<5.0 × 106 leukocytes in 95% of components
tested and ≥3.0 × 1011 platelets in the final
container and pH ≥6.2 in 90% tested units

5.7.5.20

Granulocytes
Pheresis

≥1.0 × 1010 granulocytes in at least 75% of components tested

5.7.5.21

Irradiated components

25 Gy delivered to the central portion of the container; minimum of 15 Gy at any point in the
component

5.7.4.2

*The specification is the threshold value; the standard is the percentage of tested units meeting or exceeding this threshold. The manufacturing procedures used should be validated as capable of meeting these standards before implementation and routine QC. The number of units tested during routine QC should be such as to have a high level of assurance
that conformance with these standards is being achieved.
†
Silva MA, ed. Standards for blood banks and transfusion services. 23rd ed. Bethesda, MD: AABB, 2005.

Chapter 9: Molecular Biology in Transfusion Medicine

Chapter 9

Molecular Biology in
Transfusion Medicine
9

ROTEINS ARE MACROMOLECULES
composed of amino acids, the sequences of which are determined
by genes. Lipids and carbohydrates are
not encoded directly by genes; genetic determination of their assembly and functional structures results from the action of
different protein enzymes. Blood group
antigens can be considered gene products, either directly, as polymorphisms of
membrane-associated proteins, or indirectly, as carbohydrate configurations catalyzed by glycosyltransferases.

From DNA to mRNA to
Protein
Structure of DNA
A gene consists of a specific sequence of
nucleotides located at a specific position
(locus) along a chromosome. Each chromosome consists of long molecules or

strands of deoxyribonucleic acid (DNA).
DNA is composed of the sugar deoxyribose,
a phosphate group, the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T) and cytosine (C).
The combination of a sugar, a phosphate
group, and a base is called a nucleotide. A
double strand of DNA consists of two complementary (nonidentical) single strands
held together by hydrogen bonds between
specific base pairings of A-T and G-C. The
two strands form a double helix configuration with the sugar-phosphate backbone on the outside and the paired bases
on the inside (see Fig 9-1). DNA synthesis
is catalyzed by DNA polymerase, which
adds a deoxyribonucleotide to the 3′ end
of the existing chain. The 3′ and 5′ notation refers to the carbon position of the
deoxyribose linkage to the phosphate
group. Phosphate groups bridge the sugar
groups between the fifth carbon atom of
one deoxyribose molecule and the third
carbon atom of the adjacent deoxyribose
203

204

AABB Technical Manual

Figure 9-1. Schematic representation of the base
pairing of double-stranded DNA.

molecule and thus create the backbone of
the DNA strand. DNA polymerase-dependent synthesis always occurs in the direction of 5′ to 3′.

DNA Transcription
Linear sequences of nucleotides along the
DNA strands constitute the genes. Genes
occupy a constant location (locus) in the
DNA of a specific chromosome; the loci of
most known genes have been identified due
to the human genome project. For protein
synthesis to occur (see Fig 9-2), the information encoded in the DNA sequence
must be copied into RNA (transcription)
and transported to the cytoplasmic organelles called ribosomes where protein as-

sembly takes place (translation). Transcription is done by copying one strand of
the DNA into a primary ribonucleic acid
transcript, which is then modified into
messenger ribonucleic acid (mRNA). The
mRNA represents a single stranded linear
sequence of nucleotides that differs from
DNA in the sugar present in its backbone
(ribose instead of deoxyribose) and the replacement of thymine by uracil (U), which
also pairs with adenine. DNA transcription is catalyzed by the enzyme RNA polymerase. RNA polymerase binds tightly to a
specific DNA sequence called the promoter, which contains the site at which
RNA synthesis begins (see Fig 9-3). Proteins called transcription factors are required for RNA polymerase to bind to
DNA and for transcription to occur. Regulation of transcription can lead to increased, decreased, or absent expression
of a gene. For instance, a single base-pair
mutation in the transcription factor binding site of the Duffy gene promoter impairs the promoter activity and is respon1
sible for the Fy(a–b–) phenotype.
After binding to the promoter, RNA polymerase opens up the double helix of a local
region of DNA, exposing the nucleotides on
each strand. The nucleotides of one exposed DNA strand act as a template for complementary base pairing; RNA is synthesized by the addition of ribonucleotides to
the elongating chain. As RNA polymerase
moves along the template strand of DNA,
the double helix is opened before it and
closes behind it like a zipper. The process
continues, usually 0.5 to 2 kb downstream
of the poly-A signal, whereupon the enzyme halts synthesis and releases both the
DNA template and the new RNA chain.

mRNA Processing
Shortly after the initiation of transcription, the newly formed chain is capped at

Chapter 9: Molecular Biology in Transfusion Medicine

205

↓
↓
Figure 9-2. Model of a nucleotide sequence. The sequence is fictitious.

its 5′ end by the addition of a methylated
G nucleotide. The 5′ cap is important for
initiating protein synthesis and possibly
for protecting the mRNA molecule from
degradation during its transport to the cytoplasm. At the 3′ end of the mRNA, a
multiprotein cleavage-polyadenylation
complex carries out a two-step process
that cleaves the new RNA at a specific sequence and then attaches 100 to 200 cop-

ies of adenylic acid, called the poly-A tail.
The poly-A tail functions in the export of
mature mRNA from the cell nucleus to the
cytoplasm, in the stabilization of the mRNA,
and as a ribosomal recognition signal required for efficient translation.
In eukaryotic cells, the nucleotide sequence of a gene often contains certain regions that are represented in the mRNA and
other regions that are not represented. The

Figure 9-3. The promoter sequence (• ) contains the starting site for RNA synthesis. RNA polymerase
binds to the promoter and opens up a local region of the DNA sequence. One strand of DNA (the lower
one in this figure) acts as a template for complementary base pairing. The RNA polymerase copies the
DNA in a 5′ to 3′ direction until it encounters a stop signal (■). Lowercase letters represent nucleotides in introns; uppercase letters represent coding bases in exons. The sequence is fictitious.

206

AABB Technical Manual

regions of the gene that are represented in
mRNA are called exons, which specify the
protein-coding sequences and the sequences of the untranslated 5′ and 3′ regions. The regions that are not represented
in the mRNA are called intervening sequences or introns. In the initial transcription of DNA to RNA, the introns and exons
are copied in their entirety, and the resulting product is known as the primary RNA
transcript or pre-mRNA. Processing occurs
while pre-mRNA is still in the nucleus, and
the introns are cut out by a process known
as RNA splicing (see Fig 9-4).
RNA splicing depends on the presence of
certain highly conserved sequences consisting of GU at the 5′ splice site (donor site)
and AG at the 3′ splice site (acceptor site).
Additionally, an adenosine residue within a
specific sequence in the intron participates
in a complex reaction along with a very
large ribonucleoprotein complex called the
spliceosome. The reaction results in cleavage and joining of the 5′ and 3′ splice sites,
with release of the intervening sequence as
a lariat. Substitution of any of these highly
conserved sequences can result in inaccurate RNA splicing (see Fig 9-4). Splicing of

pre-RNA is highly regulated during differentiation and is tissue-specific. For example,
acetylcholinesterase, which bears the Cartwright blood group antigen, is spliced in a
manner to produce a glycosylphosphatidylinositol-linked protein in red cells, but it
is a transmembrane protein in nerve cells.2
Alternative splicing may also occur in a single tissue type and may result in the production of more than one protein from the
same gene; an example of this type is the
production of glycophorins C and D from a
single glycophorin C (GPC) gene.3

Translation of mRNA
The bases within a linear mRNA sequence
are read (or translated) in groups of three,
called codons. Each three-base combination codes for one amino acid. There are
only 20 amino acids commonly used for
protein synthesis, and there are 64 (4 × 4 ×
4) possible codons. Most amino acids can
be specified by each of several different
codons, a circumstance known as “degeneracy” or “redundancy” in the genetic
code. For example, lysine can be specified

Figure 9-4. Substitution of two nucleotides (boldface) in the intron sequences flanking exon 3 of GYPB
prevents normal splicing. Instead, all nucleotides between the 5′ donor site of the second intron and
the 3′ acceptor site of the third intron are excised. Because exon 3 is not translated, it is called a
pseudoexon (Ψ).

Chapter 9: Molecular Biology in Transfusion Medicine

by either AAA or AAG. Methionine has
only the single codon AUG. Three codons
(UAA, UGA, and UAG) function as stop signals; when the translation process encounters one of them, peptide synthesis
stops.
For the translation of codons into amino
acids, cytoplasmic mRNA requires the assistance of transfer RNA (tRNA) molecules.
The tRNA molecules interact with the
mRNA through specific base pairings and
bring with them the amino acid specified
by the mRNA codon. Thus, the amino acids
are linked in amino to carboxyl peptide
bonds forming a growing polypeptide
chain. Proteins are synthesized such that
the initial amino acid has an unlinked
amine (NH2) group and ends with a terminal carboxylic acid (COOH) group. Protein
synthesis occurs on ribosomes, which are
large complexes of RNA and protein molecules; ribosomes bound to the rough endoplasmic reticulum are the site of synthesis
of membrane and secretory proteins, whereas free ribosomes are the site of synthesis of
cytosolic proteins. The ribosome binds to
the tRNA and to the mRNA, starting at its 5′
end (amino terminal). Therefore, protein
synthesis occurs from the amino-terminal
end toward the carboxyl-terminal end. The
protein is produced sequentially until a stop
codon is reached, which terminates the
translation process and releases the newly
synthesized protein.
Many of the steps in the pathway of RNA
synthesis to protein production are closely
regulated at different levels to control gene
expression. Control steps include: initiation
of transcription, proofreading of the transcription process, addition of the poly-A tail,
transportation of mRNA to the cytosol, initiation of translation, and elongation of the
polypeptide chain. Moreover, tissue-specific
and differentiation or stage-specific transcription factors, as well as hormone response elements and regulatory elements

207

at the 5´ and 3´ ends of mRNA, can affect
gene expression.

Genetic Mechanisms that
Create Polymorphism
Despite the redundancy inherent in degeneracy of the genetic code, molecular
events such as substitution, insertion, or
deletion of a nucleotide may have farreaching effects on the protein encoded.
Some of the blood group polymorphisms
observed at the phenotypic level can be
traced to small changes at the nucleotide
level. The sequence in Fig 9-2, which is
not meant to represent a known sequence,
can be used to illustrate the effects of
minute changes at the nucleotide level,
discussed below.

Nucleotide Substitution
Nucleotide substitutions in the genomic
DNA can have profound effects on the resultant protein. Many blood group antigens are the result of single nucleotide
changes at the DNA level. The nucleotide
changes are transcribed into the RNA,
which alters the sequence of the codon. In
some instances, the codon change results
in the incorporation of a different amino
acid. For example, a change in the DNA
sequence from a T to a C at a given position would result in an mRNA change
from U to G. Any one of three possible
outcomes can follow the substitution of a
single nucleotide:
1.
Silent mutation. For example, the
substitution of an A with a C in the
third position of the DNA coding
strand for serine (UCU) in Fig 9-2 also
codes for serine (UCG). Thus, there
would be no effect on the protein
because the codon would still be
translated as serine.

208

AABB Technical Manual

Missense response. The substitution
of a G with an A in the second position of the DNA coding strand for the
second serine changes the product
of the codon from serine (UCG) to
leucine (UUG). Many blood group
polymorphisms reflect a single amino
acid change in the underlying molecule. For example, the K2 antigen has
threonine, but K1 has methionine, as
the amino acid at position 193. This
results from a single C to T substitution in exon 6 of the Kell (KEL) gene.4
3.
Nonsense response. The substitution of a G with a T in the second position of the DNA coding strand for
serine (UCG) results in the creation of
the codon (UAG), which is one of the
three stop codons. No protein synthesis will occur beyond this point,
resulting in a shortened or truncated
protein. Depending upon where this
nonsense substitution occurs, the
synthesized protein may be rapidly
degraded or may retain some function in its abbreviated form. The
Cromer blood group antigens reside
on the decay-accelerating factor
(DAF) membrane protein. In the null
(Inab) phenotype, a G to A nucleotide substitution of the DAF gene
creates a stop codon at position 53,
and the red cell has no DAF expression.5
A nucleotide substitution outside an
exon sequence may alter splicing and lead
to the production of altered proteins, as
seen with the altered expression of glycophorin B (GPB)6 or in the failure to produce
a normal amount of protein, as in the
Dr(a–) phenotype of Cromer.7 In GYPB, the
gene that encodes GPB, substitutions of
two conserved nucleotides required for
RNA splicing and present in the
glycophorin A gene result in the excision of
exon 3 as well as introns 2 and 3 of GYPB

(see Fig 9-4). Because exon 3 is a coding
exon in GYPA but is noncoding in GYPB, it
is called a pseudoexon in GYPB.

Nucleotide Insertion and Deletion
Insertion of an entirely new nucleotide results in a frameshift, described as +1, because a nucleotide is being added. Nucleotide deletion causes a –1 frameshift. A
peptide may be drastically altered by the
insertion or deletion of a single nucleotide. For example, the insertion of an A after the second nucleotide of the noncoding
DNA strand of Fig 9-2 would change the
reading frame of the mRNA to UAU CAG
AAG CUG CCC UGG and represent the
polypeptide isoleucine-valine-phenylalanine-aspartic acid-glycine-threonine.

Genetic Variability
Gene conversion and crossing over may
occur between homologous genes located
on two copies of the same chromosome
that are misaligned during meiosis. Examples of homologous genes encoding blood
group antigens are the RHD and RHCE
genes of the Rh blood group system and
GYPA and GYPB, which encode the antigens of the MNS blood group system.

Single Crossover
A single crossover is the mutual exchange
of nucleotides between two homologous
genes. If crossover occurs in a region
where paired homologous chromosomes
are misaligned, two hybrid genes are
formed in reciprocal arrangement (see Fig
9-5). The novel amino acid sequences encoded by the nucleotides at the junction
of the hybrid gene may result in epitopes
recognized by antibodies in human serum and are known to occur in at least
three blood group systems: Rh, MNS, and
Gerbich.

Chapter 9: Molecular Biology in Transfusion Medicine

209

Figure 9-5. Single crossover: exchange of nucleotides between misaligned homologous genes. The
products are reciprocal.

Gene Conversion

Molecular Techniques

The process of gene conversion is thought
to consist of crossover, a general DNA recombination process, and DNA repair
during meiosis. The result is that nucleotides from one homologous gene are inserted into another gene without reciprocal exchange. At the site of chromosome
crossover during meiosis, a heteroduplex
joint can form; this is a staggered joint between nucleotide sequences on two participating DNA strands (see Fig 9-6). A
second type of gene conversion occurs
in meiosis when the DNA polymerase
switches templates and copies information from a homologous sequence. This
event is usually the result of mismatch
repair; nucleotides removed from one
strand are replaced by repair synthesis using the homologous strand as a template.

The development of modern molecular
techniques has greatly expanded our
knowledge of all biologic systems. These
same techniques are also applicable to the
diagnosis of disease, the practice of forensic science, the generation of recombinant proteins, and the production of functional genes for gene therapy. Many of
these processes begin with DNA typing
and analysis, techniques that are reviewed
below.

Isolation of Nucleic Acids
The first step in most molecular biology
techniques is the isolation and purification of nucleic acid, either DNA or RNA.
For applications of interest to the blood
banking community, the desired nucleic

210

AABB Technical Manual

Figure 9-6. Gene conversion: a heteroduplex joint forms between homologous sequences on two genes.
DNA polymerase repairs the double strands. Any excess single-stranded DNA is degraded by nucleases, producing a hybrid gene on one chromosome but not on the other.

acid is typically human genomic DNA and
mRNA. Genomic DNA is present in all nucleated cells and can be isolated from peripheral blood white cells or from buccal
tissue obtained by a simple cheek swab.
Both nucleated cells and reticulocytes are
cell-specific sources of mRNA.
Manufacturers offer kits for the isolation
of human genomic DNA from whole blood,

cells, and tissues. These kits vary in the
quantity and quality of the DNA isolated, in
rough proportion to the cost and ease of
use of the kit. High-quality DNA is of high
molecular weight and is relatively free of
contamination by protein or RNA. DNA purity is assessed by the ratio of its optical
density (OD) at 260 nm to that at 280 nm,
with the OD 260/280 ratio for pure DNA be-

Chapter 9: Molecular Biology in Transfusion Medicine

ing 1.8. Low ratios (<1.6) indicate that the
DNA is contaminated with protein or materials used in the isolation procedure, and
high ratios (>2.0) indicate that the DNA is
contaminated with RNA. If the DNA is pure
and of sufficient concentration, it can be
quantitated by measurement of the OD at
260 nm. If the DNA is impure or in low concentration, it is best quantitated by electrophoresis in agarose gel along with DNA
standards of known concentration, followed by visualization of the DNA with
ethidium bromide staining.
For certain molecular biology techniques
such as polymerase chain reaction (PCR),
the quantity and quality of the genomic
DNA used as starting material are not crucial, and good results can be obtained with
even nanogram quantities of DNA that has
been degraded into small fragments. For
other molecular biology techniques, such
as cloning, larger quantities of high-molecular-weight DNA are required.
One nucleated cell contains about 6 pg
of genomic DNA. Based on an average
white cell count of 5000/µL, each milliliter
of peripheral blood contains about 30 µg of
DNA. Commercial DNA isolation kits typically yield in excess of 15 µg of DNA per
milliliter of whole blood processed.

Polymerase Chain Reaction
The introduction of the PCR technique has
revolutionized the field of molecular genetics.8 This technique permits specific
DNA sequences to be multiplied rapidly
and precisely in vitro. PCR can amplify, to
a billionfold, a single copy of the DNA sequence under study, provided a part of the
nucleotide sequence is known. The investigator must know at least some of the
gene sequence in order to synthesize DNA
oligonucleotides for use as primers. Two
primers are required: a forward primer (5′)

211

and a reverse primer (3′). These are designed so that one is complementary to
each strand of DNA, and, together, they
flank the region of interest. Primers can
be designed that add restriction sites to
the PCR product to facilitate its subsequent cloning or labeled to facilitate its
detection. Labels may incorporate radioactivity, or, more frequently, a nonradioactive tag, such as biotin or a fluorescent
dye. The PCR reaction is catalyzed by one
of several heat-stable DNA polymerases
isolated from bacterial species that are native to hot springs or to thermal vents on
the ocean floor. The thermostability of
these enzymes allows them to withstand
repeated cycles of heating and cooling.

Reaction Procedure
The amplification technique is simple and
requires very little DNA (typically <100 ng
of genomic DNA). The DNA under study
is mixed together with a reaction buffer,
excess nucleotides, the primers, and polymerase (see Fig 9-7). The reaction cocktail
is placed in a thermocycler programmed
to produce a series of heating and cooling
cycles that result in exponential amplification of the DNA. The target DNA is initially denatured by heating the mixture,
which separates the double-stranded DNA
into single strands. Subsequent cooling in
the presence of excess quantities of single-stranded forward and reverse primers
allows them to bind or anneal with complementary sequences on the singlestranded template DNA. The specific cooling temperature is calculated to be appropriate for the primers being used. The reaction mixture is then heated to the
optimal temperature for the thermo-stable DNA polymerase, which generates a
new strand of DNA on the single-strand
template, using the nucleotides as build-

212

AABB Technical Manual

Figure 9-7. The polymerase chain reaction results in the exponential amplification of short DNA sequences such that the target sequence is amplified over a billionfold after 20 cycles. (Taq polymerase
is used here as an example of a thermostable DNA polymerase.)

Chapter 9: Molecular Biology in Transfusion Medicine

ing blocks for elongation. The next cycle
denatures newly formed double strands,
and the single-stranded DNA copies serve as
templates for subsequent synthesis. The
number of DNA copies doubles with each
cycle, such that after 20 cycles, there is a
billionfold amplification of the target DNA.
The amplified DNA may be analyzed by
agarose gel electrophoresis in the presence
of ethidium bromide, which binds to DNA
and is visible under ultraviolet light. The
DNA will be present as a single discrete
band equivalent in length to the distance
between the 5′ ends of the primers. The
amplified DNA can also be differentiated by
size using capillary electrophoresis. Alternatively, the DNA sample can be blotted
onto a membrane and hybridized to a labeled, allele-specific probe. This is known
as a dot blot and is particularly useful when
multiple samples are being analyzed for the
same polymorphism.
Variations of the PCR have been developed to meet specific needs. For instance,
“long-distance” PCR, which has a mixture
of thermostable polymerases, can amplify
much larger targets (up to 40 kilobases in
length) than those typically amplified by
conventional PCR (up to 2 kilobases in
length). It is even possible to perform PCR
in situ, in tissue and cells. A related technology, the ligase chain reaction (LCR), uses
a thermostable DNA ligase instead of a
thermostable DNA polymerase. Rather
than amplification of a DNA target segment
located between two flanking primers using
a DNA polymerase as occurs in PCR, in the
LCR direct primer ligation occurs with no
amplification of an intervening DNA segment. Other PCR variations include multiplex PCR, in which multiple independent
segments of DNA are co-amplified in the
same reaction, and kinetic PCR, in which
the amplification product is measured in
real time in order to quantitate the amount
of starting nucleic acid.

213

Applications of PCR
Amplification of minute quantities of DNA
to detectable levels may significantly affect the practice of transfusion medicine.
In screening donor blood for infectious
agents, PCR has become the procedure of
choice and thus eliminate our reliance on
seroconversion, which occurs well after
exposure to viruses or other pathogens
(see Chapter 28). Blood centers have been
using nucleic acid amplification testing
(NAT) to identify the presence of HIV and
HCV RNA in donor samples; detection of
West Nile virus RNA has been performed
as an investigational test. Detection of viral RNA involves three steps: extraction,
amplification, and detection. Extraction
may occur following centrifugation steps
to concentrate the virus and remove contaminants. Otherwise, RNA extraction can
occur first followed by capture of viral
RNA onto a molecule that is immobilized
on a solid phase or onto magnetic particles in solution (this procedure is referred
to as target capture). Once immobilized,
the impurities may be removed by a series
of wash steps. Before amplification, viral
RNA must be converted to DNA; this is accomplished by the enzyme, reverse transcriptase (RT). Amplification of the DNA
then can occur through multiple intermediates. In the case of PCR, the amplified
product is DNA (and is synthesized using
thermostable Taq DNA polymerase),
whereas, in the case of transcription-mediated amplification, the amplified product is RNA (and is synthesized using T7
RNA polymerase) (see Fig 9-8). Detection
of the amplified product can occur by
capture of the amplified DNA on nitrocellulose or by enzyme immunoassay or
chemiluminescence. Currently, NAT for
blood donor screening has been implemented for HIV and hepatitis C; NAT for
hepatitis B is under development. NAT for

214

AABB Technical Manual

other agents (eg, human T-cell lymphotropic virus, hepatitis A virus, parvovirus
B19) is not widely available.
PCR is being used for prenatal determination of many inheritable disorders, such
as sickle cell disease, in evaluating hemolytic
disease of the fetus and newborn, to type
fetal amniocytes,9 to quantitate residual
white cells in filtered blood, and for tracing
donor leukocytes in transfusion recipients
(chimerism). Long-distance PCR is used for
cloning, sequencing, and chromosome
mapping, and reverse transcriptase (RT)PCR is used for studying gene expression
and cDNA cloning. LCR has special applicability in transfusion medicine because of
its powerful ability to detect genetic variants. In the field of transplantation, PCR using sequence-specific oligonucleotide
probes or sequence-specific primers is
used to determine HLA types (see Chapter
17).

Restriction Endonucleases
The discovery of bacterial restriction endonucleases provided the key technique for
DNA analysis. These enzymes, found in
different strains of bacteria, protect a bacteria cell from viral infection by degrading
viral DNA after it enters the cytoplasm.
Each restriction endonuclease recognizes
only a single specific nucleotide sequence,
typically consisting of four to six nucleotides. These enzymes cleave the DNA
strand wherever the recognized sequence
occurs, generating a number of DNA fragments whose length depends upon the
number and location of cleavage sites in
the original strand. Many endonucleases
have been purified from different species
of bacteria; the name of each enzyme reflects its host bacterium, eg, Eco RI is isolated from Escherichia coli, Hind III is
from Hemophilus influenzae, and Hpa I is
from Hemophilus parainfluenzae.

Restriction Fragment Length Polymorphism
Analysis
The unique properties of restriction endonucleases make analysis of restriction
fragment length polymorphism (RFLP)
suitable for the detection of a DNA polymorphism. The changes in nucleotide sequence described above (substitution, insertion, deletion) can alter the relative
locations of restriction nuclease cutting
sites and thus alter the length of DNA
fragments produced. RFLPs are detected
using Southern blotting and probe hybridization (see Fig 9-9).
The isolated DNA is cleaved into fragments by digestion with one or more restriction endonucleases. The DNA fragments are separated by electrophoresis
through agarose gel and then transferred
onto a nylon membrane or nitrocellulose
paper. Once fixed to a nylon membrane or
nitrocellulose paper, the DNA fragments
are examined by application of a probe,
which is a small fragment of DNA whose
nucleotide sequence is complementary to
the DNA sequence under study. A probe
may be an artificially manufactured oligonucleotide or may derive from cloned complementary DNA. The probe is labeled with
a radioisotope or another indicator that
permits visualization of the targeted DNA
restriction fragments and is then allowed
to hybridize with the Southern blot. Unbound excess probe is washed off, and hybridized DNA is visualized as one or more
bands of specific size, dictated by the specific nucleotide sequence. If several individuals are analyzed for polymorphism,
several different banding patterns may be
observed.
RFLP analysis has been used in gene
mapping and analysis, linkage analysis,
characterization of HLA genes in transplantation, paternity testing, and forensic science.

Chapter 9: Molecular Biology in Transfusion Medicine

Step 1:
Step 2:
Step 3:
Step 4:
Step 5:
Step 6:
Step 7:
Step 8:
Step 9:
Step 10:
Step 11:
Step 12:

Promoter-primer binds to rRNA target.
Reverse transcriptase (RT) creates DNA copy of rRNA target.
RNA:DNA duplex.
RNAse H activities of RT degrade the rRNA.
Primer 2 binds to the DNA and RT creates a new DNA copy.
Double-stranded DNA template with a promoter sequence.
RNA polymerase (RNA Pol) initiates transcription of RNA from DNA template.
100-1000 copies of RNA amplicon are produced.
Primer 2 binds to each RNA amplicon and RT creates a DNA copy.
RNA:DNA duplex.
RNAse H activities of RT degrade the rRNA.
Promoter-primer binds to the newly synthesized DNA. RT creates a double-stranded DNA and the autocatalytic cycle
repeats, resulting in a billion-fold amplification.

Figure 9-8. Transcription-mediated amplification cycle.

215

216

AABB Technical Manual

Figure 9-9. Southern blotting: a technique for the detection of polymorphism by gel-transfer and hybridization with known probes.

DNA Profiling
Regions of DNA that show great allelic variability (“minisatellites” and “microsatellites”) can be studied by the application of
RFLP mapping and/or PCR analysis (a process sometimes called DNA profiling, DNA
typing, or DNA fingerprinting). Minisatellites or variable number of tandem repeat
(VNTR) loci consist of tandem repeats of a
medium-sized (6-100 base-pair) sequence,
whereas microsatellites or short tandem
repeat (STR) loci consist of tandem repeats of a short (typically four base pair)
sequence. These regions are almost always found in the noncoding regions of
DNA. Variability stems from differences in
the number of repeat units contained
within the fragments. There is so much

variation between individuals that the
chances are very low that the same numbers of repeats will be shared by two individuals, even if related. The VNTR and/or
STR patterns observed at four to eight different loci may be unique for an individual and thus constitute a profile or “fingerprint” that identifies his or her DNA.
When DNA profiling was developed,
testing was performed by RFLP analysis.
DNA profiling is now increasingly done by
amplification of selected, informative
VNTR and/or STR loci using locus-specific
oligonucleotide primers, followed by measurement of the size of the PCR products
produced. PCR products can be separated
by size by electrophoresis through polyacrylamide gel and detected by silver staining, or, if the PCR products incorporate a

Chapter 9: Molecular Biology in Transfusion Medicine

fluorescent tag, by fluorescent detection
systems including those designed for automated DNA sequencing. Comparison of the
VNTR and STR PCR products with a standard-size ladder distinguishes the alleles
present in the sample.
DNA profiling is a technique that is extremely powerful for identifying the source
of human DNA; therefore, it has applications in forensic and paternity testing, as well
as in the documentation of chimerism, which
is of special importance in monitoring
allogeneic hematopoietic transplantation.

DNA Cloning
PCR may also be used for analysis of mRNA,
which is an especially useful source of genetic material because only the exons of
the gene are present. By a modification of
PCR called RT-PCR, the single-stranded
mRNA is converted to double-stranded
DNA. The enzyme RT is used to generate
a single strand of DNA, which serves as a
template for a second strand generated by
DNA polymerase. The product is complementary DNA (cDNA) and it is the DNA
molecule of choice for cloning and sequencing.
In gene cloning, the DNA containing the
gene of interest is inserted into a vector,
which is a self-replicating genetic element
such as a virus (eg, the bacteriophage lambda
gt11) or a plasmid. Plasmids are small circular molecules of double-stranded DNA
that occur naturally in bacteria and typically confer antibiotic resistance. After the
gene has been inserted into the DNA of the
vector, the recombinant DNA can be introduced into a bacterial host where it undergoes replication. Because many vectors
carry genes for antibiotic resistance, this
characteristic can be exploited by growing
the host bacteria in the presence of the appropriate antibiotic; only bacteria that have

217

successfully incorporated the recombinant
vector will survive to form colonies or
clones. Each individual vector potentially
contains a different cDNA sequence. The
sum of bacterial clones harboring recombinant vectors is called a DNA library. Libraries can be obtained from many commercial
sources or can be produced by the individual investigator. The library can be probed
through a technique similar to Southern
blotting, with an oligonucleotide probe
based on part of a known sequence. Positive clones can be selected and a pure culture grown in large quantities. Once purified, the cloned DNA can be recovered for
use as a probe or for detailed molecular
characterization.
The ability to insert genes into the
genomes of virtually any organism, including bacteria, plants, invertebrates (such as
insects), and vertebrates (such as mammals), permits not only gene characterization but also genetic engineering, including
the production of recombinant proteins (see
below) and gene therapy. Although still in
the developmental stages, gene therapy
promises to have a role in the management
of disorders as diverse as inherited genetic
diseases, human immunodeficiency virus,
and cancer, and in the development of
novel vaccines.10,11

DNA Sequencing
A major worldwide scientific effort called
the Human Genome Project has obtained
the complete nucleotide sequence of the
human genome as well as the genomes of
several other key organisms. The initiative
also improved DNA sequencing technology. Realization of both goals has had a
positive impact on transfusion practice.
The identification of all human genes12,13
provides a complete blueprint of the proteins that are relevant in transfusion medicine. In turn, this information has helped

218

AABB Technical Manual

in the development of recombinant proteins for use as transfusion components and
in-vitro test reagents. It also plays a role in
clarifying the disorders that afflict transfusion recipients.
Advances in DNA sequencing have taken
the field a long way from the cumbersome
manual techniques common in research
laboratories until recently.14 Automated
DNA sequencers using laser detection of
fluorescently labeled sequencing products
detect all four nucleotide bases in a single
lane on polyacrylamide gel and can be optimized for specialty applications such as
heterozygote detection and sizing of PCR
fragments. Automated DNA sequencers using capillary electrophoresis are especially
useful for the rapid sequencing of short
DNA templates. DNA sequence can also be
obtained using mass spectrometry. Automated sequencers will become increasingly
common in clinical laboratories as this
technology evolves. If it can be made
cost-effective for routine use, then DNA sequencing could become a routine genotyping method.

DNA Microarrays
The complexity of the human genome requires that the differential expression of
multiple genes be analyzed at once to understand normal biologic processes as well
as changes in diseases. A powerful technique to accomplish this goal is DNA
15
microarrays or gene chips. In this method,
tens of thousands of separate DNA molecules are spotted or synthesized on a small
area of a solid support, often a glass slide.
The DNA can be generated by PCR or oligonucleotide synthesis. This microarray is
then probed, in a process analogous to
Southern blotting, using cDNA created
from the total mRNA expressed at a given
time by a cell or tissue. The result is a picture of the gene expression profile of the

tissue for all of the genes on the microarray. Also, microarrays can be used for
comparative genomics and genotyping, an
application for blood groups.

Recombinant Proteins
The technology to make recombinant
proteins includes in-vitro systems in bacteria, yeast, insect cells, and mammalian
cells, as well as in-vivo systems involving
transgenic plants and animals.16 First, a
source of DNA corresponding in nucleic
acid sequence to the desired protein is
prepared, typically by cloning the cDNA
and ligating it into a suitable expression
vector. Then, the expression vector containing the DNA of interest is transfected
into the host cell, and the DNA of interest
is transcribed under the control of the
vector promoter. Next, the resulting mRNA
is translated into protein by the host cell.
Posttranslational modifications such as
the addition of carbohydrates to the new
protein will be carried out by the host cell.
If specific posttranslational modifications
required for the new protein’s function cannot be carried out by the host cell, then it
may be necessary to endow the host cell
with additional capabilities; for example, by
cotransfection with the cDNA for a specific
enzyme. In some cases, posttranslational
modification may not be crucial for a recombinant protein to be effective; for
instance, granulocyte colony-stimulating
factor (G-CSF) is produced in a nonglycosylated form in E. coli (filgrastim) and in a
glycosylated form in yeast (lenograstim).
Recombinant proteins are finding multiple uses in transfusion medicine, as therapeutic agents and vaccine components, in
component preparation, in virus diagnosis,
and in serologic testing. Recombinant human erythropoietin,17 G-CSF and GM-CSF,18
interferon-alpha, interleukin-2, interleu19
20
21
kin-11, and Factors VIII, IX, VII, and VIIa

Chapter 9: Molecular Biology in Transfusion Medicine

are all available and finding clinical acceptance. For instance, recombinant erythropoietin can be used to increase red cell production in anemic patients before surgery,
reducing the need for allogeneic blood.22 It
can also be used to increase the amount of
autologous red cells that can be withdrawn
before surgery from non-anemic patients.23
Moreover, it has revolutionized the management of renal transplant candidates
whose kidneys are too impaired to produce
endogenous erythropoietin.
Recombinant human thrombopoietin
may be of value in augmenting platelet
yields from apheresis donors but is unlikely
to significantly reduce the need for platelet
transfusions when given to thrombocytopenic patients.24 In the coagulation
arena, recombinant proteins such as protein C, antithrombin, and hirudin are approved by the Food and Drug Administration for use, and tissue factor pathway
inhibitor, Factor XIII, and von Willebrand
factor appear promising. Recombinant
myeloid growth factors such as G-CSF are
used to enhance yields of progenitor cells
during apheresis and to support patients
following chemotherapy and hematopoietic transplantation.25
Recombinant proteins can be used as
transfusion components.26 Recombinant
human hemoglobin has been produced in
a number of in-vitro expression systems
and in vivo in transgenic swine and may be
useful as a noninfectious blood substitute.27
Recombinant human serum albumin has
been produced in yeast. Alphagalactosidase, an enzyme that is capable of converting group B cells into group O cells, has
been produced in a recombinant form that
can modify group B units for transfusion to
group A and O recipients.28 These recombinant proteins and other products under development will undoubtedly affect the variety of transfusion components that will
become available in the future.

219

Recombinant proteins corresponding to
proteins from clinically relevant viruses,
bacteria, and parasites, some of which may
be transmitted by blood transfusion, may
be used as vaccine components29 and as
antigens in test kits for the detection of antibodies. Cells transfected with appropriate
vectors can be induced to express recombinant proteins on the membrane surface
and, as such, may become useful as genetically engineered reagent cells for in-vitro
testing.

Gene Therapy
Gene therapy refers to the introduction of
nonself genetic material into cells to treat
or prevent disease. At present, gene therapy is restricted to somatic cells because
of ethical concerns about the transfer of
genes to germ-line cells. More than 3500
patients have been administered gene
therapy in clinical trials,30 but results overall have been disappointing largely due to
problems with delivery (transfection) of
the genetic material (transgene) into cells
and the limited lifespan of the successfully transfected cells.
There are different types of vectors (genedelivery vehicles) that deliver genes to cells,
including viral vectors (retrovirus, adenovirus, adeno-associated virus, vaccinia, herpes simplex), naked DNA, and modified DNA.
Genetic material can also be transferred to
cells by physical means such as electroporation (use of an electric field). As methods for gene delivery improve, it is anticipated that the benefits of gene therapy will
become more apparent.
Gene therapy can be used to replace a
defective gene, leading to increased production of a specific protein as in the replacement of Factor VIII or IX for patients
with hemophilia,31 or it can be used to
downregulate (reduce) expression of an undesirable gene. The latter can be accom-

220

AABB Technical Manual

plished by the introduction of DNA sequences (antisense oligonucleotides)
corresponding to the antisense strand of
the mRNA, which then interferes with
translation of the mRNA into protein.

Protein and RNA Targeted Inactivation
More recently, novel pharmacologic agents
have been developed that interfere with
specific molecules produced by cancer
cells or infectious particles. One such example is imatinib mesylate (Gleevec,
Novartis Pharmaceuticals, East Hanover,
NJ), which binds specifically to the BcrAbl protein and blocks its tyrosine kinase
activity in malignant white cells. It specifically blocks the binding site for adenosine triphosphate on the kinase, thus
inhibiting its ability to phosphorylate
intracellular proteins.32 The inactivation
prevents Bcr-Abl-induced malignant cell
proliferation and anti-apoptosis. Normal
kinase signaling pathways are largely unaffected.
Another promising therapy is RNA interference, which has its historical roots as a
research tool used to characterize the function of known genes. RNA interference is
based on an antiviral mechanism in which
dsRNA is delivered to a cell and is subsequently processed into small (21-25 bp) interfering RNA (siRNA) molecules. The siRNA
molecules silence the expression of a target
gene in a sequence-specific manner. More
important, RNA interference has potential
as a therapeutic strategy to silence cancer-related genes or infectious diseases like viral
hepatitis.33

10.
11.

12.

13.

14.

15.

16.

References
1.

Tournamille C, Colin Y, Cartron JP, Le Van
Kim C. Disruption of a GATA motif in the
Duffy gene promoter abolishes erythroid
gene expression in Duffy-negative individuals. Nat Genet 1995;10:224-8.

17.

Li Y, Camp S, Taylor P. Tissue-specific expression and alternative mRNA processing of the
mammalian acetylcholinesterase gene. J Biol
Chem 1993;268:5790-7.
Le Van Kim C, Mitjavila MT, Clerget M, et al.
An ubiquitous isoform of glycophorin C is
produced by alternative splicing. Nucleic Acids Res 1990;18:3076.
Lee S, Wu X, Reid M, et al. Molecular basis of
the Kell (K1) phenotype. Blood 1995;85:91216.
Lublin DM, Mallinson G, Poole J, et al. Molecular basis of reduced or absent expression of
decay-accelerating factor in Cromer blood
group phenotypes. Blood 1994;84:1276-82.
Kudo S, Fukuda M. Structural organization of
glycophorin A and B genes: Glycophorin B
gene evolved by homologous recombination
at Alu repeat sequences. Proc Natl Acad Sci
U S A 1989;86:4619-23.
Lubin DM, Thompson ES, Green AM, et al.
Dr(a–) polymorphism of decay accelerating
factor. Biochemical, functional, and molecular characterization and production of allelespecific transfection. J Clin Invest 1991;87:
1945-52.
Erlich HA. Principles and applications of the
polymerase chain reaction. Rev Immunogenet 1999;1:127-34.
Bennett PR, Le Van Kim C, Colin Y, et al. Prenatal determination of fetal RhD type by DNA
amplification. N Engl J Med 1993;329:607-10.
Friedmann T. Overcoming the obstacles to
gene therapy. Sci Am 1997;276:80-5.
Hillyer CD, Klein HG. Immunotherapy and
gene transfer in the treatment of the oncology patient: Role of transfusion medicine.
Transfus Med Rev 1996;10:1-14.
International Human Genome Sequencing
Consortium. Initial sequencing and analysis
of the human genome. Nature 2001;409:860921.
Venter JC, Adams MD, Myers EW, et al. The
sequence of the human genome. Science
2001;291:1304-51.
Griffin HG, Griffin AM. DNA sequencing. Recent innovations and future trends. Appl Biochem Biotechnol 1993;38:147-59.
Duggan DJ, Bittner M, Chen Y, et al. Expression profiling using cDNA microarrays. Nat
Genet 1999;21:S10-14.
Lubon H, Paleyanda RK, Velander WH,
Drohan WN. Blood proteins from transgenic
animal bioreactors. Transfus Med Rev 1996;
10:131-43.
Cazzola M, Mercuriali F, Brugnara C. Use of
recombinant human erythropoietin outside
the setting of uremia. Blood 1997;89:4248-67.

Chapter 9: Molecular Biology in Transfusion Medicine

18.

19.

20.

21.

22.

23.

24.
25.
26.

27.

28.

29.

30.
31.

32.

33.

34.

Ganser A, Karthaus M. Clinical use of hematopoietic growth factors. Curr Opin Oncol 1996;
8:265-9.
VanAken WG. The potential impact of recombinant factor VIII on hemophilia care and the
demand for blood and blood products. Transfus Med Rev 1997;11:6-14.
White GC II, Beebe A, Nielsen B. Recombinant factor IX. Thromb Haemost 1997;78:
261-5.
Lusher JM. Recombinant factor VIIa (NovoSeven) in the treatment of internal bleeding
in patients with factor VIII and IX inhibitors.
Haemostasis 1996;26(Suppl 1):124-30.
Braga M, Gianotti L, Gentilini O, et al. Erythropoietic response induced by recombinant human erythropoietin in anemic cancer
patients candidate to major abdominal surgery. Hepatogastroenterology 1997;44:685-90.
Cazenave JP, Irrmann C, Waller C, et al. Epoetin alfa facilitates presurgical autologous
blood donation in non-anaemic patients
scheduled for orthopaedic or cardiovascular
surgery. Eur J Anaesthesiol 1997;14:432-42.
Kuter DJ. What ever happened to thrombopoietin? Transfusion 2002;42:279-83.
Ketley NJ, Newland AC. Haemopoietic growth
factors. Postgrad Med J 1997;73:215-21.
Growe GH. Recombinant blood components:
Clinical administration today and tomorrow.
World J Surg 1996;20:1194-9.
Kumar R. Recombinant hemoglobins as
blood substitutes: A biotechnology perspective. Proc Soc Exp Biol Med 1995;208:150-8.
Lenny LL, Hurst R, Zhu A, et al. Multiple-unit
and second transfusions of red cells enzymatically converted from group B to group O: Report on the end of phase 1 trials. Transfusion
1995;35:899-902.
Ellis RW. The new generation of recombinant
viral subunit vaccines. Curr Opin Biotechnol
1996;7:646-52.
Mountain A. Gene therapy: The first decade.
Trends Biotechnol 2000;18:119-28.
Kaufman RJ. Advances toward gene therapy
for hemophilia at the millennium. Hum Gene
Therapy 1999;10:2091-107.
Druker BJ, Tamura S, Buchdunger E, et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive
cells. Nat Med 1996;2:561-6.
Radhakrishnan SK, Layden TJ, Gartel AL. RNA
interference as a new strategy against viral
hepatitis. Virology 2004;323:173-81.
Farese AM, Schiffer CA, MacVittie TJ. The impact of thrombopoietin and related mpl-lig-

35.

36.

37.

38.

39.

40.
41.

42.

43.

44.

221

ands on transfusion medicine. Transfus Med
Rev 1997;11:243-55.
Barbara JA, Garson JA. Polymerase chain reaction and transfusion microbiology. Vox
Sang 1993;64:73-81.
Power EG. RAPD typing in microbiology—a
technical review. J Hosp Infect 1996;34:24765.
Larsen SA, Steiner BM, Rudolph AH, Weiss JB.
DNA probes and PCR for diagnosis of parasitic infections. Clin Microbiol Rev 1995;8:121.
Weiss JB. DNA probes and PCR for diagnosis
of parasitic infections. Clin Microbiol Rev
1995;8:113-30.
Majolino I, Cavallaro AM, Scime R. Peripheral
blood stem cells for allogeneic transplantation. Bone Marrow Transplant 1996;18(Suppl
2):171-4.
Gretch DR. Diagnostic tests for hepatitis C.
Hepatology 1997;26:43S-7S.
Pena SD, Prado VF, Epplen JT. DNA diagnosis
of human genetic individuality. J Mol Med
1995;73:555-64.
van Belkum A. DNA fingerprinting of medically important microorganisms by use of PCR.
Clin Microbiol Rev 1994;7:174-84.
Siegel DL. Research and clinical applications
of antibody phage display in transfusion
medicine. Transfus Med Rev 2001;15:35-52.
Hyland CA, Wolter LC, Saul A. Identification
and analysis of Rh genes: Application of PCR
and RFLP typing tests. Transfus Med Rev
1995;9:289-301.

Suggested Reading
Denomme G, Lomas-Francis C, Storry RJ, Reid ME.
Approaches to molecular blood group genotyping
and their applications. In: Stowell C, Dzid W, eds.
Emerging technologies in transfusion medicine.
Bethesda, MD: AABB Press, 2003:95-129.
Garratty G, ed. Applications of molecular biology
to blood transfusion medicine. Bethesda, MD:
AABB, 1997.
Lewin B. Genes VII. Oxford: Oxford University Press,
2000.
Sheffield WP. Concepts and techniques in molecular biology—an overview. Transfus Med Rev 1997;
11:209-23.

222

AABB Technical Manual

Appendix 9-1. Molecular Techniques in Transfusion Medicine
Technique

Applications

Examples

PCR

Infectious disease
testing

Viruses, bacteria,
parasites

35
36
37, 38

Polymorphism detection

HLA, blood group
antigens

1, 39

Prenatal detection

Recombinant proteins/ Therapy
DNA cloning

Erythropoietin,
thrombopoietin,
G-CSF

9
17, 18, 34

Apheresis

G-CSF

Infectious disease
testing

Viral testing

Recombinant
components

Coagulation factors

Hemoglobin

Component processing Alpha-galactosidase

DNA profiling

References

Vaccine production

Hepatitis, malaria, HIV

Human identification

Chimerism, forensic
science
Bacterial typing

Bacterial identification

27-29

DNA sequencing

Polymorphism detection, heterozygote
detection

HLA

Phage display/
repertoire cloning

Monoclonal antibody
production

Anti-D

RFLP

Polymorphism
detection

HLA, blood group
antigens

PCR = polymerase chain reaction; G-CSF = granulocyte colony-stimulating factor; HIV = human immunodeficiency virus; RFLP = restriction fragment length polymorphism.

Chapter 10: Blood Group Genetics

Chapter 10

Blood Group Genetics

ANDSTEINER’S DISCOVERY OF
the ABO blood group system demonstrated that human blood expressed inheritable polymorphic structures.
Shortly after the discovery of the ABO system, red cells proved to be an easy and accessible means to test for blood group
polymorphisms in individuals of any age.
As more blood group antigens were described, blood group phenotyping provided a wealth of information about the
polymorphic structures expressed on proteins, glycoproteins and glycolipids on
red cells, and the genetic basis for their
inheritance.

Basic Principles
Inheritance of transmissible characteristics or “traits,” including blood group antigens, forms the basis of the science of
genetics. The genetic material that determines each trait is found in the nucleus

of a cell. This nuclear material is called
chromatin and is primarily made up of DNA
(see Chapter 9). When a cell divides, the
chromatin loses its homogenous appearance and forms a number of rod-shaped
organelles called chromosomes. Encoded
in the chromatin or chromosomal DNA
are units of genetic information called
genes. The genes are arranged in a specific order along a chromosome with the
precise gene location known as the locus.

Chromosomes
The number of chromosomes and chromosome morphology are specific for each
species. Human somatic cells have 46
chromosomes that exist as 23 pairs (onehalf of each pair inherited from each parent). Twenty-two of the pairs are alike in
both males and females and are called
autosomes; the sex chromosomes, XX in
females and XY in males, are the remaining pair.
223

224

AABB Technical Manual

Each chromosome consists of two arms
joined at a primary constriction, the centromere. The two arms are usually of different
lengths: the short, or petite, arm is termed
“p,” and the long arm is termed “q.” The
arms of individual chromosomes are indicated by the chromosome number followed
by a p or q (ie, Xp is the short arm of the X
chromosome; 12q is the long arm of chromosome 12). When banded and stained,
each chromosome displays a unique pattern of bands, which are numbered from
the centromere outward (see Fig 10-1).
Chromosomes are identified by the location of the centromere and their banding
patterns. The locations of individual genes
along the chromosome may be physically
“mapped” to specific band locations.

Lyonization
In the somatic cells of females, only one X
chromosome is active. Inactivation of one
of the X chromosomes is a random process that occurs within days of fertilization. Once an X chromosome has become
inactivated, all of that cell’s clonal descendants have the same inactive X. Hence, inactivation is randomly determined but
once the decision is made, the choice is
permanent. This process is termed lyonization.

Mitosis and Meiosis
Cells must replicate their chromosomes
as they divide so that each daughter cell
receives the full complement of genetic
information. Cell division is of two kinds:
mitosis and meiosis. Mitosis is the process whereby the body grows or replaces
dead or injured somatic cells. This process
consists of five stages: prophase, prometaphase, metaphase, anaphase, and
telophase. The end result after cytokinesis
is two complete daughter cells, each with
a nucleus containing all the genetic infor-

Figure 10-1. Diagram of Giemsa-stained normal
human chromosome 7. With increased resolution
(left to right), finer degrees of banding are evident. Bands are numbered outward from the
centromere (c), which divides the chromosome
into p and q arms.1

mation of the original parent cell (Fig
10-2).
Meiosis occurs only in primordial cells
destined to mature into haploid gametes.
Diploid cells that undergo meiosis give rise
to haploid gametes (sperm and egg cells with
only 23 chromosomes). Hence, somatic
cells divide by mitosis, giving rise to diploid
cells that have a 2N chromosome complement. Gametes formed following meiosis
are haploid, with a 1N chromosome complement. It is during meiosis that genetic
diversity occurs. One type of diversity is the

Chapter 10: Blood Group Genetics

225

Figure 10-2. The two types of cell division are mitosis and meiosis.

independent assortment of maternal and
paternal homologous chromosomes that
occurs during division I of meiosis. By the
end of meiosis, gametes are formed with an
assortment of maternal and paternal chromosomes. The other type of diversity occurs when homologous pairs of chromosomes line up during the first prophase.
The homologous chromosomes genetically
recombine in a process called chromosomal crossing over (see below). Therefore,
not only are the chromosomes shuffled
during meiosis, but also portions of chromosomes are recombined to shuffle the
genes of an individual chromosome.

Genetics and Heredity
Alleles
Alternative forms of genes, any one of
which may occupy a single locus on homologous chromosomes, are called al-

leles. The ISBT terminology distinguishes
between the alleles for blood group antigens (ie, the genetic polymorphisms) and
the antigens that they encode. For example, the major antigens of the ABO system
are A, B, and O, yet the alleles are A1, B1,
and O1. In the Kell system, two alleles, K
and k, determine the K and k antigens, respectively. Individuals who have identical
alleles at a given locus on both chromosomes are homozygous for the allele (eg,
A1/A1 or K/K or k/k). In the heterozygous
condition, the alleles present at the particular locus on each chromosome are
nonidentical (eg, A1/O1 or A1/B1 or K/k). Table 10-1 summarizes genotypes and chromosomal locations for the 29 blood group
antigen systems.
Individuals who are homozygous for an
allele in some blood group systems may
have more antigen expressed on their red
cells than persons who are heterozygous for
that allele. For example, red cells from a

226

AABB Technical Manual

Table 10-1. Chromosomal Locations of Human Blood Group System Genes*
Gene(s)
Designation
(ISGN)

Location

System

ISBT No.

ISBT
Symbol

ABO

001

ABO

9q34.2

MNS

002

MNS

GYPA, GYPB, GYPE

4q28.2-q31.1

003

22q11.2-qter

004

RHD, RHCE

1p36.13-p34.3

Lutheran

005

19q13.2

Kell

006

KEL

7q33

Lewis

007

FUT3

19p13.3

Duffy

008

DARC

1q22-q23

Kidd

009

SLC14A1

18q11-q12

Diego

010

SLC4A1

17q21-q22

011

ACHE

7q22.1

012

Xp22.32

Scianna

013

1p34

Dombrock

014

12p12.3

Colton

015

AQP1

7p14

Landsteiner-Wiener

016

19p13.3

Chido/Rodgers

017

CH/RG

C4A, C4B

6p21.3

018

FUT1

19q13.3

019

Xp21.1

Gerbich

020

GYPC

2q14-q21

Cromer

021

CROM

DAF

1q32

Knops

022

CR1

1q32

Indian

023

CD44

11p13

024

CD147

19p13.3

RAPH

025

MER2

11p15.5

JMH

026

JMH

SEMA7A

15q22.3-q23

027

CGNT2

6p24

Globoside

028

B3GALT3

3q25

GIL

029

GIL

AQP3

9q13

*Modified from Zelinski ; Garratty et al ; and Denomme et al.

Chapter 10: Blood Group Genetics

person whose phenotype is Jk(a+b–) have a
“double dose” of the Jka allele and, as a result, express more Jka antigen on the red
cell surface than an individual whose phenotype is Jk(a+b+) (a single dose of the Jka
allele). The difference in amount of antigen
expressed on the red cell membrane between a homozygous and a heterozygous
phenotype can often be detected serologically and is termed the dosage effect. For
example, some anti-Jka sera may give the
following pattern of reactivity:

Antibody
Anti-Jka

Phenotype of RBC Donor
Jk(a+b–)
Jk(a+b+)
3+
2+

Dosage effect is not seen with all blood
group antigens or even with all antibodies
of a given specificity. Antibodies that typically demonstrate dosage include those in
the Rh, MNS, Kidd, and Duffy blood group
systems.
Alleles arise by genetic changes at the
DNA level and may result in a different expressed phenotype. Some of the changes
found among blood group alleles may result from:
■
Missense mutations (a single nucleotide substitution leading to the
coding of a different amino acid)
■
Nonsense mutations (a single nucleotide substitution leading to the
coding of a stop codon)
■
Mutations in motifs involved in transcription
■
Mutations leading to alternate RNA
splicing
■
Deletion of a gene, exon, or nucleotide(s)
■
Insertion of an exon or nucleotide(s)
■
Alternate transcription initiation site
■
Chromosome translocation
■
Gene conversion or recombination
■
Crossing over

227

Mutations may result in the creation of
new polymorphisms associated with the
altered gene. Figure 10-3 illustrates how
mutations in the genes that code for the
MNS blood group antigens have resulted
in the creation of various low-incidence
MNS system antigens.

Allele (Gene) Frequencies
The frequency of an allele (or its gene frequency) is the proportion that it contributes to the total pool of alleles at that locus within a given population at a given
time. This frequency can be calculated
from phenotype frequencies observed
within a population. The sum of allele frequencies at a given locus must equal 1.

The Hardy-Weinberg Law
The Hardy-Weinberg law is based on the
assumption that genotypes are distributed in proportion to the frequencies of
individual alleles in a population and will
remain constant from generation to generation if the processes of mutation, migration, etc do not occur. For example, the
Kidd blood group system is basically a
two-allele system (Jka and Jkb; the silent Jk
allele is extremely rare) that can be used
to illustrate the calculation of gene frequencies. This calculation uses the HardyWeinberg equation for a two-allele system. If p is the frequency of the Jka allele
and q is the frequency of the Jkb allele,
then the frequencies of the three combinations of alleles can be represented by
the equation p2 + 2pq + q2 = 1 where:
p
q
2
p
2pq
2
q

=
=
=
=
=

frequency
frequency
frequency
frequency
frequency

of
of
of
of
of

Jk
Jkb
a
a
Jk /Jk
a
b
Jk /Jk
b
b
Jk /Jk

allele
allele
genotype
genotype
genotype

228

AABB Technical Manual

Figure 10-3. How crossover, recombination, and nucleotide substitution (nt subs) result in variations
of genes producing glycophorin A and B. The changes are associated with the presence of various
low-incidence MNS system antigens. (Modified from Reid.4 )

Using the observation that 77% of india
viduals within a population express Jk antigen on their red cells, then:
2

p + 2pq

=
q = 1 – (p + 2pq) =
2

q2 = 1 – 0.77

frequency of
persons who
are Jk(a+) and
carry the Jka allele
0.77
frequency of
persons who
are Jk(a–)
(homozygous for
the Jkb allele)
0.23
0.23
0.48 (allele
b
frequency of Jk )

Because the sum of frequencies of both
alleles must equal 1.00,

p+q =

1–q

1 – 0.48

0.52 (allele frequency of Jka)

Once the allele frequencies have been
calculated, the number of Jk(b+) individuals (both homozygous and heterozygous)
can be calculated as:
2pq + q2 =
=
=

frequency of Jk(b+)
2 (0.52 × 0.48) + (0.48)2
0.73

If both anti-Jka and anti-Jkb sera are available, allele frequencies can be determined
more easily by direct counting. As shown in
Table 10-2, the random sample of 100 peoa
b
ple tested for Jk and Jk antigens possess a
total of 200 alleles at the Jk locus (each person inherits two alleles, one from each parent). There are two Jka alleles inherited by

Chapter 10: Blood Group Genetics

229

Table 10-2. Gene Frequencies in the Kidd Blood Group System Calculated Using
Direct Counting Method*
Gene Frequencies (%)
Phenotype

No. of
Individuals

No. of
Kidd Genes

Jka

Jk>

Jk(a+b–)

Jk(a+b+)

Jk(a–b+)

100

200

105

Totals
Gene Frequency

0.525

0.475

*Assumes absence of silent Jk allele.

each of the 28 individuals who phenotype
as Jk(a+b–), for a total of 56 alleles. There
are 49 Jka alleles in the individuals who are
Jk(a+b+), for a total of 105 alleles or a gene
frequency of 0.52 (105 ÷ 200). The frequency of Jkb is 95 ÷ 200 = 0.48.
The Hardy-Weinberg law is generally
used to calculate allele and genotype frequencies in a population when the frequency of one genetic trait (eg, antigen
phenotype) is known. However, it relies on
certain assumptions: no mutation; no migration (in or out) of the population; lack of
selective advantage/disadvantage of a particular trait; and a large enough population
so that chance alone cannot alter an allele
frequency. If all of these conditions are
present, the gene pool is in equilibrium and
allele frequencies will not change from one
generation to the next. If these assumptions
do not apply, changes in allele frequencies
may occur over a few generations and can
explain many of the differences in allele
frequencies between populations.

Segregation
The term segregation refers to the concept that the two members of a single
gene pair (alleles) are never found in the
same gamete but always segregate and

pass to different gametes. In blood group
genetics, this can be illustrated by the inheritance of the ABO alleles (Fig 10-4). In
this example, the members of the parental generation (P1) are homozygous for an
A allele and an O allele. All members of
the first filial generation (F1) will be heterozygous (A/O) but will still express the
blood group A antigen (O is a silent allele). If an F1 individual mates with an A/O
genotypic individual, the resulting progeny [termed the second filial generation:
(F2)] will blood group as A (either heterozygous or homozygous) or group O. If the
F1 individual mated with a heterozygous
group B person (B/O), the offspring could
have the blood group A, B, AB, or O.

Independent Assortment
Mendel’s law of independent assortment
states that genes determining various
traits are inherited independently from
each other. For example, if one parent is
group A (homozygous for A) and K+k+,
and the other parent is group B (homozygous for B) and K–k+ (homozygous for k),
all the F1 children would be group AB; half
would be K+k+ and half K–k+ (Fig 10-5). A
second filial generation could manifest
any of the following phenotypes: group A,

230

AABB Technical Manual

Figure 10-4. Mendel’s law of independent segregation demonstrated by the inheritance of ABO genes.

K+k+; group AB, K+k+; group B, K+k+;
group A, K–k+; group AB, K–k+; group B,
K–k+. The proportions would be
1:2:1:1:2:1.
Independent assortment applies if the
genes are on different chromosomes or on
distant portions of the same chromosome.
One exception to this rule is that closely
linked genes on the same chromosome do
not sort independently but often remain together from one generation to another. This
observation is termed linkage.

Linkage
Genetic linkage is defined as the tendency
for alleles close together on the same
chromosome to be transmitted together.
During mitosis, each pair of homologous
chromosomes undergoes a series of recombinations. The resultant reciprocal
exchange of segments between the chro-

matids is termed crossing over (Fig 10-6).
Genes close together on a chromosome
tend to be transmitted together during
these recombinations and their alleles,
therefore, do not segregate independently.
Sometimes, the linkage is very tight so that
recombination rarely occurs. The strength
of linkage can be used as a unit of measurement to estimate the distance between
different loci. This type of analysis can
help in identifying, mapping, and diagnosing the genes responsible for certain
inherited diseases.
The demonstration of linkage between
the gene controlling ABH secretion (Se) and
the expression of Lutheran blood group antigens (Lua, Lub) was the first recognized ex5
ample of autosomal linkage in humans.
Analysis of this relationship also provided
the first evidence in humans of recombination due to crossing-over and helped dem-

Chapter 10: Blood Group Genetics

231

Figure 10-5. Mendel’s law of independent assortment demonstrated by the inheritance of ABO and Kell
genes.

onstrate that crossing-over occurs more
often in females than in males.

be the product of the frequencies of the individual alleles. However, the frequencies
observed are not those expected:

Linkage Disequilibrium
When two loci are closely linked, alleles at
those loci tend to be inherited together
and are said to constitute a haplotype.
Again, the close linkage between the loci
controlling expression of M and N and of
S and s is an example of linkage disequilibrium. The approximate frequencies of
each of the four alleles are:
M = 0.53
N = 0.47

S = 0.33
s = 0.67

If the alleles of the M, N, S, and s antigens segregated independently, the expected frequency of each haplotype would

MS
Ms
NS
Ns
Total

=
=
=
=

Expected
Observed
Frequency
Frequency
0.53 × 0.33 = 0.17
0.24
0.53 × 0.67 = 0.36
0.28
0.47 × 0.33 = 0.16
0.08
0.40
0.47 × 0.67 = 0.31
1.00
1.00

This is an example of linkage disequilibrium: the tendency of specific combinations of alleles at two or more linked loci
to be inherited together more frequently
than would be expected by chance.
Another commonly cited example of
linkage disequilibrium occurs in the HLA
system (see Chapter 17). The combination

232

AABB Technical Manual

Figure 10-6. Very closely linked loci are rarely affected by crossing over so that alleles of those loci
are inherited together (N and S, M and s in the example shown). Loci on the same chromosome that
are not closely linked (the Ss locus and the Zz locus shown) can demonstrate crossing over. Crossing
over is one kind of recombination. It occurs between homologous chromatids during meiosis, resulting
in segregation of alleles on the same chromosome.

of HLA-A1 with HLA-B8 occurs in some
populations approximately five times more
frequently than would be expected based
on the frequencies of the individual alleles,
an example of positive linkage disequilibrium. Linkage disequilibrium may be positive or negative, and it may indicate a selective advantage of one haplotype over another.
Over many generations, the alleles of even
closely linked loci may reach equilibrium
and associate according to their individual
frequencies in the population.
When there is linkage equilibrium, the
alleles at two loci associate with frequencies that reflect their individual frequencies.
For example, if alleles in the population
have the following frequencies:

then the frequencies of the combination
should be the product of the frequency of
each allele:
YZ
Yz
yZ
yz
Total

0.53 × 0.3
0.53 × 0.7
0.47 × 0.3
0.47 × 0.7

=
=
=
=

0.16
0.37
0.14
0.33
1.00

In such a case, the alleles are in linkage
equilibrium because they are inherited
independently.

Patterns of Inheritance
Dominant and Recessive Traits

Y
y
Total

0.53
0.47
1.00

Z
z

0.30
0.70
1.00

Traits are the observed expression of genes.
A trait that is observable when the determining allele is present is called domi-

Chapter 10: Blood Group Genetics

nant; when different alleles on homologous
chromosomes each produce an observable
trait, the term co-dominant is used. A recessive trait is observable only when the
allele is not paired with a dominant allele
(two recessive alleles are present). Describing traits as dominant and recessive
depends on the method used to detect gene
products. Observable traits are called phenotypes. Thus, blood group antigen typing
using antisera identifies a phenotype. In
some cases, genotypes may be inferred
from the phenotype, especially when
family studies are performed, but genotypes are not usually determined directly
by typing red cells.

Autosomal Dominant Trait
An autosomal dominant trait shows a
characteristic pattern of inheritance. The
trait appears whenever an individual possesses the allele. Figure 10-7(A) presents a
pedigree showing the pattern of autosomal dominant inheritance. Typically,
each person with the trait has at least one
parent with the trait, continuing backward through generations.

Autosomal Recessive Trait
People who exhibit a recessive trait are
homozygous for the encoding allele. Their
parents may or may not express the trait.
However, parents who lack the trait must
be carriers, ie, heterozygotes for an allele
whose presence is not phenotypically
apparent.
If the frequency of the variant allele is
low, the recessive trait will be rare and generally will occur only in members of one
generation, not in preceding or successive
generations unless consanguineous mating
occurs. Blood relatives are more likely to
carry the same rare allele than unrelated
persons from a random population. When
offspring are homozygous for a rare allele

233

(frequency: <1:10,000) and display the trait,
the parents are often blood relatives [Fig
10-7(B)]. Recessive traits may remain unexpressed for many generations, so that the
appearance of a rare recessive trait does not
necessarily imply consanguinity, although
family ethnicity and geographic origin may
be informative. A higher frequency for a recessive allele indicates the less likelihood of
consanguinity. Traits inherited in either
autosomal dominant or autosomal recessive fashion typically occur with equal frequency in males and females.

Sex-Linked Dominant or Co-dominant
Trait
A male always receives his single X chromosome from his mother. The predominant feature of X-linked inheritance, of
either dominant or recessive traits, is absence of male-to-male (father-to-son)
transmission of the trait. Because a male
passes his X chromosome to all his daughters, all daughters of a man expressing
a dominant X-linked trait also possess the
allele and the trait. If a woman expresses a
dominant trait, but is heterozygous, each
child, male or female, has a 50% chance of
inheriting that allele and thus the trait
[Fig 10-7(C)]. If the mother possesses the
determining allele on both X chromosomes, all her children will express the
trait. X-linked dominant traits tend to appear in each generation of a kindred, but
without male-to-male transmission. A
sex-linked dominant trait of interest in
blood group genetics is the Xg blood
group system.

Sex-Linked Recessive Trait
Hemophilia A provides a classic example
of X-linked recessive inheritance [Fig
10-7(D)]. Males inherit the trait from carrier mothers or, very rarely, from a mother
who is homozygous for the allele and

234

AABB Technical Manual

Figure 10-7. Four pedigrees showing different patterns of inheritance.

therefore expresses the trait. In the mating of a normal male and a carrier female,
one half of the male offspring are affected
and one half of the females are carriers.
Among the children of an affected male
and a female who lacks the determining
allele, all sons are normal and all daughters are carriers.
If the recessive X-linked allele is rare, the
trait will be exhibited almost exclusively in
males. If the X-linked allele occurs more
frequently in the population, affected fe-

males will be seen because the likelihood
increases that an affected male will mate
with a carrier female and produce daughters, half of whom will be homozygous for
the abnormal allele.

Blood Group Co-dominant Traits
Blood group antigens, as a rule, are expressed as co-dominant traits: heterozygotes express the products of both alleles.
If an individual’s red cells type as both K+
and k+, the K/k genotype may be inferred.

Chapter 10: Blood Group Genetics

235

Figure 10-8. Inheritance and co-dominant expression of Kidd blood group antigens.

Figure 10-8 shows the inheritance patterns
of the two active alleles of the Kidd blood
a
b
group system (Jk and Jk ) and the codominant phenotypic expression of the
two respective antigens Jka and Jkb.
In the ABO system, the situation is more
complex. The genes of the ABO system do
not code for membrane proteins but control production of enzymes termed glycosyltransferases. These enzymes add specific
sugars to a precursor structure on the red
cell membrane, resulting in specific antigen
expression. In an A1/A2 heterozygote, the
phenotype is A1; the presence of the A2 al1
lele cannot be inferred. Although the A al2
lele appears dominant to that of the A allele by simple cell typing, techniques that
identify the specific transferases reveal that
an A1/A2 heterozygote does generate the
products of both alleles, ie, both A1 and A2

transferases. Similarly, in an A /O person, A
is dominant to O. The O allele codes for a
specific protein, but this protein (transferase) is nonfunctional. The presence of
ABO genes can be demonstrated by molecular techniques (see Chapter 13).

Chromosomal Assignment
The loci of all major blood group genes
have been mapped to one or another of
the 22 pairs of autosomes, as shown in Table 10-1. The Xg and XK loci are the only
blood group genes mapped to the X chromosome.
Interaction among alleles or the products of different genes may modify the expression of a trait. The terms “suppressor”
and “modifier” are used to describe genes
that affect the expression of other genes;

236

AABB Technical Manual

however, the mechanism of these postulated gene interactions is not always fully
understood. Some observations in blood
group serology have been explained by
gene interaction: weakening of the D antigen expression when the C allele is present
in cis (on the same chromosome) or in
6
trans (on the paired chromosome), and the
suppression of Lutheran antigen expression
by the dominant modifier gene, In(Lu).7
When products of two different genes
are important in the sequential development of a biochemical end product, the
gene interaction is called epistasis. Failure
to express A or B antigens if H substance
has not first been produced (absence of the
H gene) is an example of epistasis. A mutation database of gene loci encoding common and rare blood group antigens has
been established (Blood Group Antigen
Mutation Database) and is available on the
Internet (see http://www.bioc.aecom.yu.
edu/bgmut/index.htm).

Population Genetics
Some understanding of population genetics is essential for parentage testing and
helpful in such clinical situations as predicting the likelihood of finding blood
compatible with a serum that contains
multiple antibodies. Calculations use
published phenotype frequencies.

Phenotype Frequencies
The frequencies of blood group phenotypes are obtained by testing many randomly selected people of the same race or
ethnic group and observing the proportion of positive and negative reactions
with a specific blood group antibody. In a
blood group system, the sum of phenotype frequencies should equal 100%. For
example, in a Caucasian population, 77% of
randomly selected individuals are Jk(a+).

The frequency of Jk(a–) individuals should
be 23%. If blood is needed for a patient
with anti-Jka, 23% or approximately one in
four ABO-compatible units of blood should
be compatible.

Calculations for Combined Phenotypes
If a patient has multiple blood group antibodies, it may be useful to estimate the
number of units that will have to be tested
in order to find units of blood negative for
all the antigens. For example, if a patient
has anti-c, anti-K, and anti-Jka, how many
ABO-compatible units of blood would
have to be tested to find 4 units of the appropriate phenotype?

c–
K–
Jk(a–)

Phenotype Frequency (%)
20
91
23

To calculate the frequency of the combined phenotype, the individual frequencies are multiplied because the phenotypes
are independent of one another. Thus, the
proportion of persons who are c– is 20%. Of
the 20% of c– individuals, 91% are K–;
hence, 18% (0.20 × 0.91 = 0.18) are c– and
K–. Of this 18% of c–K– individuals, 23%
will be Jk(a–); therefore, only 4% of individuals will have c–K–Jk(a–) blood (0.2 ×
0.91 × 0.23 = 0.04). Therefore, of 100 units
tested, 4 compatible units should be found.
Calculations such as this influence decisions about asking for assistance from the
local blood supplier or reference laboratory
when trying to find compatible blood for an
alloimmunized patient.

Parentage Testing
Blood group antigens, many of which are
expressed as co-dominant traits with simple Mendelian modes of inheritance, are
useful in parentage analyses. If one as-

Chapter 10: Blood Group Genetics

sumes maternity and that test results are
accurate, paternity can be excluded in
either of two ways:
1.
Direct exclusion of paternity is established when a genetic marker is
present in the child but is absent
from the mother and the alleged father. Example:
Blood Group Phenotype
Child
Mother
Alleged Father
B
O
O
The child has inherited a B gene, which
could not be inherited from either the
mother or the alleged father, assuming that
neither the mother nor the alleged father is
of the rare Oh phenotype. Based on the phenotypes of mother and child, the B gene
must have been inherited from the biologic
father and is called a paternal obligatory gene.
2.
Exclusion is indirect when the child
lacks a genetic marker that the alleged
father (given his observed phenotype) must transmit to his offspring.
Example:
Blood Group Phenotype
Child
Mother
Alleged Father
Jk(a+b–) Jk(a+b–) Jk(a–b+)
In this case, the alleged father is presumably homozygous for Jkb and should have
b
transmitted Jk to the child.
Direct exclusion is more convincing than
indirect exclusion when trying to establish
parentage. Apparent indirect exclusion can
sometimes result from the presence of a silent allele. In the example above, the alleged father could have one silent allele
(Jk), which was transmitted to the child.
a
The child’s genotype could be Jk Jk instead
a
a
of the far more common Jk Jk . Interpretation of phenotypic data must take into account all biologic and analytic factors
known to influence results.

237

When the alleged father cannot be excluded from paternity, it is possible to calculate the probability of paternity. The
probability that the alleged father transmitted the paternal obligatory genes is compared with the probability that any other
randomly selected man from the same ethnic/racial population could have transmitted the genes. The result is expressed as a
likelihood ratio (paternity index) or as a
percentage (posterior probability of paternity given some prior probability). Methods
for parentage analysis often include the
study of many genetic systems other than
red cell blood groups [ie, HLA and short
tandem repeat (STR) systems]. Many parentage testing laboratories employ the STR
method of DNA analysis (see Chapter 9) as
a means of evaluating cases of disputed
parentage. The AABB has developed standards for laboratories that perform parentage studies.8

Chimerism
A chimera is one whose cells are derived
from more than one distinct zygotic line.
Although rare, this may occur when an
anastomosis occurs within the vascular
tissues of twin embryos, or when two fertilized zygotes fuse to form one individual. This condition, although not hereditary, leads to dual (multiple) phenotypic
populations of cells within one individual.
Blood types of such rare individuals may
demonstrate a mixed-field appearance,
with distinct populations of cells of the
person’s true genetic type, as well as cells
of the implanted type. Chimeras also
demonstrate immune tolerance: a genetically group O person with implanted A
cells does not produce anti-A. More commonly, chimeras are artificial and arise
from the transfer of actively dividing cells,
eg, through hematopoietic transplantation (see Chapter 25).

238

AABB Technical Manual

Blood Group Nomenclature
The terminology and notations for blood
group systems embody many inconsistencies because blood group serologists
failed to follow conventions of classic
Mendelian genetics. Listed below are a
few examples of the confusion engendered by many decades of uncoordinated
scientific publications.
1.
An allele that determines a dominant
trait often is signified by a capital
letter; one that determines a recessive trait is denoted by both lowercase letters. The A and B co-dominant genes of the ABO system are
signified by a capital letter. The O
gene is also given a capital but does
not present as a dominant trait.
Without prior knowledge, it would
be impossible for one to recognize
that these notations represent allelic
products in a blood group system.
2.
Some co-dominant traits have been
designated with capital letters and
allelic relationships with lowercase
letters; for example, K and k of the
Kell blood group system and C and c
of the Rh system.
3.
Some co-dominant traits have identical base symbols but different superscript symbols, such as Fya and
b
a
b
Fy (Duffy system) and Lu and Lu
(Lutheran system).
4.
In some allelic pairs, the lower frequency antigen is expressed with an
“a” superscript (Wra has a lower freb
quency than Wr ). In other allelic
pairs, the “a” superscript denotes the
higher incidence antigen (Coa has a
b
higher frequency than Co ).
5.
Some authors have denoted the absence of a serologic specificity with a
base symbol devoid of superscripts,
and others use a lowercase version
of the base symbol. In the Lutheran

system, the assumed amorphic gene
is called Lu, not lu, whereas the
amorph in the Lewis system is le.
6.
Numeric terminology was introduced for some blood group systems, resulting in mixtures of letters
and numbers for antigen designaa
tions, eg, K, Kp , and K11.
Colloquial use of these terminologies,
even in some published articles and texts,
has compounded their improper use. Early
model computers or printers also did not
easily accept certain terminologies (eg, superscripts, subscripts, unusual fonts).
In recent years, concerted attempts have
been made to establish rational, uniform
criteria for the notations used to designate
phenotype, genotype, and locus information for blood group systems. Issitt and
Crookston9 and Garratty et al2 presented
guidelines for the nomenclature and terminology of blood groups. The International
Society of Blood Transfusion (ISBT) Working Party on Terminology for Red Cell Surface Antigens has provided a standardized
system for classifying blood group antigens
(see Appendix 6). Similar international
committees have established principles for
assigning nomenclature of the hemoglobins, immunoglobulin allotypes, histocompatibility antigens, clusters of differentiation, STR sequences, and other serum
protein and red cell enzyme systems. Although many of the older terminologies
must be retained to avoid even further
confusion, common conventions now exist
for correct usage.
The ISBT terminology for red cell antigens was devised as a numeric nomenclature suitable for computerization. A sixdigit designation indicates each blood
group specificity. The first three numbers
identify the blood group system and the
last three numbers identify the individual
specificity. This numeric terminology is designed mainly for computer databases and

Chapter 10: Blood Group Genetics

is not necessarily intended to supplant more
common usage.
For ISBT classification, each defined
blood group system must be genetically
distinct. Assignment of antigens to a specific blood group system is dependent on
genetic, serologic, and/or biochemical relationships. Gene cloning has made the task
of assignment more definitive and has allowed some designations previously unproved by traditional family studies (ie, the
expansion of the Diego system to include a
number of low-incidence antigens).
Some antigens, however, have not yet
been proven to be part of a recognized system. Collections (termed the 200 series) are
apparently related sets of antigens for which
definitive genetic information is lacking.
Other isolated antigens of high (901 series)
or low (700 series) incidence are listed together until genetic information becomes
available. In recent years, the number of
antigens in these three series has dramatically declined as further genetic and biochemical data allow reassignment.

Correct Terminology
The following are accepted conventions
for expressing red cell antigen phenotypes and genotypes.2
1.
Genes encoding the expression of
blood group antigens are written in
italics (or underlined if italics are not
available). If the antigen name includes a subscript (A1), generally the
encoding gene is expressed with a
superscript (A1).
2.
Antigen names designated by a sua
perscript or a number (eg, Fy , Fy:1)
are written in normal (Roman) script.
Numeric designations are written on
the same line as the letters. Superscript letters are lowercase. (Some
exceptions occur, based on historic
usage: hrS, hrB.)

239

When antigen phenotypes are expressed using single letter designations, results are usually written as +
or –, set on the same line as the letter(s) of the antigen: K+ k–.
4.
To express phenotypes of antigens
designated with a superscript letter,
that letter is placed in parentheses
on the same line as the symbol defining the antigen: Fy(a+) and Fy(a–).
5.
For antigens designated by numbers, the symbol defining the system
is notated in capital letters followed
by a colon, followed by the number
representing the antigen tested. Plus
signs do not appear when test results are positive (K:1), but a minus
sign is placed before negative test results: K:1, K:–1. If tests for several antigens in one blood group have been
done, the phenotype is designated
by the letter(s) of the locus or blood
group system followed by a colon,
followed by antigen numbers separated by commas: K:–1,2,–3,4. Only
antigens tested are listed; if an antibody defining a specific antigen was
not tested, the number of the antigen is not listed: K:–1,–3,4.
Although numeric terminology has been
devised for various systems and antigens, it
should not be assumed that it must replace
conventional terminology. The use of conventional antigen names is also acceptable.
In some systems, notably Rh, multiple terminologies exist and not all antigens within
the system have names in each type.

References
1.

Zelinski T. Chromosomal localization of human blood group genes. In: Silberstein LE, ed.
Molecular and functional aspects of blood
group antigens. Bethesda, MD: AABB, 1995:
41-73.
Garratty G, Dzik W, Issitt PD, et al. Terminology for blood group antigens and genes—his-

240

AABB Technical Manual

torical origins and guidelines in the new millennium. Transfusion 2000;40:477-89.
Denomme G, Lomas-Francis C, Storry JR, Reid
ME. Approaches to blood group molecular
genotyping and its applications. In: Stowell C,
Dzik W, eds. Emerging diagnostic and therapeutic technologies in transfusion medicine.
Bethesda, MD: AABB Press, 2003: 95-129.
Reid ME. Molecular basis for blood groups and
functions of carrier proteins. In: Silberstein
LE, ed. Molecular and functional aspects of
blood group antigens. Bethesda, MD: AABB,
1995:75-125.
Mohr J. A search for linkage between the Lutheran blood group and other hereditary

characters. Acta Path Microbiol Scand 1951;
28:207-10.
Araszkiewicz P, Szymanski IO. Quantitative
studies on the Rh-antigen D effect of the C
gene. Transfusion 1987;27:257-61.
Crawford NM, Greenwait TJ, Sasaki T. The
phenotype Lu(a–b–) together with unconventional Kidd groups in one family. Transfusion
1961;1:228-32.
Gjertson D, ed. Standards for parentage testing laboratories. 6th ed. Bethesda, MD: AABB,
2004.
Issitt PD, Crookston MC. Blood group terminology: Current conventions. Transfusion 1984;
24:2-7.

Chapter 10: Blood Group Genetics

241

Appendix 10-1. Glossary of Terms in Blood Group Genetics
Allelic:
Centromere:
Chromatid:

Chromatin:
Chromosome:
Co-dominant:

Crossing over:

Dominant:

Gene:
Locus:
Lyonization:

Meiosis:

Mitosis:
Recessive:
Sex-linked:
Somatic cell:
X-linked:

Pairs of genes located at the same site on chromosome pairs.
A constricted region of a chromosome that connects the chromatids during cell division.
One of the two potential chromosomes formed by DNA replication
of each chromosome before mitosis and meiosis. They are joined
together at the centromere.
The deeply staining genetic material present in the nucleus of a cell
that is not dividing.
A linear thread made of DNA in the nucleus of the cell.
A gene that expresses a trait regardless of whether or not an alternative allele at the same locus is also expressed on the other parental chromosome.
The process of breaking single maternal and paternal DNA double
helices in each of two chromatids and rejoining them to each other
in a reciprocal fashion, which results in the exchange of parts of homologous chromosomes.
A gene that expresses a trait that does not allow the expression of a
trait encoded by an alternative allele at the same locus on the other
parental chromosome.
The basic unit of heredity, made of DNA. Each gene occupies a specific location on a chromosome.
The site of a gene on a chromosome.
The inactivation of one of the female X chromosomes during
embryogenesis. This inactivated chromosome forms the Barr body
in the cell nucleus.
A process of two successive cell divisions producing cells, egg, or
sperm that contain half the number of chromosomes found in somatic cells.
Division of somatic cells resulting in daughter cells containing the
same number of chromosomes as the parent cell.
A gene that in the presence of its dominant allele does not express
itself. A recessive trait is apparent only if both alleles are recessive.
A gene contained within the X or Y chromosome.
Nonreproductive cells or tissues.
A gene on the X chromosome for which there is no corresponding
gene on the Y chromosome.

Chapter 11: Immunology

Chapter 11

Immunology

HE IMMUNE RESPONSE is a
highly evolved innate and adaptive system that is fundamental for
survival. It has a sophisticated ability to
distinguish self from nonself and provides
a memory bank that allows the body to
rapidly respond to recurring foreign organisms. A healthy immune response can
recognize foreign material or pathogens
that invade the body and can initiate a series of events to eliminate these pathogens
with minimal or no prolonged morbidity
to the host.
The numerous components of the immune system work in delicate balance to
ensure a state of health. This may include
destruction of abnormal/malignant cells,
removal of harmful bacteria or viruses,
and/or an inflammatory response to promote healing. If the immune system becomes hyperreactive, the body may attack
its own tissue or organs (autoimmune disease), or allergies may develop. If hyporeac-

tive, the host may be susceptible to a wide
variety of infectious agents or proliferation
of malignant cells. The ultimate goal of immune activity is to maintain this delicate
balance.

Immune Response
The immune response can be classified
into two categories: the innate response
and the adaptive (acquired) response. Innate responses are indiscriminate: the
same mechanisms can be deployed against
invasive organisms or harmful stimuli. In
contrast, the adaptive response recognizes
specific features of the harmful stimuli
and provides a customized response based
on previous experiences (Fig 11-1). The
adaptive response is a late evolutionary
development, found only in vertebrates.
Innate immunity, on the other hand, uses
universal properties and processes such as
epithelial barriers, proteolytic enzymes,
243

244

AABB Technical Manual

Figure 11-1. Examples of the factors used in innate and adaptive immunity and examples of the two
types of immunity.

cellular phagocytosis, and inflammatory
reactions. It is important to note that innate and adaptive immunity are complementary—not mutually exclusive—immune
responses. Regardless of the classification,
the immune response reflects the complex
interaction of cells, tissues, organs, and
soluble factors. Appendix 11-1 describes
some frequently used immunology terms.

Immunoglobulin Superfamily
Molecules that belong to the immunoglobulin superfamily (IgSF) of receptors
play a critical role in the recognition of
foreign antigens. Antigen recognition is
accomplished through receptors that are
found in a soluble form and on the surface of lymphoid cells. Figure 11-2 illustrates the similar structure of some of
these immunology receptors. The overall
structure of these receptors is very similar, and it has been proposed that they
arose from a common ancestral gene. The
basic structure is similar to the domains
of the immunoglobulin molecule; hence,

these receptors have been grouped into a
single superfamily of molecules. Examples
of these receptors include: immunoglobulins, T-cell receptor (TCR), major histocompatibility complex (MHC) Class I and
Class II molecules, and receptors for growth
factors and cytokines. There are several
hundred members of the IgSF (ie, immunoglobulin receptors, integrins, etc).2

Major Histocompatibility Complex
The MHC is a large cluster of genes. In
humans, the MHC includes the genes of
the HLA system and genes that encode
other proteins such as tumor necrosis factor ( TNF), some complement components, and some heat shock proteins.3 The
genes that encode MHC molecules are located on the short arm of chromosome 6.
There are three classes of these molecules.

MHC Class I Molecules
MHC Class I molecules are found on almost all cells in the body. The molecules
are defined by three major Class I genes

Chapter 11: Immunology

Figure 11-2. Structures of some receptors found on various cells in the immune system.

designated HLA-A, -B, and -C. Hence,
each individual will express six distinct
Class I HLA molecules: two HLA-A, two
HLA-B, and two HLA-C. Each locus has
many alleles; more than 50 have been defined for HLA-A; more than 75 for HLA-B;
and more than 30 for HLA-C.
The structure of a Class I HLA molecule
is illustrated in Fig 11-2. Each molecule
contains a heavy chain (45 kDa) and a
smaller (12 kDa) peptide chain called
β2-microglobulin. The heavy chain has a cytoplasmic tail, a transmembrane region,
and three extracellular immunoglobulin-like domains. β2-microglobulin is noncovalently associated with the heavy chain
and is not a transmembrane protein. This
protein is required for Class I MHC expres-

245

sion and function on the cell surface. The
antigen-binding groove of the Class I molecule is formed by the α1 and α2 domains of
the heavy chain. The structure of the antigen-binding groove consists of a platform
made up of eight parallel β strands that is
supported by two α helices.4 The peptides
displayed in the antigen-binding groove are
8 to 12 amino acids long and represent hydrolyzed proteins that have been synthesized within the antigen-presenting cell;
hence, they are referred to as endogenous
antigens. The endogenous source of proteins indicates that the genes encoding the
protein also must reside in the cell. These
proteins could be the product of host genes
including tumorigenic genes or genes from
viruses or intracellular bacteria.

246

AABB Technical Manual

MHC Class II Molecules
There are three major Class II gene loci:
HLA-DR, -DQ, and -DP. As with the Class I
molecules, there are many different alleles that could occupy each locus. Each
individual expresses four DR molecules, four
DQ molecules, and two DP molecules, for
a total of 10 forms of Class II HLA molecules.
Class II molecules are heterodimeric
structures consisting of a heavy α chain (30
to 34 kDa) and a light β chain (26 to 29
kDa). Each chain has a cytoplasmic tail, a
transmembrane region, and an extracellular portion. There are two immunoglobulin-like domains on each chain (α1 and α2
and β1 and β2). The α2 and β2 domains for
each gene have a constant structure; the α1
and β1 domains are diverse. The antigenbinding groove is located within the α1, β1
domains and is similar in structure to the
Class I molecules. However, the groove is
larger, accommodating peptides that are 12
to 20 amino acids in length. Class II molecules are expressed on monocytes, macrophages, dendritic cells, and B lymphocytes.
The antigenic peptides displayed in the
groove of Class II HLA molecules come
from proteins that have been phagocytosed
or endocytosed by antigen-presenting cells.
These proteins are termed exogenous antigens and include most bacteria, parasites,
and viral particles released from other
cells.5

MHC Class III Molecules
There are approximately 20 genes in the
Class III region of the MHC. These genes
code for proteins of the complement system and proinflammatory molecules such
as TNF.

Cluster of Differentiation (CD) Molecules
The CD designation is a nomenclature
system used to describe numerous mole-

cules expressed on cells and components
of the blood and lymphoid organs. Over
200 CD markers have been described to
3
date. Some of the major CD markers on
cells of the immune system are summarized in Table 11-1.

Cell Adhesion Molecules
For normal immune function to occur,
leukocytes must be able to attach to extracellular matrices and each other. Three
families of adhesion molecules facilitate
this attachment process: selectins, integrins, and IgSF adhesion molecules. The
members of the selectin family (L-selectin, E-selectin, and P-selectin) are found
on leukocytes and participate in the process of leukocyte rolling along the vascular endothelium. The integrins ( VAL,
LFA-1, and MAC-I) and the IgSF adhesion
molecules (ICAMs, VCAMs, LFA-2, and
LFA-3) are required to stop leukocyte rolling and mediate leukocyte aggregation
and transendothelial migration.6 Most adhesion molecules have CD designations
(eg, LFA-2 is CD2 and LFA-3 is CD58).7

Signal Transduction
Signal transduction is the process of sending signals between or within cells that
results in the initiation or inhibition of
gene transcription. Cell surface activation
signals associated with the immune response are initiated by extracellular interaction of various ligands and receptors.
Many of these ligands and receptors have
CD designations. Any defect or deficiency
in the signal transduction process can have
significant consequences on the normal
functioning of the immune system. For
example, severe combined immunodeficiency disease can result from a deficiency of Jak3, which impairs signal
transduction of a specific cytokine receptor subunit.7

Table 11-1. Some Major CD Antigens on Cells of the Immune System
Cell
Population

Other Cells
with Antigen

CD1

Cortical thymocytes

Some APCs, some B cells

Strength of expression is inverse to
expression of TCR/CD3

CD2

Pan-T marker, present on early
thymocytes

NK cells

This is a sheep-cell rosette receptor;
activation and adhesion function
(LFA-2)

CD3

T lymphocytes

——

Functions as a signal transduction
complex

CD4

Developing and mature thymocytes
and on 2/3 of peripheral T cells

T helper cells and some macrophages

Adhesion molecule that mediates MHC
restriction; signal transmission; HIV
receptor

CD5

Pan-T marker, from late cortical stage

B cells of chronic lymphocytic leukemia; possibly long-lived
autoreactive B cells

Function unknown; possibly involved
in costimulatory effects of
cell-to-cell adhesion

CD8

Developing and mature thymocytes
and cytotoxic T lymphocytes

None

Adhesion molecule that mediates MHC
restriction; signal transmission

CD14

Monocytes

——

LPS receptor

CD16

Macrophages, neutrophils, NK cells

——

FcγRIII (low-affinity Fc receptor for
IgG)

CD19

B lymphocytes

——

Signaling (also called B4)

CD20

B lymphocytes

——

Signaling (also called B1)

Comments

(cont’d)

Chapter 11: Immunology

CD
Designation

247

248

CD
Designation

Cell
Population

Other Cells
with Antigen

CD21

Mature B cells

Possibly macrophages

This is a receptor for C3d (CR2); also
receptor for EBV

CD25

Activated T and B cells

Macrophages; virally transformed cells

High-affinity IL-2 receptor, earlier
called TaC

CD34

Stem cells

Hematopoietic cells; endothelial cells

Called “stem cell antigen”; used in the
laboratory to isolate hematopoietic
precursor cells; physiologic function unknown

CD35

Mature and activated B cells

Red cells, macrophages, granulocytes,
dendritic cells

This is a receptor for C3b (CR4)

CD45

Immature and mature B and T cells

All cells of hematopoietic origin except
red cells

Also called leukocyte common antigen
(LCA); different leukocytes have different isoforms

CD56

NK cells

NKT cells

NK cell adhesion and lineage marker
for NK cells; innate immunity

CD71

Early thymocytes; activated T and B
cells

Activated hematopoietic cells; proliferating cells of other somatic lines;
reticulocytes

This is the transferrin receptor

Comments

CD = clusters of differentiation; APCs = antigen-presenting cells; NK = natural killer; TCR = T-cell receptor; MHC = major histocompatibility complex; HIV = human immunodeficiency virus; IL = interleukin; NKT = natural killer T.

AABB Technical Manual

Table 11-1. Some Major CD Antigens on Cells of the Immune System (cont’d)

Chapter 11: Immunology

Organs of the Immune
System
Numerous organs are involved in the immune system. The central organs include
the marrow, liver, and thymus. Peripheral
organs are the lymph nodes and spleen.
The gastrointestinal tract-associated lymphoid tissue and bronchus-associated
lymphoid tissue also play an important
role involving both central and peripheral
functions.

Cells of the Immune System
The primary cells involved in the adaptive
immune system are lymphocytes—B cells,
T cells, and antigen-presenting cells.
Other cells such as macrophages are involved in the induction of the immune response, and both macrophages and polymorphonuclear leukocytes participate in
various inflammation responses associated
with the immune response. All of these cells
are of hematopoietic origin (ie, marrowderived) and are formed from a single progenitor called a pluripotent hematopoietic
stem cell. As illustrated in Fig 11-3, the
pluripotent stem cell can give rise to two
lineages: myeloid and lymphoid. Granulocytes (neutrophils, basophils, and eosinophils), platelets, mast cells, red cells, and
macrophages arise from myeloid progenitors. T and B lymphocytes are formed from
lymphoid progenitors. Dendritic cells and
natural killer (NK) cells are also derived
from the pluripotent stem cell; however,
their precise origin is unknown.

Lymphocytes
The two major lineages of lymphocytes
are B cells and T cells. B cells are derived
from the marrow and T cells are derived
from T-cell precursors produced in the
marrow, which migrate to the thymus. B

249

cells are the precursors of the cells that
make antibody (plasma cells). T cells consist of subpopulations that either help in
antibody formation (helper T cells), kill
target cells (cytotoxic T cells), induce inflammation (delayed hypersensitivity T
cells), or inhibit the immune response
(regulatory T cells).

B Lymphocytes
Each B cell can recognize one (or a limited set) of antigen epitopes through receptors on the cell surface. These receptors are similar to IgM and IgD molecules,
which are produced and transported to
the membrane surface. When the immunoglobulin receptor binds to a specific
antigen, the cell is stimulated to divide
and differentiate into a plasma cell. The
plasma cell can secrete a soluble form of
the immunoglobulin “receptor” known as
antibody. The antibody secreted is specific
for the same antigen that interacted with
the cell-bound immunoglobulin receptor.
The B-cell receptor (BCR) has one additional heavy chain domain (CH4) compared
to secreted immunoglobulin. This domain
is required to anchor the receptor to the cell
membrane. Several accessory molecules on
the surface of B cells are closely associated
with the BCR and are important for signal
transduction. Some of these accessory molecules include Igα, Igβ, MHC Class II molecules, complement receptors, and some CD
markers (see Table 11-2). All of these components are part of the BCR antigen complex (some of these structures are illustrated in Fig 11-2).
The challenge for the body is to have B
lymphocytes that can recognize each of the
thousands of different foreign antigens encountered over a lifetime. The immune system has a unique approach to ensure this
diversity. The human genome is known to
contain approximately 40,000 genes. The B

250

AABB Technical Manual

Table 11-2. Receptors/Markers Present on Macrophages, Monocytes, and B
Lymphocytes
Marker

Function

Macrophages and Monocytes
Complement receptors
CR1 (C3b receptors, CD35)
CR3 (C3bi receptor, CDIIb)
Cytokine receptors (IL-1, IL-4, IFNγ)
and migration-inhibition factor
Fc receptors
FcγRI (CD64)
FcγRII (CD32)
FcγRIII (CD16)
FcγRII (CD23)
Leukocyte function antigen
(LFA1 or CD11a)
Mannose/fucose receptors
P150,95 (cD11c)
B Lymphocytes
CD5
CD19, 20, and 22
CD72-78
Complement receptors
C3b (CR1, CD35); C3d (CR2, CD21)
Igα
Igβ
MHC Class II (DP, DQ, DR)

cells in the body probably make more than
100 million different antibody proteins that
are expressed as immunoglobulin receptors. Because the human body does not
have enough genes to code for the millions
of different foreign proteins that the immune system must have the capability to
recognize, a process called gene rearrangement is used to create the required diver9
sity.

Binds to cells coated with C3b
Adhesion and activation
Receptors that bind cytokines signaling
activation and other cell functions
High affinity for IgG
Medium affinity for IgG
Low affinity for IgG
Low-affinity receptor for the Fc of IgE
Adhesion and activation
Binds sugars on microorganisms
Adhesion and activation

Cell marker that identifies a subset of B cells
predisposed to autoantibody production
Primary cell markers used to distinguish
B cells
Other cell markers that identify B cells
Play a role in cell activation and “homing”
of cells
Transport and assemble IgM monomers in
the cell membrane
Accessory molecules that interact with the
transmembrane segments of IgM
Present on antigen-presenting cells and is
critical for initiating T-cell-dependent
immune responses

Several genes code for the heavy chain
and light chain that make up the immunoglobulin receptor on B cells. The loci for
genes that code for the heavy chain are on
chromosome 14 and the loci that code for
the light chains are on chromosome 2 (κ
light chain) or chromosome 22 (λ light
chain). Three gene loci contribute to the diversity of the immunoglobulin receptor: V
(variable), D (diversity), and J (joining). The

Chapter 11: Immunology

251

Figure 11-3. The pluripotent stem cell, in the upper middle part of the diagram, gives rise to the lymphoid stem cell and to the myeloid stem cell, from which all other lines of blood cells derive.
Cytokines from marrow stromal cells influence the replication and differentiation of stem and later
cells. Cytokines from activated members of the highly differentiated T-cell and macrophage lines exert
major effects at all stages of myeloid and lymphoid development. (Used with permission from Goldsby
et al.8 )

252

AABB Technical Manual

fourth locus codes for the C (constant) region
of the immunoglobulin receptor and defines the expression of isotypes. The C region
does not affect antigen binding but codes
for the biologic functions associated with
the immunoglobulin such as complement
activation and binding to specific receptors.
Isotype switching (changing from IgM or
IgD to one of the other isotypes) is a T-celldependent process that occurs at the DNA
level. The process of isotype switching allows the specificity of antibody molecule to
be maintained regardless of the heavy chain
isotype.9,10 Table 11-3 summarizes the biologic properties of the different immunoglobulin isotypes.
At the pre-B-cell stage of development,
one heavy chain gene is randomly selected
from each of the four segments (VH, DH, JH,
4
and CH). There are over 10 possible combinations because of the large number of
possible genes at each segment. The light
chain gene has only three segments (VL, JL,
and CL), and one gene is selected from each
of these segments in a process similar to
the heavy chain gene selection. This process results in over 1000 possible light chain
combinations. When the heavy chain and
light chains are combined, approximately
10 million different combinations could be
formed, each one representing an immunoglobulin receptor with unique antigen
specificity. In addition to gene rearrangement, several other processes contribute to
this diversity. These processes include somatic mutations that occur at the time of
B-cell activation, combinatorial shuffling
that occurs when the heavy and light
chains are assembled, and the addition of
random DNA bases to the end of the genes
during the joining process. The combination of all of these processes ensures that B
cells have immunoglobulin receptors specific for any foreign antigen that could be
encountered (approximately 1011 antigen
specificities).9-11

Because the formation of the B-cell immunoglobulin receptor occurs through
random rearrangement, some of the receptors produced will react to the body’s own
cells. To prevent autoimmune disease, the
body must eliminate or downregulate these
B cells. This is accomplished through a process of negative selection.12 When a B cell is
formed, it encounters large quantities of self
antigen. If a B cell binds strongly to self antigen, the immunoglobulin receptor sends
a signal that activates enzymes within the
cell to cleave nuclear DNA. This causes the
cell to die, a process termed apoptosis (programmed cell death). Only 25% of the B cells
that mature in the marrow reach the circulation. The majority of B cells undergo
apoptosis. This process results in B cells
that have low affinity to self antigen but still
bind to foreign antigens that enter the
body. When mature B cells enter the peripheral blood circulation, they bind foreign antigens that are specific for their immunoglobulin receptors. When specific
antigen binds to the immunoglobulin receptor, a signal occurs causing the receptor/antigen complex to be internalized. Inside the cell, the antigen is degraded into
small peptides that bind to MHC Class II
molecules within the cell. This MHC-peptide complex is transported to the outer
membrane of the cell where it can interact
with the TCR. This interaction signals the
cell to produce various cytokines, causing
the B cell to proliferate into a memory cell
or a plasma cell. The antibodies produced
by a plasma cell are always of the same immunoglobulin class. However, each time
B-cell proliferation occurs, somatic mutations result in slight differences in the binding affinity of the immunoglobulin receptor. Because immunoglobulin receptors
with the highest binding affinity will be the
ones most likely to encounter antigen, this
process preferentially results in proliferation of B cells with the highest affinity for

Chapter 11: Immunology

253

Table 11-3. Characteristics and Biologic Properties of Human Immunoglobulins
Class

IgG

IgA

IgM

IgD

IgE

γ
4
κ,λ
150,000

α
2
κ,λ
180,000500,000

µ
1
κ,λ
900,000

δ
?
κ,λ
180,000

ε
?
κ,λ
200,000

Yes

Electrophoretic mobility

between γ
and β

fast γ

Sedimentation constant
(in Svedberg units)

6-7S

7-15S

19S

Gm allotypes (H chain)

Km allotypes (Kappa L chain:
formerly Inv)

Am allotypes

1000-1500

200-350

85-205

0.01-0.07

Total immunoglobulin (%)

<0.1

Synthetic rate (mg/kg/day)

6-7

<0.4

<0.02

Serum half-life (days)

2-8

1-5

Distribution (% of total
in intravascular space)

Present in epithelial secretions

Yes

Antibody activity

Yes

Probably
no

Yes

Usually
nonagglutinating

Usually
agglutinating

Fixes complement

Yes

Crosses placenta

Yes

Structure
H-chain isotype
Number of subclasses
L-chain, types
Molecular weight (daltons)
Exists as polymer

Serum concentration (mg/dL)

Serologic characteristics

antigen. This preferential selection is termed
a focused immune response.5,13,14

T Lymphocytes
There are two major types of T cells: cytotoxic T cells and helper T cells. These two
cell types can be differentiated by the pre-

sence of specific CD markers on the cell.
Cytotoxic T cells are positive for the surface marker CD8 and negative for CD4
and make up approximately one-third of
the circulating T cells in the peripheral
blood. Helper T cells are CD4 positive and
CD8 negative and represent approximately

254

AABB Technical Manual

two-thirds of the circulating T cells. Helper
T cells recognize antigen presented by
Class II HLA molecules, whereas cytotoxic
T cells recognize antigen in the context of
Class I HLA molecules. Approximately 5%
of peripheral blood T cells are negative for
both CD4 and CD8.
T cells go through a process of positive
and negative selection in the thymus during T-cell ontogeny. If a T cell is able to recognize self MHC antigens, it survives (positive selection) and migrates to the medulla
of the thymus. In the medulla, the T cells
undergo a process (negative selection) that
deletes T cells with high affinity for self
MHC antigens. T cells that fail to recognize
self MHC antigens undergo apoptosis. The
primary goal is to select T cells that recognize self MHC molecules that have foreign
peptides in their groove. The process is so
exquisite that approximately 10% of T cells
have the ability to react with foreign MHC
complexes, which forms the major basis of
transplantation rejection. In the end, only
5% of the cells in the thymus survive both
positive and negative selection and become
mature T cells.5,13,15
The receptor on the T cell that is responsible for MHC/peptide recognition is the TCR
(see Fig 11-2). There are two major types of
TCRs: those that express α and β chains or γ
and δ chains as part of the TCR complex.
Approximately 90% of all T cells bear α, β
chains. Each transmembrane chain has two
domains (one variable, the other constant).
These chains are produced through a process of gene rearrangement in a manner
similar to MHC and immunoglobulin receptors. The number of possible TCR α and
β specificities is estimated at 1015. The TCRs
bearing γ and δ are expressed on 5% to 15%
of T cells, predominantly by those T cells in
the mucosal endothelium. These T cells appear to play an important role in protecting
the mucosal surfaces of the body from foreign bacteria.

The TCR is noncovalently associated with
the CD3 complex, which is made up of three
pairs of dimers. This CD3 complex is responsible for signal transduction once peptide is recognized by the TCR.16
Recognition by Cytotoxic T Cells.
Cytotoxic T cells recognize peptides associated with Class I MHC molecules. As discussed previously, these peptides may be
derived from self proteins or proteins from
intracellular viruses or microbes. The
TCR-2 receptor on the cytotoxic T cell recognizes the peptide-MHC Class I molecule
in combination with a co-receptor (CD8) on
the T cell. These interactions signal the cell
to produce proteins (eg, perforin) that disrupt the integrity of the target cell membrane, resulting in cell death. During this
process, cytokines [TNF-α and interferon-γ
(IFNγ)] are also produced. These cytokines
prevent replication of virus that may be shed
from the cell during cell death; hence, the
infection is stopped through these processes.
Although the process is an extremely effective mechanism for killing cells infected with
virus, the process can be harmful to the host.
The cytokines produced to prevent viral replication can cause adverse effects including
the damage or destruction of healthy host
tissue. Liver damage associated with hepatitis B infection is an example of morbidity
caused by the cytotoxic T-cell response.5,11
Stimulation of B Cells. B-cell activation
can occur through activation by T cells or by
a mechanism independent of T-cell interaction.
These two mechanisms are described below.
T-Cell-Dependent Stimulation. Helper
T-cell receptors recognize foreign peptides
in the antigen-binding groove of Class II
MHC molecules in combination with CD4.
This TCR-CD4 interaction with MHC Class
II upregulates the expression of CD80/86
on the surface of antigen-specific B cells.
CD80/86 reacts with the ligand CD28 on
the T-cell surface, causing upregulation of
the CD40 ligand (CD40L), which engages

Chapter 11: Immunology

with CD40 on B cells. This cascade of signals is important for cytokine production,
which results in the isotype switch response
by the B cell. The production of interleukin
(IL)-4 causes B cells to switch from IgM to
IgG4 and IgE. The production of transforming growth factor β (TGF-β) and IL-10
causes the B cell to switch to IgA1 and IgA2.
If there is an absence or impaired function
of the ligand interaction isotype, switching
can be affected. For example, if the CD40LCD40 interaction is impaired, isotype
switching will not occur and only IgM antibody is produced. This clinical situation is
termed hyper-IgM immune deficiency.7
T-Cell-Independent Stimulation. B cells
can be activated to produce antibodies
by polysaccharides, lipopolysaccharides,
and polymeric proteins independent of
T-cell interaction. The B cells react directly
with these molecules, producing a rapid
immune response to pathogens. However,
there are disadvantages to this mechanism:
the process is ineffective for the production
of memory B cells; antibody affinity maturation is poor; and isotype switching is not
induced. The T-cell-independent process
can also cause antibody production by B
cells whose immunoglobulin is specific for
antigens other than those found on the
pathogen; hence, both protective antibodies
and autoantibodies may be produced. When
autoantibodies are produced through this
mechanism, transient clinical symptoms of
autoimmune disease may occur.7

NK Cells
NK cells do not express T-cell receptors or
B-cell receptors and represent approximately 10% of the lymphocyte population. These cells have the ability to kill
some cells infected with viruses and some
tumor cells. NK cells do this by a mechanism termed antibody-dependent cellular
cytotoxicity or ADCC. ADCC occurs when

255

viral proteins are expressed on the surface
of an infected cell, usually as a result of
viral budding. The proteins are recognized as foreign by the immune system
and antibodies are produced, which later
bind to the viral proteins expressed on the
infected cell. NK cells recognize the presence of antibody bound on the surface of
the cells via their Fcγ receptors. The NK
cells produce perforins, which cause lysis
of the virus-infected cells by a mechanism
that is not entirely understood.5

Phagocytic Cells
Phagocytes include cells such as monocytes
and polymorphonuclear granulocytes
(eosinophils, basophils, and neutrophils).
Some monocytes migrate into tissues
(liver, lungs, spleen, kidney, lymph nodes,
and brain) and become tissue macrophages.
The polymorphonuclear granulocytes are
rapidly produced and live only for a short
time (several days). The neutrophil is the
most abundant granulocyte. These cells
respond to chemotactic agents such as
complement fragments and cytokines,
causing them to migrate to the site of inflammation. Eosinophils represent only
2% to 15% of the white cells and play an
important role in regulating the inflammatory response by releasing an antihistamine. These cells may also play a role in
phagocytosing and killing microorganisms. Basophils make up less than 0.2% of
the total leukocyte pool. These cells also
respond to chemotactic factors and are
involved in the allergic response. Historically, this network of phagocytic cells was
called the reticuloendothelial system but is
now termed the mononuclear phagocytic
system.
The ability of cells to phagocytose is accomplished through the presence of receptors on the cell membrane. There are many
types of receptors including: mannose-fucose

256

AABB Technical Manual

receptors that bind to sugars on the surface
of microorganisms, Fc receptors that bind
to IgG, and complement receptors. A summary of some membrane receptors found
on macrophages and monocytes is found
in Table 11-2. The internalization and processing of particulate matter occur through
the production of enzymes (peroxidase and
acid hydrolases).5 Under optimal cytokine
conditions, macrophages and monocytes
can become formal antigen-presenting
cells; therefore, their ability to ingest and
process foreign molecules is an essential
part of the adaptive immune response.

Soluble Components of the
Immune Response
There are three major soluble components
of the immune response: immunoglobulins, complement, and cytokines.

Immunoglobulins

of the immunoglobulin molecule are held
together by disulfide bonds. The polypeptide chains (both heavy and light) are
looped, forming globular structures called
the immunoglobulin domain. On each
chain, there is a variable domain in which
the amino acid sequence is diverse, giving
the immunoglobulin its specificity. The epitopes expressed in this region are termed
idiotypes. The remaining domains on both
the heavy and light chains are called constant domains and have similar amino acid
sequences, depending on the isotype. The
hinge area of the molecule (between CH1
and CH2, as shown in Fig 11-4) gives the
molecule flexibility, allowing the two antigen-binding components to operate independently. The biologic functions of the
molecule are associated with the constant
domains on the heavy chain. These functions include: placental transfer, macrophage binding, and complement activation.4

Interchain Bonds

Immunoglobulins are the proteins that can
be cell bound and serve as antigen receptors on B cells (see section on B lymphocytes) or can be secreted in a soluble form
as antibodies. The molecular development
of the immunoglobulin molecule is discussed in the section on B lymphocytes.
The structures of the different types of
immunoglobulin are discussed below.
Each monomeric immunoglobulin molecule consists of two identical heavy chains
and two identical light chains. The heavy
chains consist of approximately 450 amino
acids with a molecular weight of approximately 50 to 77 kDa. There are five heavy
chain classes termed isotypes. They include
mu (µ), gamma (γ), alpha (α), delta (δ), and
epsilon (ε). The light chains are smaller (approximately 210 amino acids; molecular
weight 25 kDa) and can be either kappa (κ)
or lambda (λ). The heavy and light chains

Each light chain is joined to one heavy chain
by a disulfide bond. One or more disulfide
bonds link the two heavy chains at a point
between CH1 and CH2, in an area of considerable flexibility called the hinge region. It is these interchain disulfide bonds
that are the target of reducing agents used
to produce “chemically modified” anti-D
reagent.

Fab and Fc Fragments
Polypeptides can be cleaved at predictable
sites by proteolytic enzymes. Much information about immunoglobulin structure
and function is derived from the study of
cleavage fragments generated by papain
digestion of Ig molecules. Papain cleaves
the heavy chain at a point just above the
hinge, creating three separate fragments.
Two are identical, each consisting of one

Chapter 11: Immunology

257

Figure 11-4. The basic four-chain immunoglobulin unit. Idiotypic specificity resides in the variable domains of heavy and light chains (VH and VL ). Antigen-binding capacity depends on intact linkage between one light chain (VL and CL ) and the amino-terminal half of one heavy chain (VH and CH 1), the Fab
fragments of the molecule. Disulfide bonds in the hinge region join carboxy-terminal halves of both
heavy chains (CH 1 and CH 3, plus CH 4 for and heavy chains), to form the Fc fragment. (Used with permission from Goldsby et al. 8 )

light chain linked to the N-terminal half
of one heavy chain; the other fragment
consists of the C-terminal halves of the
heavy chains, still joined to one another
by the hinge-region disulfide bonds. The
two identical N-terminal fragments,
which retain the specificity of the antibody, are called Fab fragments. The joined
C-terminal halves of the heavy chains
constitute a nonantibody protein fragment capable of crystallization, called the
Fc fragment.

Immunoglobulin Polymers
Disulfide bonds may also join some Ig
monomeric units to one another to form
larger polymeric molecules; only IgM and
IgA can form polymers. IgG, IgE, and IgD

exist only in the monomeric form; there
are no polymeric forms of these Ig classes.
The IgM synthesized by unstimulated B
cells and expressed on the membrane as
the immunoglobulin receptor is expressed in the monomeric form. As mentioned previously, the µ heavy chain has
four constant domains in the membrane
form of IgM. The fourth domain allows
the IgM monomer to bind to the cell
membrane as the immunoglobulin receptor. Following clonal expansion and differentiation to a plasma cell, the activated
cell produces µ chains with one less constant domain. While still in the plasmacell cytoplasm, five IgM monomers are
joined by the formation of disulfide bonds
between the CH3 domains and CH4 do-

258

AABB Technical Manual

mains. The result is a pentamer that is
secreted to the exterior of the cell and
constitutes the form in which IgM accumulates in body fluids.
Secreted IgA exists in both monomeric
and polymeric forms. Monomeric forms
predominate in the bloodstream, but dimers
and trimers that are secreted by B cells in
mucosal surfaces and exocrine tissue are
the biologically active form.

Other Chains
Pentameric IgM and the dimers and trimers of IgA contain a 15-kDa polypeptide
called the J chain. Before the polymer
leaves the plasma-cell cytoplasm, this
chain attaches to the terminal constant
domain of two adjacent monomers. No
matter how many monomers constitute
the polymer, there will be only one J chain.
Its function is not fully understood.
The polymeric Ig molecules present in
epithelial secretions also exhibit a subunit
called the secretory component. The secretory component appears to protect the biologically important surface antibodies from
proteolysis in the enzyme-rich secretions of
respiratory and alimentary tracts.

Individual Immunoglobulin Classes
IgM. IgM is the first Ig class produced by
the maturing B cell. It is the first to appear
in the serum of maturing infants and the
first to become detectable in a primary
immune response. Secreted pentameric
IgM normally constitutes 5% to 10% of the
immunoglobulin in normal serum. Very
few of these large molecules diffuse into
interstitial fluid.
Although the five monomers comprise
10 antigen-combining sites, only five sites
are readily available to combine with most
antigens; hence, IgM is described as pentavalent. Because of their large size and

multivalency, IgM molecules readily bind to
antigens on particulate surfaces, notably
those on red cells or microorganisms. IgM
antibodies can crosslink cells expressing a
specific antigen, forming a clump of cells
(agglutination). Although extremely useful
as a laboratory endpoint, agglutination
probably plays a relatively minor role in
biologic events.
The most important biologic effect of IgM
is its ability to activate the complement
cascade, which enhances inflammatory
and phagocytic defense mechanisms and
may produce lysis of antigen-bearing cells.
IgG. Immunoglobulin G exists only as a
monomer and accounts for 75% to 80% of
the immunoglobulins present in serum. It
is equally distributed in the intravascular
and extravascular compartments. In vivo,
cells or particles coated with IgG undergo
markedly enhanced interactions with cells
that have receptors for the Fc portion of γ
chains, especially neutrophils and macrophages.
IgG molecules can be classified into four
subclasses: IgG1, IgG2, IgG3, and IgG4.
Structurally, these subclasses differ primarily in the characteristics of the hinge region
and the number of inter-heavy-chain
disulfide bonds. Biologically, they have significantly different properties. IgG3 has the
greatest ability to activate complement, followed by IgG1 and, to a much lesser extent,
IgG2. IgG4 is incapable of complement activation. IgG1, IgG3, and IgG4 readily cross
the placenta. IgG1, IgG2, and IgG4 have a
half-life of 23 days, significantly longer than
that of other circulating immunoglobulins;
however, the half-life of IgG3 is only slightly
longer than IgA and IgM. IgG1 and IgG3
readily interact with the Fc receptors on
phagocytic cells, but IgG4 and IgG2 have
low affinity for these receptors with the exception that IgG2 has an affinity similar to
IgG1 and IgG3 for an allotype of Fcγ receptor IIa.

Chapter 11: Immunology

IgA. Although there is a large body content of IgA (10% to 15% of serum immunoglobulin concentration), relatively little is
found in the blood. Most of the IgA exists in
mucosal secretions. Secretory IgA protects
the underlying epithelium from bacterial
and viral penetration. Polymeric IgA is
thought to combine with environmental
antigens, forming complexes that are eliminated as surface secretions are excreted.
This process may be important in controlling the development of hypersensitivity.
The heavy chain of IgA has no complement-binding site; hence, IgA cannot activate complement through the classical
pathway. IgA can activate complement
through the alternative pathway (see below).
IgE. The concentration of serum IgE is
measured in nanograms, compared with
milligram levels for other immunoglobulins. Even when patients with severe allergies have markedly elevated serum concentrations of IgE, the absolute level is minimal
compared with other immunoglobulins.
Most IgE is present as monomers tightly
bound to the membrane of basophilic
granulocytes or mast cells. IgE is responsible
for immediate hypersensitivity events, such
as allergic asthma, hay fever, and systemic
anaphylactic reactions. Although IgE appears to be involved in reactions to protozoal parasites, no specific protective mechanisms have been identified.
IgD. Serum contains only trace amounts
of IgD. Most IgD exists as membrane immunoglobulin on unstimulated B cells. The
function of IgD is unknown, but it may be
important for lymphocytic differentiation,
which is triggered by antigen binding, and
in the induction of immune tolerance.

Complement
Complement is the term applied to a system of 25 to 30 serum and membrane

259

proteins that act in a cascading manner—
similar to the coagulation, fibrinolytic,
and kinin systems—to produce numerous
biologic effects. The participating proteins remain inactive until an event initiates the process, following which the
product of one reaction becomes the catalyst for the next step (see Fig 11-5). Each
evolving enzyme or complex can act on
multiple substrate molecules, creating the
potential for tremendous amplification of
an initially modest or localized event.
Complement has three major roles: promotion of acute inflammatory events; alteration of surfaces so that phagocytosis is enhanced; and the modification of cell membranes, which leads to cell lysis. These actions cause destruction of bacteria, protect
against viral infection, eliminate protein
complexes, and enhance development of
immune events. However, activation of
complement can also initiate inflammatory
and immune processes that may harm the
host and mediate destruction of host cells,
especially those in the blood.
17
Different mechanisms exist for activating the complement cascade: the classical
pathway, which is initiated by interaction
between an antibody and its antigen, and
the alternative pathway, which is usually not
antibody mediated.

The Classical Pathway
For the classical pathway of complement
activation to occur, an immunoglobulin
must react with target antigen. Combination with antigen alters the configuration
of the Fc portion of the immunoglobulin,
rendering accessible an area in one of the
heavy-chain constant domains that interacts with the C1 component of complement. C1 can combine only with Ig molecules having an appropriate heavy-chain
configuration. This configuration exists in

AABB Technical Manual

Classical Pathway

260

Figure 11-5. Diagram illustrating the activation of complement by the classical and alternative pathways. (Used with permission from Heddle. 1 )

the µ heavy chain and in the γ chains in
IgG subclasses 1, 2, and 3.
The C1 component of complement attaches to activation sites on the Fc portion of
two or more Ig monomers. The pentameric
IgM molecule provides an abundance of
closely configured Fc monomers; hence, a
single IgM molecule can initiate the complement cascade. For IgG antibodies to activate the sequence, two separate molecules
3
must attach to antigen sites close together.
Hence, complement activation by IgG antibodies depends not only on the concentration and avidity of the antibody, but also on
the topography of the antigen.
Circulating C1 is a macromolecule consisting of three distinct proteins (C1q, C1r,
and C1s). When an antigen-antibody reaction occurs, two or more chains of C1q attach to the CH2 domain of IgG or the CH3
domain of IgM. This causes a conforma-

tional change, resulting in activation of two
C1r molecules that then cleave two C1s
molecules into activated C1s (a strong
serine esterase). The C1r, C1s, and C1q
complex is stabilized by Ca2+ ions. In the ab2+
sence of Ca , the complex dissociates.
Thus, chelating agents often used in the
laboratory, such as citrate and oxalate anticoagulants, prevent the stabilization of the
C1 complex and subsequent activation of
complement proteins.
Activated C1s works on two substrates.
C1s cleaves C4 into two fragments: a large
fragment (C4b) and a smaller fragment
(C4a) that has modest anaphylatoxic activity. Most of the C4b generated is inactivated; however, some C4b binds to the cell
surface and acts as a binding site for C2.
C1s also cleaves the bound C2, releasing a
fragment called C2b. The C2a fragment that
remains bound to C4 forms an activated

Chapter 11: Immunology

complex C4b2a, also known as C3 convertase. Each C3 convertase can cleave more
than 200 native C3 molecules, splitting the
molecule into two fragments. A small fragment (C3a) that has anaphylatoxic activity
is released into the plasma; the larger fragment (C3b) attaches to proteins and sugars
on the cell surface.
The classical pathway and the alternative
pathway are both means of generating a C3
convertase. Once C3 has been cleaved, the
same events occur in the two pathways.

The Alternative Pathway
The alternative pathway allows complement activation in the absence of an antigen/antibody interaction. This pathway is
a first-line antimicrobial defense for vertebrates and a mechanism whereby prevertebrates can enhance their inflammatory effectiveness. The alternative pathway
is a surface-active phenomenon that can
be triggered by such initiators as dialysis
membranes; the cell wall of many bacteria, yeasts, and viruses; protein complexes, including those containing antibodies that do not bind complement;
anionic polymers such as dextran; and
some tumor cells. The alternative pathway of complement activation can also
occur spontaneously in the plasma at a
slow but steady rate (tickover activation)
(see Fig 11-5).
Four proteins participate in the alternative pathway: factor B, factor D, properdin
(factor P), and C3. Fluid-phase C3 undergoes continuous but low-level spontaneous
cleavage, resulting in C3i that is rapidly inactivated by fluid-phase control proteins. If
C3i encounters factor B, a complex called
C3iB is formed and additional interactions
can occur. Factor D acts on bound factor B,
generating a C3iBb complex capable of
cleaving C3 into C3a and C3b. Most of the
C3b generated in the fluid phase is inacti-

261

vated; however, if C3b binds to a foreign
surface such as a bacteria cell wall, the activation of the complement cascade can be
accelerated.
When C3b binds to a cell surface, factor
B is bound to give C3bB. Factor D can also
react with this cell-bound substrate, releasing the small Ba fragment leaving cellbound C3bBb. This complex will dissociate
unless it is stabilized by properdin, resulting in the complex C3bBbP. Like the C4b2a
complex of the classical pathway, C3bBbP
is capable of converting more C3 into C3b.
In summary, both the classical and alternative pathways result in C3b generation. The
C3 convertase of the classical pathway is
C4b2a, whereas the C3 convertases of the
alternative pathway are C3iBb in the fluid
phase and C3bBbP when cell bound.

The Membrane Attack Complex
The final phase of activation is called the
membrane attack complex and can occur
once C3b has been cleaved through either
the classical or alternative pathway. C3
convertase cleaves C5 into two fragments:
a small peptide (C5a) having potent anaphylatoxin activity and a larger fragment
(C5b). The C5b fragment binds C6, C7,
C8, and up to 14 monomers of C9, resulting in a lytic hole in the membrane. Small
amounts of lysis can occur when C8 is
bound; however, the binding of C9 facilitates cell lysis.
The binding of C3b to a cell membrane
is the pivotal stage of the pathway. The
cell-bound C3b can proceed to activate the
membrane attack complex (C5-C9) and
cause cell lysis; alternatively, inhibitors may
stop the activation sequence, leaving the
cell coated with C3b. Factor I is an inhibitor
that can cleave cell-bound C3b, leaving
iC3b on the membrane. These two subcomponents of C3 (C3b, iC3b) can facilitate
phagocytosis by acting as opsonins. How-

262

AABB Technical Manual

ever, C3b can be further cleaved, leaving a
small fragment called C3dg. It is the C3dg
molecule that is detected by the anti- C3d
component in anticomplement reagents
used for the direct antiglobulin test.

Complement Receptors
Some phagocytic cells have receptors that
can bind to C3 on the cell. Four different
complement receptors have been identified on phagocytic cells: CR1, CR2, CR3,
and CR4.18
CR1. CR1 is found on a variety of cells.
On red cells and platelets, the CR1 receptor
plays an important role in clearing immune
complexes. On phagocytic cells and B lymphocytes, it is an opsonic receptor that is
involved in lymphocyte activation. CR1 also
plays a regulatory role in complement activation by assisting factor I in cleaving C3b
into iC3b and C3dg.
CR2. CR2 is found on B cells, some epithelial cells, and follicular dendritic cells. It
plays an important role in mediating B-cell
activation. It is also the receptor for interferon α and the Epstein-Barr virus.
CR3. CR3 is found on cells of the myeloid
lineage. CR3 mediates phagocytosis of particles coated with iC3b and is also an important adhesion molecule capable of binding
to certain types of bacteria and yeast.
CR4. CR4 is found on both lymphoid
and myeloid cells. Its function is not well
characterized, but it appears to have opsonic activity for iC3b, and it plays a role in
adhesion.

decay and destruction of convertases, and
control of membrane attack complexes.19

Physiologic Effects of Complement
Activation
Opsonization. Neutrophils and macrophages phagocytose any particle that protrudes from the surface of a cell or microorganism with no regard to the nature of
the material. Phagocytosis is more intense
if the particle adheres firmly to the membrane of the phagocytic cell. To achieve
adherence, phagocytic cells have various
receptor molecules such as the Fc receptors for certain immunoglobulins and
receptors for C3b. The enhancement of
phagocytosis resulting from antibody or
complement coating of cells is called
opsonization.
Anaphylatoxins Promote Inflammation.
Complement fragments C3a and C5a are
anaphylatoxins and they play an important
role in acute inflammation. These anaphylatoxins bind to receptors on mast cells
and basophils, causing them to release histamine and other biologic response modifiers that can be associated with anaphylaxis.
More frequently, anaphylatoxins affect vascular permeability, membrane adhesion
properties, and smooth-muscle contraction
that constitute a large part of the acute inflammatory response. C5a and C3a also
cause neutrophils and macrophages to migrate to the site of complement activation.

Cytokines

Regulation of Complement Activation
There is a need to control or regulate the
enzyme and activation factors of the complement cascade. The regulatory actions
of these control proteins prevent damage
to host tissue. These control systems
(Table 11-4) act by several different mechanisms: direct inhibition of serine proteases,

Throughout this chapter, inference has
been made to a number of soluble mediators termed cytokines. Cytokines are a
diverse group of intracellular signaling
peptides and glycoproteins that have molecular weights ranging from 6,000 to
60,000 daltons. Each cytokine is secreted
by particular cell types in response to dif-

Chapter 11: Immunology

263

Table 11-4. Summary of the Inhibitors of Complement Activation
Complement Control Proteins
Inhibition of Serine Protease
C1 inhibitor
Decay/Destruction of Convertases
Factor I plus C4 binding protein (C4-bp)
C4-bp
Decay accelerating factor
(DAF, CD55)
Complement receptor 1 (CR1, CD35)
Membrane cofactor protein (MCP)
Factor H

Factor I

Membrane Attack Complex (MAC)
S protein (Vitronectin)
C8-binding protein (CD59)
MAC-inhibiting protein (MIP)

ferent stimuli and has been implicated in
a wide variety of important regulatory biologic functions such as inflammation,
tissue repair, cell activation, cell growth,
fibrosis, and morphogenesis. One cytokine
can have several different functions, depending on the type of cell to which it
binds. Several hundred different cytokines
have been described. Some of the cytokines involved in immune functions are
1,5
summarized in Table 11-5. Cytokines
have been implicated in a variety of clinically important factors of transfusion
medicine (hemolytic and febrile transfu-

Function
A serine protease inhibitor that binds and activates Clr and Cls
Catabolizes C4 in the fluid phase
Causes dissociation of C2a from C4b2a
Inhibits the binding of C2 to C4b
Causes dissociation of C2 from C4b
Accelerates the dissociation of C3bBb
Prompts catabolism of C4b by factor I and is a
cofactor with factor I to cleave C3b
Causes dissociation of Bb from C3I and C3b and
is a cofactor to factor I for catabolism of C3i
and C3b
Cleaves and degrades C3b using one of three
cofactors: factor H (plasma), CR1, or MCP
(membrane-bound)
Binds to the C5b67 complex preventing insertion
into the lipid bilayer
This cell-bound protein binds to C8 preventing
C9 from inserting into the membrane
Inhibitor of C9, also known as homologous
restriction factor (HRF)

sion reactions, immunomodulation, stem
cell collection, etc) (see Chapter 27).

Immunology Relating to
Transfusion Medicine
Several examples of how the immune system relates to situations encountered in
transfusion medicine are described below,
including red cell alloimmunization, platelet alloimmunization, immune-mediated red cell destruction, and reagent antibody production.

264

AABB Technical Manual

Table 11-5. Summary of Some Cytokines Involved in the Immune System, Their
1
Site of Production, and Function
Cytokine

Produced by

Primary Functions

IL-1

Many cells (eg, APCs,
endothelial cells, B cells,
fibroblasts)

T-cell activation, neutrophil
activation, stimulates
marrow, pyrogenic, acutephase protein synthesis

IL-2

Activated TH cells

T-cell growth, chemotaxis,
macrophage activation

IL-4

Activated TH cells

B-cell activation, B-cell
differentiation, T-cell
growth, TH2 differentiation

IL-6

Many cells (eg, T cells,
APCs, B cells, fibroblasts,
and endothelial cells)

B-cell differentiation,
pyrogenic, acute-phase
protein synthesis

IL-8

Many cells (eg, macrophages
and endothelial cells)

Inflammation, cell migration/
chemotaxis

IL-10

Activated TH2 cells

Suppression of TH1 cells,
inhibits antigen
presentation, inhibits
cytokine production (IL-1, IL-6,
TNFα, and IFN)

TNF

Macrophages and
lymphocytes

Neutrophil activation,
pyrogenic, acute-phase
protein synthesis

Interferon γ (IFNγ)

T cells

Phagocyte activation

Transforming growth
factor-β (TGF-β)

Various cells

Stimulates connective tissue growth
and collagen formation, inhibitory function

Colony-stimulating
factors (GM-CSF,
M-CSF, G-CSF)

Various cells

Growth and activation of phagocytic
cells

Interleukins

Others

APCs = antigen-presenting cells; G-CSF = granulocyte colony-stimulating factor; GM-CSF = granulocyte macrophage–
colony-stimulating factor; M-CSF = macrophage colony-stimulating factor; TNF = tumor necrosis factor.

Chapter 11: Immunology

Red Cell Alloimmunization
The mechanism of red cell alloimmunization is not well understood. When allogeneic red cells are transfused, some of
the red cells fragment as they age or when
they pass through the spleen, thus releasing membrane-bound red cell proteins
into the bloodstream as the fragments degrade. Both formal antigen-presenting
cells and B lymphocytes can present antigen either as a primary immune response
(formal antigen presentation by dendritic
cells) or in the secondary immune response by B cells.

HLA Alloimmunization to Platelets
Leukocyte reduction of red cells and pla6
telets to a threshold of 5 × 10 per product
has been shown to reduce HLA alloimmunization, possibly by the following
mechanism. Donor leukocytes express
both Class I and Class II MHC antigens.
Some of the donor Class II MHC antigens
will contain peptides that originated from
the MHC Class I antigens on the donor’s
cells. Most of the transfusion recipient’s T
cells recognize self-MHC-carrying foreign
peptides. However, as mentioned previously, a small percentage of T cells (<10%)
are able to recognize foreign-MHC-carrying foreign peptides. When platelets are
transfused, the recipient’s T helper cells
recognize the foreign MHC Class II complex on the donor leukocytes. If a signal
occurs, the T cells activate recipient B
cells, which have also bound donor MHC
Class I antigen fragments to their Ig receptor, resulting in cell proliferation and
MHC Class I (HLA) antibody production
(Fig 11-6). In this case, the donor’s leukocytes serve as the antigen-presenting
cells. This major mechanism is termed
“direct allorecognition” because the recipient’s T cells are directly stimulated by donor antigen-presenting cells.20 Alterna-

265

tively, the process of antigen presentation
and immune recognition of foreign HLA
peptides can occur by the recipient’s own
immune system. This alternative form of
allorecognition is termed “indirect” and is
the classical alloimmune response seen for
most foreign antigens. Leukocyte reduction appears to be effective in reducing
HLA alloimmunization because antigen
presentation by donor leukocytes is reduced and the amount of HLA Class I antigens transfused is greatly reduced.

Immune-Mediated Red Cell Destruction
The activation of complement and/or the
presence of IgG on the red cell surface can
trigger red cell destruction by predominantly two mechanisms: intravascular
hemolysis and extravascular red cell destruction (Fig 11-7). Intravascular hemolysis
occurs when complement is activated,
resulting in activation of the membrane
attack complex. As the integrity of the
membrane is disrupted, hemoglobin is released into the plasma. Free hemoglobin
binds to haptoglobin and is excreted in
the urine. The heme portion of hemoglobin binds to albumin, forming methemalbumin, which can be visually detected in
plasma by a brownish green discoloration. At times, the inhibitors of complement stop the cascade, leaving C3b on the
red cells. This complement fragment has
chemotactic activity for phagocytic cells
and these cells have receptors for C3b.
C3b-coated red cells adhere to phagocytic
cells but are relatively ineffective at triggering phagocytosis. Enzymes can cleave
the cell-bound C3b, leaving a small fragment (C3dg) present on the cells. C3dgsensitized red cells survive normally because phagocytic cells do not have receptors for C3dg. If IgG is present on the red
cells, binding to Fcγ receptors will occur,
resulting in phagocytosis. If both IgG and

266

AABB Technical Manual

Figure 11-6. Diagram illustrating the major mechanism of HLA alloimmunization due to leukocytes
present in platelet transfusion. (Used with permission from Heddle. 1 )

C3b are present on the red cells, clearance
of the cells may be enhanced, probably
because of the chemotactic function of
C3b and its adherence capability.

Reagent Antibodies
Heterogeneous antibodies are not optimal as reagents for use in serologic testing
because they can vary in concentration,
serologic properties, and epitope recognition and can contain other antibodies of
unwanted specificity. The ideal serum for
serologic testing is a concentrated suspension of highly specific, well-characterized, uniformly reactive, immunoglobulin
molecules. Until the 1970s, the only way
to obtain reagents was to immunize animals or humans with purified antigens and
then perform time-consuming and sometimes unpredictable separation techni-

ques in an attempt to purify the resulting
sera. Monoclonal antibody production
provided an alternative to human and animal sources of these proteins. Using this
approach, a single B-cell clone is propagated in cell culture and the supernatant
fluid from the culture contains antibody
of a single specificity. However, there are
problems with this approach. Normal B
cells reproduce themselves only a limited
number of times; hence, the cultured cell
lines survive only a short time.
21
In 1976, Köhler and Milstein provided a
solution to this problem. Plasma cells of
normal antibody-producing capacity were
fused to neoplastic plasma cells of infinite
reproductive capacity (ie, myeloma cells).
Techniques had previously been developed
that cause cell membranes to merge, allowing the cytoplasm and the nucleus of two

Chapter 11: Immunology

267

*Ineffective mediator of phagocytosis.
†

Due to the chemotactic ability of C3b, extravascular destruction may be enhanced when both IgG and C3b are present on the

red cells.

Figure 11-7. Summary of intravascular and extravascular red cell destruction.
different kinds of cells to fuse into a single
cell. These plasma cell/myeloma cell hybrids can be maintained in cell culture for
prolonged periods, producing large quantities of the selected antibody.
The exquisite specificity of monoclonal
antibodies is both an advantage and a disadvantage for reagent use. An antibody that
gives strong and specific reactions with one
epitope of a multivalent antigen molecule
may fail to react with cells whose antigen
expression lacks that particular configuration. Thus, reagent preparations typically
used in the laboratory are blends of several
different monoclonal products, thereby increasing the range of variant phenotypes
that the antiserum can identify. Single or
blended monoclonal preparations often re-

act more strongly than immune-serum
preparations when tested against cells with
weakly expressed antigens.
Phage technology is under investigation
as an approach for producing genetically
engineered antibodies for a variety of therapeutic treatments,22 as well as for use as
23
typing reagents.

References
1.

Heddle NM. Overview of immunology. In:
Reid ME, Nance SJ, eds. Red cell transfusion.
A practical guide. Totowa, NJ: Humana Press,
1998:13-37.
Barclay AN. Membrane proteins with immunoglobulin-like domains—a master superfamily of interaction molecules. Semin
Immunol 2003;15:215-3.

268

5.
6.

7.
8.

9.
10.

11.
12.

13.
14.

15.

16.

17.

18.
19.
20.

AABB Technical Manual

Marsh SG, Albert ED, Bodmer WF, et al. Nomenclature for factors of the HLA system, 2002.
Hum Immunol 2002;63:1213-68.
Santos-Aguado J, Barbosa JA, Biro A, Strominger JL. Molecular characterization of serologic recognition sites in the human HLAA2 molecule. J Immunol 1988;141:2811-18.
Roitt I, Brostoff J, Male D. Immunology. 5th
ed. St. Louis: Mosby, 1998.
Ono SJ, Nakamura T, Miyazaki D, et al.
Chemokines: Roles in leukocyte development, trafficking, and effector function. J Allergy Clin Immunol 2003;111:1185-99.
Huston DP. The biology of the immune system. JAMA 1997;278:1804-14.
Goldsby RA, Kindt TJ, Osborne BA. Kuby immunology. 4th ed. New York: WH Freeman
and Company, 2000.
Tonegawa S. Somatic generation of antibody
diversity. Nature 1983;302:575-81.
Alt FW, Backwell TK, Yancopoulos GD. Development of the primary antibody repertoire.
Science 1987;238:1079-87.
Janeway CA Jr. How the immune system recognizes invaders. Sci Am 1993;269(3):73-9.
Russell DM, Dembic Z, Morahan G, et al. Peripheral deletion of self-reactive B cells. Nature 1991;354:308-11.
Weissman IL, Cooper MD. How the immune
system develops. Sci Am 1993;269(3):64-71.
Paul WE. Infectious diseases and the immune
system. When bacteria, viruses and other
pathogens infect the body, they hide in different places. Sci Am 1993;269(3):90-7.
Marrack P, Lo D, Brinster R, et al. The effects
of thymus environment on T cell development and tolerance. Cell 1988;53:627-34.
Clevers H, Alarcon B, Willeman T, Terhorst C.
The T cell receptor/CD3 complex: A dynamic
protein ensemble. Annu Rev Immunol 1988;
6:629-62.
Sakamoto M, Fujisawa Y, Nishioka K. Physiologic role of the complement system in host
defense, disease, and malnutrition. Nutrition
1998;14:391-8.
Roitt I. Essential immunology. 8th ed. Oxford:
Blackwell Scientific Publications, 1994.
Devine DV. The regulation of complement on
cell surfaces. Transfus Med Rev 1991;5:123-31.
Semple JW, Freedman J. Recipient antigenprocessing pathways of allogeneic platelet
antigens: Essential mediators of immunity.
Transfusion 2002;42:958-61.

21.

22.

23.

Köhler G, Milstein C. Derivation of specific
antibody-producing tissue culture and tumor
lines by cell fusion. Eur J Immunol 1976;6:
511-19.
Marks C, Marks JD. Phage libraries—a new
route to clinically useful antibodies. N Engl J
Med 1996;335:730-3.
Siegel DL. Phage display tools for automated
blood typing (abstract). Transfusion 2004;
44(Suppl):2A.

Suggested Reading
Abbas AK, Lichtman AH, Pober JS, eds. Cellular
and molecular immunology. 4th ed. Philadelphia:
WB Saunders, 2000.
Anderson KC, Ness PM, eds. Scientific basis of
transfusion medicine. 2nd ed. Philadelphia: WB
Saunders, 1999.
Barclay AN, Brown MH, Law SKA, et al. The leukocyte antigen factsbook. 2nd ed. San Diego, CA: Academic Press, 1997.
Carroll MC. The role of complement and complement receptors in induction and regulation of immunity. Annu Rev Immunol 1998;16:545-68.
Janeway CA. Immunobiology: The immune system
in health and disease. 5th ed. New York: Garland
Publishing, 2001.
Marchalonis JJ, Schluter SF, Bernstein RM, et al.
Phylogenetic emergence and molecular evolution
of the immunoglobulin family. Adv Immunol 1998;
70:417-506.
Muller D. The molecular biology of autoimmunity.
Immunol Allerg Clin North Am 1996;16:659-82.
Paul WE. Fundamental immunology. 5th ed. Philadelphia: Lippincott Williams & Wilkins, 2003.
Paul W, Raghavan M, Bjorkman PJ. Fc receptors
and their interactions with immunoglobulins.
Annu Rev Cell Dev Biol 1996;12:181-220.
Stites DP, Terr AI, Parslow TG, eds. Medical immunology. 10th ed. Stamford, CT: Appleton & Lange, 2001.
Vamvakas EC, Blajchman MA, eds. Immunomodulatory effects of blood transfusion. Bethesda, MD:
AABB Press, 1999.

Chapter 11: Immunology

269

Appendix 11-1. Definitions of Some Essential Terms in Immunology
Adhesion molecule: Any of the many membrane
molecules expressed on white cells and endothelial cells that allow cells to come into
close apposition with each other.
Allotypic: Variations in the amino acid structure
of heavy and light chains unrelated to antibody specificity. Present in some but not all
members of the species.
Antibody: Immunoglobulin secreted by the
plasma-cell progeny of B lymphocytes after
stimulation by a specific immunogen. Immunoglobulin molecules on the surface of unstimulated lymphocytes serve as antigen
receptors.
Antigen: Any material capable of specific combination with antibody or with cell-surface receptors of T lymphocytes. Often used as a
synonym for “immunogen,” although some
antigens that react with products of the immune response are not capable of eliciting
an immune response.
Antigen-presenting cell: A cell capable of incorporating antigenic epitopes into MHC Class II
molecules and displaying the epitope-MHC
complex on its membrane.

Immune system: A collective term for all the
cells and tissues involved in immune activity.
It includes, in addition to lymphocytes and
cells of monocyte/macrophage lineage, the
thymus, lymph nodes, spleen, marrow, portions of the liver, and the mucosa-associated
lymphoid tissue.
Immunogen: A material capable of provoking an
immune response when introduced into an
immunocompetent host to whom it is foreign.
Ligand: A molecule, either free in a fluid milieu
or present on a membrane, whose three-dimensional configuration allows it to form a
tightly fitting complex with a cell-surface
molecule (its receptor) of complementary
shape.
MHC Class I molecules: Heterodimeric membrane
proteins determined by genes in the MHC,
consisting of a highly polymorphic α chain
linked noncovalently with the nonpolymorphic
β 2 -microglobulin chain; these molecules
present antigen to CD8+ T cells and are the
site of HLA antigens of the HLA-A, HLA-B,
and HLA-C series.

Cytokine: A low-molecular-weight protein, secreted from an activated cell, that affects the
function or activity of other cells.

MHC Class II molecules: Heterodimeric membrane proteins determined by genes in the
MHC, consisting of two transmembrane
polypeptide chains; these molecules present
antigen to CD4+ T cells and exhibit the
HLA-DP, HLA-DQ, and HLA-DR series of antigens.

Epitope: The small portion of an immunogen,
usually 5 to 15 amino acids or 3 to 5 glycosides, that combines specifically with the antigen receptor of a T or B lymphocyte.

Phagocytosis: The process whereby macrophages and granulocytes ingest particulate
material present in the surrounding milieu
and subject it to intracellular alteration.

Idiotype: The molecular configuration unique to
the variable portion of an antigen-receptor
molecule, reflecting the DNA rearrangement
occurring in earliest lymphocyte differentiation and conferring upon the cell its specificity of antigen recognition.

Receptor: A cell-membrane protein molecule
whose three-dimensional configuration allows it to form a tightly fitting complex with
another molecule (called its ligand) of complementary shape.

Clone: A population of genetically identical cells
derived from successive divisions of a single
progenitor cell.

Chapter 12: Red Cell Antigen-Antibody Reactions and Their Detection

Chapter 12

Red Cell Antigen-Antibody
Reactions and Their
Detection

EMONSTRATION OF RED cell antigen-antibody reactions is key to
immunohematology. The combination of antibody with antigen may produce a variety of observable results. In
blood group serology, the most commonly observed reactions are agglutination, hemolysis, and precipitation.
Agglutination is the antibody-mediated
clumping of particles that express antigen
on their surface. Agglutination of red cells
occurs because antibody molecules bind to
antigenic determinants on multiple adjacent red cells, linking them together to
form a visible aggregate. Agglutination is
the endpoint for most tests involving red
cells and blood group antibodies and is the
primary reaction type discussed in this
chapter. In some tests, antibody directly
bridges the gap between adjacent cells; in
others, antibody molecules attach to, but
do not agglutinate, the red cells, and an additional step is needed to induce visible ag-

glutination or to otherwise measure the
reaction.
Hemolysis is the rupture of red cells with
release of intracellular hemoglobin. In-vitro
antibody-mediated hemolysis depends on
activity of the membrane attack unit of
complement. Hemolysis does not occur if
the antigen and antibody interact in serum
that lacks complement, or in plasma in
which the anticoagulant has chelated the
cations (calcium and magnesium) necessary for complement activation. In tests for
antibodies to red cell antigens, hemolysis is
a positive result because it demonstrates
the union of antibody with antigen that activates the complement cascade. (The actions of complement are described in
Chapter 11.) Pink or red supernatant fluid
in a test system of serum and red cells is an
important observation that may indicate
that antigen-antibody binding has taken
place. Some antibodies that are lytic in vitro
(eg, anti-Vel, anti-Lea, and anti-Jka) may
271

272

AABB Technical Manual

cause intravascular hemolysis in a transfusion recipient.
Precipitation is the formation of an insoluble, usually visible, complex when soluble antibody reacts with soluble antigen.
Such complexes are seen in test tubes as a
sediment or ring and in agar gels as a white
line. Precipitation is the endpoint of procedures such as immunodiffusion and immunoelectrophoresis.
Precipitation may not occur, even when
soluble antigen and its specific antibody
are present. Precipitation of the antigen-antibody complex requires that antigen and
antibody be present in optimal proportions. If antibody is present in excess, too
few antigen sites exist to crosslink the molecules and a lattice structure is not formed.
Antigen-antibody complexes form but do
not accumulate sufficiently to form a visible lattice. This phenomenon is called a
prozone.
The combination of soluble antigen with
soluble antibody may also result in a full or
partial neutralization of the antibody. Although a visible precipitate is often not
produced, such inhibition can be useful in
antibody identification procedures by selectively eliminating specific antibodies.

Stage One: Sensitization
Initially, the antigen and antibody must
come together and interact in a suitable
spatial relationship. The chance of association between antibody and antigen can
be enhanced in a number of ways, such as
agitation or centrifugation, or by varying
the relative concentration of antibody and
antigen. As shown in Fig 12-1, antibody
and antigen must complement each other
with both a structural (steric) and a chemical fit.
For sensitization to occur, a noncovalent
chemical bond must form between antigen
and antibody. The forces holding antigens
and antibodies together are generally weak
(compared with covalent bonds that hold
molecules together) and are active only
over a very short distance. The antigen-antibody combination is reversible, and random bonds are constantly made and broken until a state of equilibrium is attained.

Factors Affecting Red Cell
Agglutination
Agglutination is a reversible chemical reaction and is thought to occur in two
stages: 1) sensitization, the attachment of
antibody to antigen on the red cell membrane; and 2) formation of bridges between the sensitized red cells to form the
lattice that constitutes agglutination. Various factors affect these two stages and
can be manipulated to enhance (or decrease) agglutination. The effects of enhancement techniques on the two stages
1
cannot always be clearly differentiated.

Figure 12-1. Antigen-antibody “goodness of fit.”
For maximum complementarity, both structural
fit and complementary distribution of chemical
groups must be achieved. (A) Good structural fit
with complementary chemical attraction.
(B) Chemical groups are complementary, but
structural fit is poor. (C) Good structural fit, but
chemical groupings are not attractive and may
repel each other. (Reprinted with permission
from Moore.2 )

Chapter 12: Red Cell Antigen-Antibody Reactions and Their Detection

273

Chemical Bonding
Various types of chemical bonds are responsible for the binding of antibody to
antigen, including hydrogen bonds, hydrophobic bonds, electrostatic or ionic
bonds, and van der Waals forces. These
types of chemical bonds are relevant to
immunohematology because different types
of bonds have different thermodynamic
characteristics; they are either exothermic
or endothermic. Thermodynamic characteristics, in turn, may affect the serologic
phenomena observed in the test system.
For example, carbohydrate antigens tend
to form exothermic hydrogen bonds with
the antibody-combining site, so the bond
is stronger at lower temperatures. In contrast, hydrophilic bonds formed with protein antigens are endothermic, so these
bonds are enhanced at higher reaction
temperatures.

Equilibrium (Affinity) Constant of the
Antibody
The equilibrium constant or affinity constant (Ko) of a reaction is determined by
the relative rates of association and dissociation (see Fig 12-2). For each antigenantibody reaction, the Ko varies. The Ko reflects the degree to which antibody and
antigen associate and bind to one another
(“goodness of fit”) and the speed of the reaction. The higher the Ko value, the better
the association or “fit.” When the Ko is
large, the reaction occurs more readily
and is more difficult to dissociate; such
antibodies may have a greater clinical importance. When the Ko is small, a higher
ratio of antibody to antigen may be required for detection.
The degree of antigen-antibody “fit” is
influenced by the type of bonds predominating. Hydrophobic bonds are usually associated with higher Ko than hydrogen
bonds. The Ko is also affected by physical

All chemical reactions are reversible. Antigen (Ag)-antibody (Ab) reactions may be
expressed as
Ag + Ab→ AgAb
←

The reaction proceeds until a state of
equilibrium is reached. This is controlled
by the rate constants of association (ka)
and dissociation (kd).
ka

Ag + Ab→ AgAb
←
kd

By the law of mass action, the speed of
the reaction is proportional to the concentrations of the reactants and their product. The equilibrium constant (Ko) is a
function of these intrinsic association
constants for the antibody being tested.
[AbAg] k a
=
=Ko
[Ab][Ag] k d
Figure 12-2. The law of mass action and the equilibrium constant.

conditions such as the temperature at
which the reaction occurs, the pH and ionic
strength of the suspending medium, and
the relative antigen-to-antibody concentrations. In laboratory tests that use agglutination as an endpoint, altering the physical
conditions of the system can increase or
decrease the test’s sensitivity.

Temperature
Most blood group antibodies react within
restricted temperature ranges. Typically,
these antibodies fall into two broad categories: those optimally reactive at “cold”
temperatures (eg, 4 to 25 C) and those optimally reactive at “warm” temperatures
(eg, 30 to 37 C). Antibodies that react in
vitro only at temperatures below 30 C
rarely cause destruction of transfused an-

274

AABB Technical Manual

tigen-positive red cells and are generally
considered clinically insignificant. Many
of these “cold-reactive” antibodies have been
found to be IgM, whereas their “warm-reactive” counterparts have often been found
to be IgG. This has led to the mistaken
conclusion by many that antibody class
determines the temperature of bonding
(and clinical significance). Instead, however, the temperature of optimal antigenantibody reactivity has more to do with
the type of reaction and the chemical nature of the antigen than with the antibody
class. Carbohydrate antigens are more
commonly associated with “cold-reactive”
antibodies and protein antigens with
“warm-reactive” antibodies.

pH
Changes in pH can affect electrostatic
bonds. For most clinically significant
blood group antibodies, optimal pH has
not been determined but is assumed to
approximate the physiologic pH range.
Occasional antibodies, notably some examples of anti-M, react best at a lowered
pH. For most routine testing, a pH around
7.0 should be used. Stored saline often has
a pH of 5.0 to 6.0. Buffered saline is an alternative and may be particularly helpful
3
in solid-phase testing.

Incubation Time
The time needed to reach equilibrium differs for different blood group antibodies.
Significant variables include temperature
requirements, immunoglobulin class, and
specific interactions between antigen
configuration and the Fab site of the antibody. The addition of enhancement agents
to the system can decrease the incubation
time needed to reach equilibrium.
For saline systems in which antiglobulin
serum is used to demonstrate antibody at-

tachment, 30 to 60 minutes of incubation at
37 C is adequate to detect most clinically
significant antibodies. For some weakly reactive antibodies, association may not
reach equilibrium at 30 minutes and extending the incubation time may increase
sensitivity of the test. Prolonging the incubation time beyond 60 minutes has few disadvantages except for the delay before results are available.
Incubation time at 37 C can usually be
reduced to 10 to 15 minutes if a low-ionicstrength saline (LISS) solution is used (including LISS additive solutions). The use of
water-soluble polymers such as polyethylene glycol (PEG) can also reduce the necessary incubation time, although for different
reasons. (See section on Enhancement of
Antibody Detection and Method Section 3.)

Ionic Strength
+

–

In normal saline, Na and Cl ions cluster
around and partially neutralize opposite
charges on antigen and antibody molecules. This hinders the association of antibody with antigen. By lowering the ionic
strength of the reaction medium, however, this shielding effect can be weakened and electrostatic attractions enhanced.
Reducing the salt concentration of the serum-cell system increases the rate at
which antibody and antigen come into
proximity and may increase the amount
of antibody bound. Extending the incubation time in LISS systems may result in a
loss of sensitivity.4 (See section on LISS and
LISS Additives.)

Antigen-Antibody Proportions
An excess of antigen to antibody should
result in increased antibody uptake. For
inhibition or adsorption tests, such an excess of antigen is desirable. For most red
cell tests, however, antigen excess reduces
the number of antibody molecules bound

Chapter 12: Red Cell Antigen-Antibody Reactions and Their Detection

per red cell, limiting their ability to agglutinate. Antibody excess is, therefore, desirable in most routine test systems. A
commonly used ratio in red cell serology
is 2 drops of serum to 1 drop of a 2% to
5% red cell suspension. If the antibody is
weakly reactive, increasing the quantity of
antibody present can increase the test’s
sensitivity. Very rarely, significant antibody excess may inhibit direct agglutination, producing a prozone phenomenon
comparable to what occurs with precipitation reactions. Usually, however, increasing antibody concentration enhances the sensitivity of agglutination
tests. Reducing the concentration of red
cells from 5% to 2% or 3% doubles the serum-to-cell ratio, as does adding 4 drops
of serum to the standard cell suspension.
Sometimes, it is useful to increase the volume of serum to 10 or even 20 drops, particularly during an investigation of a
hemolytic transfusion reaction in which
routine testing reveals no antibody. Alterations in the volume of serum or plasma
significantly affect the ionic strength of
test systems in which LISS has reduced
the dielectric constant, so procedures
must be modified so that the appropriate
ratio of serum to LISS is maintained.
Chapters 18 and 19 give more details
about antibody detection and pretransfusion testing.

Stage Two: Agglutination
Once antibody molecules attach to antigens on the red cell surface, the sensitized
cells must be linked into a lattice. This allows visualization of the reaction. The size
and physical properties of the antibody
molecules, the concentration of antigen
sites on each cell, and the distance between cells all have an effect on the development of agglutinates.

275

The bridges formed between antibodies
interlinked to antigen sites on adjacent red
cells usually result from chance collision of
the sensitized cells. Under isotonic conditions, red cells cannot approach each other
closer than a distance of 50 to 100 Å.1 IgG
molecules characteristically fail to bridge
this distance between red cells and cause
sensitization without lattice formation. For
larger, multivalent IgM molecules, however,
direct agglutination occurs easily. The location and density of antigen sites on the cells
may also allow some IgG antibodies to
cause direct agglutination; A, B, M, and N
antigens, for example, are on the outer
edges of red cell glycoproteins and have relatively high densities, allowing IgG antibodies to crosslink.
Red cells suspended in saline have a net
negative charge at their surface and therefore repel one another. Negatively charged
molecules on the red cell membrane cause
mutual repulsion of red cells. This repulsion may be decreased by various laboratory manipulations and by inherent or altered red cell membrane characteristics.
Various strategies are used to overcome
this repulsion and to enhance agglutination. Centrifugation physically forces the
cells closer together. The indirect antiglobulin test (IAT) uses antiglobulin serum
to crosslink the reaction. Other methods include reducing the negative charge of surface molecules (eg, proteolytic enzymes),
reducing the hydration layer around the cell
(eg, albumin), and introducing positively
charged macromolecules (eg, Polybrene®)
1
that aggregate the cells.

Inhibition of Agglutination
In agglutination inhibition tests, the presence of either antigen or antibody is detected by its ability to inhibit agglutination in a system with known reactants
(see Chapter 19). For example, the saliva

276

AABB Technical Manual

from a secretor contains soluble blood
group antigens that combine with anti-A,
-B, or -H. The indicator system is a standardized dilution of antibody that agglutinates the corresponding cells to a known
degree. If the saliva contains blood group
substance, incubating saliva with antibody will wholly or partially abolish agglutination of cells added to the incubated mixture. The absence of expected
agglutination indicates the presence of
soluble antigen in the material under test.
Agglutination of the indicator cells is a
negative result.

Enhancement of Antibody
Detection
Albumin Additives
Although used routinely for many years as
an enhancement medium, albumin itself
probably does little to promote antibody
uptake (Stage 1). Much of the enhancement effect attributed to albumin may be
due to its attributes as a low-ionic-strength
buffer. Albumin may influence agglutination by reducing the repulsion between
cells, thus predisposing antibody-coated
cells to agglutinate. Bovine serum albumin is available as solutions of 22% or
30% concentration.

Enzymes
The proteolytic enzymes used most often
in immunohematology laboratories are
bromelin, ficin, papain, and trypsin. While
enhancing agglutination by some antibodies, the enzymes destroy certain red
cell antigens, notably M, N, S, Fya, and Fyb.
Proteolytic enzymes reduce the red cell
surface charge by cleaving polypeptides
containing negatively charged sialic acid

molecules from polysaccharide chains.
Sialic acid is a major contributor to the net
negative charge at the red cell surface. Any
mechanism that reduces the net charge
should enhance red cell agglutination, and
red cells pretreated with proteolytic enzymes often show enhanced agglutination
by IgG molecules.

Polyethylene Glycol
PEG is a water-soluble linear polymer
used as an additive to potentiate antigen-antibody reactions.5 It has been suggested that PEG promotes antibody uptake through steric exclusion of water
molecules in the diluent, such that antigens and antibody molecules come into
closer proximity, resulting in increased
cell-antibody collisions and subsequent
antibody binding. Multiple studies have
shown that PEG increases the detection of
potentially clinically significant antibodies and decreases the detection of clinically insignificant antibodies.6
Anti-IgG is usually the antihuman globulin (AHG) reagent of choice with PEG testing, to avoid false-positive reactions with
some polyspecific AHG reagents. Commercially available PEG reagents may be prepared in a LISS solution. PEG can be used
in tests with eluates, as well as with serum
or plasma. PEG can enhance warm-reactive
autoantibodies and thus may be advantageous in detecting weak serum autoantibodies for diagnostic purposes. On the
other hand, this enhancement may be disadvantageous when trying to detect alloantibodies in the presence of autoantibodies. In such cases, testing the serum by
LISS or a saline IAT may allow for the detection or exclusion of alloantibodies without
interference from autoantibodies.
Centrifugation of PEG with test serum
and red cells before washing should be
avoided because the nonspecific aggregates

Chapter 12: Red Cell Antigen-Antibody Reactions and Their Detection

generated by PEG may not disperse. After
incubation with PEG, test cells should be
washed immediately in saline for the
antiglobulin test.
Precipitation of serum proteins when
PEG is added has been reported; this problem appears to be related to elevated serum
globulin levels.6 This problem becomes apparent when the IgG-coated red cells are
nonreactive. At least four washes of the red
cells at the antiglobulin phase, with agitation, will ensure that the red cells are fully
resuspended and will serve to prevent this
problem from occurring.

LISS and LISS Additives
LISS (approximately 0.03 M) greatly increases the speed of antibody sensitization of red cells, compared with normal
saline (approximately 0.17 M). To prevent
lysis of red cells at such a low ionic strength,
a nonionic substance such as glycine is
incorporated in the LISS.
Most laboratories use a LISS additive reagent, rather than LISS itself. These commercially available LISS additives may contain albumin in addition to ionic salts and
buffers. LISS solutions increase the rate of
antibody association (Stage 1) when volume proportions are correct. (See Methods
3.2.2 and 3.2.3.) Increasing the volume of
serum used in a test will increase the ionic
strength of the test; hence, any alteration in
prescribed volumes of serum used requires
adjustment of the LISS volume or omission
of LISS. For this reason, the use of LISS for
routine titration studies and for some other
tests is problematic. When LISS is used as
an additive reagent, the manufacturer’s instructions must be followed.

The Antiglobulin Test
7

In 1945, Coombs, Mourant, and Race described procedures for detecting attach-

277

ment of antibodies that did not produce
agglutination. This test uses antibody to
human globulins and is known as the
antiglobulin test. It was first used to demonstrate antibody in serum, but later the
same principle was used to demonstrate
in-vivo coating of red cells with antibody
or complement components. As used in
immunohematology, antiglobulin testing
generates visible agglutination of sensitized red cells. The direct antiglobulin test
(DAT) is used to demonstrate in-vivo sensitization of red cells. An IAT is used to
demonstrate in-vitro reactions between
red cells and antibodies that sensitize, but
do not agglutinate, cells that express the
corresponding antigen.

Principles of the Antiglobulin Test
All antibody molecules are globulins. Animals injected with human globulins produce antibody to the foreign protein. After the animal serum is adsorbed to remove
unwanted agglutinins, it will react specifically with human globulins and can be
called AHG serum. AHG sera with varying
specificities can be produced, notably,
anti-IgG and antibodies to several complement components. Hybridoma techniques are used for the manufacture of
most AHG. These techniques are more
fully described in Chapter 11.
Anti-IgG combines mainly with the Fc
portion of the sensitizing antibody molecules, not with any epitopes native to the
red cell (see Fig 12-3). The two Fab sites of
the AHG molecule form a bridge between
adjacent antibody-coated cells to produce
visible agglutination. Cells that have no
globulin attached will not be agglutinated.
The strength of the observed agglutination
is usually proportional to the amount of
bound globulin.
AHG will react with human antibodies
and complement molecules that are bound

278

AABB Technical Manual

Figure 12-3. The antiglobulin reaction. Antihuman IgG molecules are shown reacting with
the Fc portion of human IgG coating adjacent red
cells (eg, anti-D coating D-positive red cells).

to red cells or are present, free, in serum.
Unbound globulins may react with AHG,
causing false-negative antiglobulin tests.
Unless the red cells are washed free of unbound proteins before addition of AHG serum, the unbound globulins may neutralize
AHG and cause a false-negative result.

cells, which are then washed to remove
unbound globulins. Agglutination that occurs when AHG is added indicates that
antibody has bound to a specific antigen
present on the red cells. The specificity of
the antibody may be known and the antigen unknown, as in blood group phenotyping with an AHG-reactive reagent such
as anti-Fya. The presence or specificity of
antibody may be unknown, as in antibody
detection and identification tests. Or, in
other applications, such as the crossmatch,
the serum and cells are unknown. This
procedure is used to determine whether
any sort of antigen-antibody interaction
has occurred.
Methods have been developed that obviate the need to wash coated red cells before
adding antiglobulin reagent. Column agglutination technology, described later in
this chapter, is an example. It uses a microcolumn filled with mixtures of either glass
beads or gel, buffer, and sometimes reagents and can be used for direct or indirect antiglobulin procedures. Density barriers allow separation of test serum (or plasma)
from red cells, making a saline washing
phase unnecessary.

Direct Antiglobulin Testing
The DAT is used to demonstrate in-vivo
coating of red cells with antibodies or
complement, in particular IgG and C3d.
Washed red cells from a patient or donor
are tested directly with AHG reagents (see
Method 3.6). The DAT is used in investigating autoimmune hemolytic anemia
(AIHA), drug-induced hemolysis, hemolytic
disease of the fetus and newborn, and
alloimmune reactions to recently transfused red cells.

Indirect Antiglobulin Testing
In indirect antiglobulin procedures, serum (or plasma) is incubated with red

Antiglobulin Reagents
Monospecific antibodies to human globulins can be prepared by injecting animals
with purified IgG, IgA, IgM, C3, or C4.
Such sera require adsorption to remove
unwanted (eg, heterophile) antibodies
from the monospecific AHG reagent.
These animal-made antisera are polyclonal in nature. Monospecific monoclonal reagents can also effectively be
prepared from hybridomas (see Chapter
11). Monospecific animal or hybridomaderived antibodies can be combined into
reagent preparations containing any desired combination of specificities, or dif-

Chapter 12: Red Cell Antigen-Antibody Reactions and Their Detection

ferent clones all recognizing the same antigen specificity can be combined into a
single reagent. Thus, reagents may be
polyclonal, monoclonal, blends of monoclonal, or blends of monoclonal and polyclonal antibodies.
The Food and Drug Administration
(FDA) has established definitions for a vari8
ety of AHG reagents, as shown in Table 12-1.
Antisera specific for other immunoglobulins
(IgA, IgM) or subclasses (IgG1, IgG3, etc) exist but are rarely standardized for routine
test tube methods and must be used with
rigorous controls.

Polyspecific AHG
Polyspecific AHG reagents are used for
DATs and, in some laboratories, for rou-

279

tine compatibility tests and antibody detection. These reagents contain antibody
to human IgG and to the C3d component
of human complement. Other complement antibodies may be present, including anti-C3b, -C4b, and -C4d. Currently
available, commercially prepared, polyspecific antiglobulin sera contain little, if
any, activity against IgA and IgM heavy
chains. However, some reagents may react with IgA or IgM molecules because the
polyspecific mixture may react with lambda
and kappa light chains, which are present
in immunoglobulins of all classes.
Because most clinically significant antibodies are IgG, the most important function of polyspecific AHG, in most procedures,
is detection of IgG. The anticomplement

Table 12-1. Antihuman Globulin Reagents
Antibody Designation on
Container Label

Definition*

(1)

Anti-IgG, -C3d;
Polyspecific

Contains anti-IgG and anti-C3d (may contain other
anticomplement and anti-immunoglobulin antibodies).

(2)

Anti-IgG

Contains anti-IgG with no anticomplement activity (not necessarily gamma chain specific).

(3)

Anti-IgG; heavy
chains

Contains only antibodies reactive against human gamma
chains.

(4)

Anti-C3b

Contains only C3b antibodies with no anti-immunoglobulin activity. Note: The antibody produced in response to immunization is usually directed against the antigenic determinant,
which is located in the C3c subunit; some persons have
called this antibody “anti-C3c.” In product labeling, this antibody should be designated anti-C3b.

(5)

Anti-C3d

Contains only C3d antibodies with no anti-immunoglobulin
activity.

(6)

Anti-C4b

Contains only C4b antibodies with no anti-immunoglobulin
activity.

(7)

Anti-C4d

Contains only C4d antibodies with no anti-immunoglobulin
activity.

*As defined by the FDA.

280

AABB Technical Manual

component has limited usefulness in crossmatching and in antibody detection because antibodies detectable only by their
ability to bind complement are quite rare.
Anti-C3d activity is important, however, for
the DAT, especially in the investigation of
AIHA. In some patients with AIHA, C3d
may be the only globulin detectable on
their red cells.9

Monospecific AHG Reagents
Licensed monospecific AHG reagents in
common use are anti-IgG and anti-C3b,
-C3d. The FDA has established labeling
requirements for other anticomplement
reagents, including anti-C3b, anti-C4b,
and anti-C4d, but these products are not
generally available. If the DAT with a
polyspecific reagent reveals globulins on
red cells, monospecific AHG reagents are
used to characterize the coating proteins.

Anti-IgG
Reagents labeled “anti-IgG” contain no
anticomplement activity. The major component of anti-IgG is antibody to human
gamma heavy chains, but unless labeled
as “heavy-chain-specific,” these reagents
may exhibit some reactivity with light
chains, which are common to all immunoglobulin classes. An anti-IgG reagent
not designated “heavy-chain-specific”
must be considered theoretically capable
of reacting with light chains of IgA or IgM.
A positive DAT with such an anti-IgG does
not definitively prove the presence of IgG,
although it is quite rare to have an in-vivo
coating with IgA or IgM in the absence of
IgG. Many workers prefer anti-IgG over
polyspecific AHG in antibody detection
and compatibility tests because anti-IgG
AHG does not react with complement

bound to red cells by cold-reactive antibodies that are not clinically significant.

Anti-C3b, -C3d
Anti-C3b, -C3d reagents prepared by animal immunization contain no activity
against human immunoglobulins and are
used in situations described for anti-C3d.
This type of anti-C3d characteristically reacts with C3b and possibly other epitopes
present on C3-coated red cells. Murine
monoclonal anti-C3b, -C3d reagent is a
blend of hybridoma-derived antibodies.

Role of Complement in Antiglobulin
Reactions
Complement components may attach to
red cells in vivo or in vitro by one of two
mechanisms:
1.
Complement-binding antibody specific for a red cell antigen may cause
attachment of complement to the cell
surface as a consequence of complement activation by the antigen-antibody complex.
2.
Immune complexes, not specific for
red cell antigens, may be present in
plasma and may activate complement components that adsorb onto
red cells in a nonspecific manner.
Attachment of complement to the
membrane of cells not involved in
the specific antigen-antibody reaction is often described as “innocent
bystander” complement coating.
Red cells coated with elements of the
complement cascade may or may not undergo hemolysis. If the cascade does not go
to completion, the presence of bound early
components of the cascade can be detected
by anticomplement reagents. The component most readily detected is C3 because
several hundred C3 molecules may be

Chapter 12: Red Cell Antigen-Antibody Reactions and Their Detection

bound to the red cell by the attachment of
only a few antibody molecules. C4 coating
also can be detected, but C3 coating has
more clinical significance.
4.

Complement as the Only Coating Globulin
Complement alone, without detectable immunoglobulin, may be present on washed
red cells in certain situations.
1.
IgM antibodies reacting in vitro occasionally attach to red cell antigens
without agglutinating the cells, as is
seen with IgM antibodies to Lewis
antigens. IgM coating is difficult to
demonstrate in AHG tests, partly because IgM molecules tend to dissociate during the washing process
and partly because polyspecific AHG
contains little if any anti-IgM activity. IgM antibodies may activate
complement, and the IgM reactivity
can be demonstrated by identifying
the several hundred C3 molecules
bound to the cell membrane near
the site of antibody attachment.
2.
About 10% to 20% of patients with
warm AIHA have red cells with a positive DAT due to C3 coating alone.10
No IgG, IgA, or IgM coating is demonstrable with routine procedures,
although some specimens may be
coated with IgG at levels below the
detection threshold for the DAT.
3.
In cold agglutinin syndrome, the
cold-reactive autoantibody can react
with red cell antigens at temperatures up to 32 C.11 Red cells passing
through vessels in the skin at this
temperature become coated with
autoantibody, which activates complement. If the cells escape hemolysis, they return to the central circulation, where the temperature is 37 C,
and the autoantibody dissociates

281

from the cells, leaving complement
components firmly bound to the red
cell membrane. The component
usually detected by AHG reagents is
C3d.
Immune complexes that form in the
plasma and bind weakly and nonspecifically to red cells may cause
complement coating. The activated
complement remains on the red cell
surface after the immune complexes
dissociate. C3 remains as the only
detectable surface globulin.

IgG-Coated Cells
The addition of IgG-coated cells to negative antiglobulin tests (used to detect IgG)
is required for antibody detection and
crossmatching procedures. 1 2 ( p 3 3 ) These
sensitized red cells should react with the
antiglobulin sera, verifying that the AHG
reagent was functional. Reactivity with
IgG-sensitized cells demonstrates that, indeed, AHG was added and it had not been
neutralized. Tests need to be repeated if
the IgG-coated cells are not reactive.
Testing with IgG-sensitized cells does
not detect all potential failures of the
antiglobulin test.13-15 Partial neutralization of
the AHG may not be detected at all, particularly if the control cells are heavily coated
with IgG. Errors in the original test, such as
omission of test serum, improper centrifuge speed, or inappropriate concentrations of test red cells, may yield negative
test results but positive results with control
cells. Oversensitized control cells may agglutinate when centrifuged.
Complement-coated cells can also be
prepared and are commercially available.
They can be used in some cases to control
tests with complement-specific reagents.
Some sources of error in antiglobulin
tests are listed in Tables 12-2 and 12-3.

282

AABB Technical Manual

Table 12-2. Sources of Error in Antiglobulin Testing—False-Negative Results
Neutralization of Antihuman Globulin (AHG) Reagent
■

Failure to wash cells adequately to remove all serum/plasma. Fill tube at least ¾ full of saline for
each wash. Check dispense volume of automated washers.

■

If increased serum volumes are used, routine wash may be inadequate. Wash additional times or
remove serum before washing.

■

Contamination of AHG by extraneous protein. Do not use finger or hand to cover tube.
Contaminated droppers or wrong reagent dropper can neutralize entire bottle of AHG.

■

High concentration of IgG paraproteins in test serum; protein may remain even after multiple
washes.13

Interruption in Testing
■

Bound IgG may dissociate from red cells and either leave too little IgG to detect or may
neutralize AHG reagent.

■

Agglutination of IgG-coated cells will weaken. Centrifuge and read immediately.

Improper Reagent Storage
■

AHG reagent may lose reactivity if frozen. Reagent may become bacterially contaminated.

■

Excess heat or repeated freezing/thawing may cause loss of reactivity of test serum.

■

Reagent red cells may lose antigen strength on storage. Other subtle cell changes may cause
loss of reactivity.

Improper Procedures
■

Overcentrifugation may pack cells so tightly that agitation required to resuspend cells breaks up
agglutinates. Undercentrifugation may not be optimal for agglutination.

■

Failure to add test serum, enhancement medium, or AHG may cause negative test.

■

Too heavy a red cell concentration may mask weak agglutination. Too light suspension may be
difficult to read.

■

Improper/insufficient serum:cell ratios.

Complement
■

Rare antibodies, notably some anti-Jka, -Jkb, may only be detected when polyspecific AHG is
used and active complement is present.

Saline
■

Low pH of saline solution can decrease sensitivity.3 Optimal saline wash solution for most
antibodies is pH 7.0 to 7.2.

■

Some antibodies may require saline to be at specific temperature to retain antibody on the cell.
Use 37 C or 4 C saline.

Chapter 12: Red Cell Antigen-Antibody Reactions and Their Detection

283

Table 12-3. Sources of Error in Antiglobulin Testing—False-Positive Results
Cells Agglutinated Before Washing
■

If potent agglutinins are present, agglutinates may not disperse during washing. Observe cells
before the addition of antihuman globulin (AHG) or use control tube substituting saline for AHG;
reactivity before the addition of AHG or in saline control invalidates AHG reading.

Particles or Contaminants
■

Dust or dirt in glassware may cause clumping (not agglutination) of red cells. Fibrin or
precipitates in test serum may produce cell clumps that mimic agglutination.

Improper Procedures
■

Overcentrifugation may pack cells so tightly that they do not easily disperse and appear positive.

■

Centrifugation of test with polyethylene glycol or positively charged polymers before washing
may create clumps that do not disperse.

Cells That Have Positive Direct Antiglobulin Test (DAT)
■

Cells that are positive by DAT will be positive in any indirect antiglobulin test. Procedures for
removing IgG from DAT-positive cells are given in Methods 2.13 and 2.14.

Complement
■

Complement components, primarily C4, may bind to cells from clots or from CPDA-1 donor
segments during storage at 4 C and occasionally at higher temperatures. For DATs, use red cells
anticoagulated with EDTA, ACD, or CPD.

■

Samples collected in tubes containing silicone gel may have spurious complement attachment.14

■

Complement may attach to cells in specimens collected from infusion lines used to administer
dextrose-containing solutions. Strongest reactions are seen when large-bore needles are used or
when sample volume is less than 0.5 mL.15

Other Methods to Detect
Antigen-Antibody Reactions
The following methods represent alternatives to traditional tube testing and use of
antiglobulin serum. Some methods do
not allow detection of both IgM and IgG
antibodies and may not provide information on the phase and temperature of reactivity of antibodies that is obtained
when traditional tube tests are used.

Solid-Phase Red Cell Adherence Tests
Solid-phase microplate techniques use
immobilized antigen or antibody. In a direct test, antibody is fixed to a microplate
well and red cells are added. If the cells
express the corresponding antigen, they
will adhere across the sides of the well; if
no antigen-antibody reaction occurs, the
red cells pellet to the bottom of the well
when centrifuged.16 An indirect test uses
red cells of known antigenic composition

284

AABB Technical Manual

bound to a well. The test sample is added
to the red-cell-coated wells and allowed
to react with the cells, after which the
plates are washed free of unbound proteins. The indicator for attached antibody
is a suspension of anti-IgG-coated red
cells. The reaction is positive if the indicator cells adhere across the sides of the
well. If they pellet to the bottom when
centrifuged, it demonstrates that no antigen-antibody reaction has occurred (see
Fig 12-4).17 In the indirect test, isolated
membrane components (eg, specific proteins), rather than intact cells, can be affixed to the microwell. Solid-phase attachment of antigen or antibody is an
integral part of other tests, such as the
monoclonal antibody-specific immobilization of erythrocyte antigens (MAIEA)
assay, discussed below. Solid-phase systems have also been devised for use in detecting platelet antibodies and in tests for
syphilis, cytomegalovirus, and hepatitis B
surface antigen.17-20

Column Agglutination Technology
Various methods have been devised in
which red cells are filtered through a column containing a medium that separates
selected red cell populations. As commercially prepared, the systems usually employ a card or strip of microtubes, rather
than conventional test tubes. They allow
simultaneous performance of several tests.
Usually, a space or chamber at the top of
each column is used for red cells alone or
to incubate red cells and serum or plasma.
As the cells pass through the column
(usually during centrifugation), the column
medium separates agglutinated from
unagglutinated red cells, based on aggregate size.17 Alternatively, in some tests,
specific antisera or proteins can be included in the column medium itself; cells
bearing a specific antigen are selectively
captured as they pass through the medium. When the column contains antiglobulin serum or a protein that specifically binds immunoglobulin, the selected

Figure 12-4. An indirect solid-phase test. A monolayer of red cells is affixed to a microwell (1). Test
serum is added. If antibody (2) is present, it binds to antigens on the affixed red cells (3). Indicator red
cells coated with IgG and anti-IgG (4) are added. The anti-IgG portion binds to any antibody attached to
the fixed red cells (5). In a positive test, the indicator red cells are effaced across the microwell. In a
negative test, the indicator cells do not bind but pellet to the center of the well when centrifuged. Weak
reactions give intermediate results.

Chapter 12: Red Cell Antigen-Antibody Reactions and Their Detection

cells will be those sensitized with immunoglobulin. By using a centrifuge time
and speed that allow red cells to enter the
column but leave serum or plasma above,
the need for saline washing for anti21
globulin tests can be eliminated.
Typically in column tests, negative test
cells pellet to the bottom of the column. In
positive tests, the cells are captured at the
top, or in the body, of the column. An advantage of most such systems is the stability of the final reaction phase, which can be
read by several individuals or, in some
cases, documented by photocopying. Column tests generally have sensitivity similar
to LISS antiglobulin methods, but such
tests have reportedly performed less well in
detecting weak antibodies, especially those
in the ABO system.22
In 1986, Lapierre et al developed a process leading to a technology that uses a column of gel particles.23 As commercially prepared, the gel test uses six microtubes
instead of test tubes, contained in what is
called a card or strip. The gel particles function as filters that trap red cell agglutinates
when the cards are centrifuged. Gels containing antiglobulin serum are used to capture sensitized, but unagglutinated, cells.
Gels with various antisera can be used for
phenotyping cells (see Fig 12-5).
In another column agglutination technology, a column of glass microbeads in a
diluent is used instead of gel. As with the
gel test, the beads may either entrap agglutinated cells or antisera, such as anti-IgG,
can be added to the diluent.24

Automated Testing Platforms
Several automated devices have been developed for the detection of antigen-antibody reactions. All steps in the testing
process, from sample aliquotting to reporting results, are performed by the system. These test systems permit the per-

285

Figure 12-5. Gel test.

formance of multiple tests, using solidphase, gel-column, and/or microtiter
plate technology. The systems are controlled by a computer program and positive sample identity can be ensured by
barcode technology. The test system’s
computer may be integrated into a laboratory’s information system for results reporting. Automated test systems may be
particularly useful in institutions performing large volumes of patient or donor
testing.

Immunofluorescence
Immunofluorescence testing allows identification and localization of antigens
inside or on the surface of cells. A fluorochrome such as fluorescein or phycoerythrin can be attached to an antibody
molecule, without altering its specificity
or its ability to bind antigen. Attachment
of fluorescein-labeled antibody to cellular
antigen makes the antibody-coated cells
appear brightly visible yellow-green or
red (depending on the fluorochrome).
Immunofluorescent antibodies can be
used in direct or indirect procedures, with
the fluorescence analogous to agglutination
as an endpoint. In a direct test, the fluorescein-labeled antibody is specific for a single
antigen of interest. In an indirect test,

286

AABB Technical Manual

fluorescein-labeled antiglobulin serum is
added to cells that have been incubated
with an unlabeled antibody of known specificity. Immunofluorescent techniques were
initially used to detect antigens in or on
lymphocytes or in tissue sections. More recently, immunofluorescent antibodies have
been used in flow cytometry. Among their
many applications, they have been used to
quantify fetomaternal hemorrhage, to identify transfused cells and follow their survival
in recipients, to measure low levels of
cell-bound IgG, and to distinguish homozygous from heterozygous expression of
blood group antigens.25

Enzyme-Linked Immunosorbent Assay
Enzyme-linked immunosorbent assays
(ELISAs) are used to measure either antigen or antibody. Enzymes such as alkaline
phosphatase can be bound to antibody
molecules without destroying either the
antibody specificity or the enzyme activity. ELISAs have been used to detect and
measure cell-bound IgG and to demonstrate fetomaternal hemorrhage. When
red cells are examined, the test often is
called an enzyme-linked antiglobulin test
(ELAT).

antibody also attached). Conjugated antihuman antibody is added, which reacts
with the bound human antibody and
gives an ELISA-readable reaction. Thus
far, this method has been used primarily
to isolate specific membrane structures
for blood group antigen studies.26,27

References
1.

Monoclonal Antibody-Specific
Immobilization of Erythrocyte Antigens
Assay
In the MAIEA assay, red cells are incubated with two antibodies. One contains
human alloantibody to a blood group antigen and the other is a nonhuman (usually mouse monoclonal) antibody that reacts with a different portion of the same
membrane protein. The red cells are lysed
and the membrane solubilized, then
added to a microwell coated with goat
antimouse antibody. This antibody then
captures the mouse antibody attached to
the membrane protein (with the human

10.

11.

van Oss CJ. Immunological and physiochemical nature of antigen-antibody interactions. In: Garratty G, ed. Immunobiology of
transfusion medicine. New York: Marcel
Dekker, Inc., 1994:327-64.
Moore BPL. Antibody uptake: The first stage
of the hemagglutination reaction. In: Bell CA,
ed. A seminar on antigen-antibody reactions
revisited. Arlington, VA: AABB, 1982:47-66.
Rolih S, Thomas R, Fisher E, Talbot J. Antibody detection errors due to acidic or unbuffered saline. Immunohematology 1993;
9:15-18.
Jørgensen J, Nielsen M, Nielsen CB, Nørmark
J. The influence of ionic strength, albumin
and incubation time on the sensitivity of the
indirect Coombs’ test. Vox Sang 1980;36:18691.
Nance SJ, Garratty G. Polyethylene glycol: A
new potentiator of red blood cell antigen-antibody reactions. Am J Clin Pathol 1987;87:
633-5.
Issitt PD, Anstee DJ. Applied blood group serology. 4th ed. Durham, NC: Montgomery
Scientific Publications, 1998:47-8.
Coombs RRA, Mourant AE, Race RR. A new
test for the detection of weak and “incomplete” Rh agglutinins. Br J Exp Pathol 1945;
26:255-66.
Code of federal regulations. Title 21 CFR
660.55. Washington, DC: US Government
Printing Office, 2004 (revised annually).
Packman CH. Acquired hemolytic anemia
due to warm-reacting autoantibodies. In:
Beutler E, Lichtman MA, Coller BS, et al, eds.
Williams’ hematology. 6th ed. New York:
McGraw Hill, 2001:639-48.
Sokol RJ, Hewitt S, Stamps BK. Autoimmune
haemolysis: An 18-year study of 865 cases referred to a regional transfusion centre. Br
Med J 1981;282:2023-7.
Packman CH. Cryopathic hemolytic syndromes. In: Beutler E, Lichtman MA, Coller

Chapter 12: Red Cell Antigen-Antibody Reactions and Their Detection

12.

13.

14.

15.

16.

17.
18.

19.

20.

BS, et al, eds. Williams’ hematology. 6th ed.
New York: McGraw-Hill, 2001:649-55.
Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.
Ylagen ES, Curtis BR, Wildgen ME, et al. Invalidation of antiglobulin tests by a high thermal amplitude cryoglobulin. Transfusion
1990;30:154-7.
Geisland JR, Milam JD. Spuriously positive direct antiglobulin tests caused by silicone gel.
Transfusion 1980;20:711-13.
Grindon AJ, Wilson MJ. False-positive DAT
caused by variables in sample procurement.
Transfusion 1981;21:313-14.
Rolih SD, Eisinger RW, Moheng JC, et al. Solid
phase adherence assays: Alternatives to conventional blood bank tests. Lab Med 1985;16:
766-70.
Walker P. New technologies in transfusion
medicine. Lab Med 1997;28:258-62.
Plapp FV, Sinor LT, Rachel JM, et al. A solid
phase antibody screen. Am J Clin Pathol 1984;
82:719-21.
Rachel JM, Sinor LT, Beck ML, Plapp FV. A
solid-phase antiglobulin test. Transfusion
1985;25:24-6.
Sinor L. Advances in solid-phase red cell adherence methods and transfusion serology.
Transfus Med Rev 1992;6:26-31.

21.
22.

23.

24.

25.

26.

27.

287

Malyska H, Weiland D. The gel test. Lab Med
1994;25:81-5.
Phillips P, Voak D, Knowles S, et al. An explanation and the clinical significance of the
failure of microcolumn tests to detect weak
ABO and other antibodies. Transfus Med
1997;7:47-53.
Lapierre Y, Rigal D, Adam J, et al. The gel test:
A new way to detect red cell antigen-antibody
reactions. Transfusion 1990;30:109-13.
Reis KJ, Chachowski R, Cupido A, et al. Column agglutination technology: The antiglobulin test. Transfusion 1993;33:639-43.
Garratty G, Arndt P. Applications of flow cytofluorometry to transfusion science. Transfusion 1994;35:157-78.
Petty AC. Monoclonal antibody-specific immobilisation of erythrocyte antigens (MAIEA).
A new technique to selectively determine antigenic sites on red cell membranes. J Immunol
Methods 1993;161:91-5.
Petty AC, Green CA, Daniels GL. The monoclonal antibody-specific antigens assay
(MAIEA) in the investigation of human redcell antigens and their associated membrane
proteins. Transfus Med 1997;7:179-88.

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens

Chapter 13

ABO, H, and Lewis Blood
Groups and Structurally
Related Antigens

HE ABO, AS well as the H, Lewis, I,
and P, blood group antigens reside
on structurally related carbohydrate
molecules. The antigens arise from the
action of specific glycosyltransferases that
add individual sugars sequentially to sites
on short chains of sugars (oligosaccharides) on common precursor substances. Interactions of the ABO, Hh, Sese, and Lele
gene products affect the expression of the
ABO, H, and Lewis antigens. Refer to Appendix 6 for ISBT numbers and nomenclature for the blood groups.

The ABO System
The ABO system was discovered when Karl
Landsteiner recorded the agglutination of
human red cells by the sera of other individuals in 19001 and, in the following year,
detailed the patterns of reactivity as three
types, now called groups A, B, and O.2,3 He

found that serum from group A individuals agglutinated the red cells from group B
individuals, and, conversely, the serum from
group B individuals agglutinated group A
red cells. A and B were thus the first red
cell antigens to be discovered. Red cells
that were not agglutinated by the serum
of either the group A or group B individuals were later called group O; the serum
from group O individuals agglutinated the
red cells from both group A and group B
individuals. Von Decastello and Sturli in
4
1902 discovered the fourth group, AB.
The importance of Landsteiner’s discovery is the recognition that antibodies to A
and B antigens are present when the corresponding antigen is missing. Routine ABO
typing procedures developed from these and
5
later studies.
The ABO antigens and antibodies remain
the most significant for transfusion practice.
It is the only blood group system in which
the reciprocal antibodies (see Table 13-1)
289

290

AABB Technical Manual

Table 13-1. Routine ABO Typing
Reaction of Cells
Tested with

Reaction of Serum
Tested Against

Interpretation

Incidence (%) in
US Population

Anti-A

Anti-B

A1 Cells

B Cells

O Cells

ABO
Group

Whites

Blacks

0
+
0
+

0
0
+
+

+
0
+
0

+
+
0
0

0
0
0
0

0
A
B
AB

45
40
11
4

49
27
20
4

+ = agglutination; 0 = no agglutination.

are consistently and predictably present in
the sera of most people who have had no
exposure to human red cells. Due to these
antibodies, transfusion of ABO-incompatible blood may cause severe intravascular
hemolysis as well as the other manifestations of an acute hemolytic transfusion reaction (see Chapter 27). Testing to detect ABO
incompatibility between a recipient and the
donor is the foundation on which all pretransfusion testing is based.

Genetics and Biochemistry
The genes for all of the carbohydrate antigens discussed in this chapter encode specific glycosyltransferases, enzymes that
transfer specific sugars to the appropriate
carbohydrate chain acceptor; thus, the antigens are indirect products of the genes.
Genes at three separate loci (H, Se, and
ABO) control the occurrence and the location of the A and B antigens. The H and Se
(secretor) loci, officially named FUT1 and
FUT2, respectively, are on chromosome
19 and are closely linked. Each locus has
two recognized alleles, one of which has
no demonstrable product and is considered an amorph. The active allele at the H
locus, H, produces a transferase that acts

at the cellular level to form the H antigen
on red cells. The amorph, h, is very rare.
The active allele at the Se locus, Se, produces a transferase that also acts to form H
antigen, but primarily in secretions such
as saliva.6 Eighty percent of individuals are
secretors. The amorphic allele is se. The
enzymes produced by H and Se alleles are
both fucosyltransferases, but they have
slightly different activity. H antigen on red
cells and in secretions is the substrate for
the formation of A and B antigens.
There are three common alleles at the
ABO locus on chromosome 9, A, B, and O.7
The A and B alleles encode glycosyltransferases that produce the A and B antigens
respectively; the O allele does not encode a
functional enzyme.8 The red cells of group
O individuals lack A and B antigens but carry
an abundant amount of H antigen, the unconverted precursor substance on which A
and B antigens are built.
The carbohydrate chains (oligosaccharides) that carry ABH antigens can be attached to either protein (glycoprotein),
sphingolipid (glycosphingolipid), or lipid
(glycolipid) carrier molecules. Glycoproteins and glycosphingolipids carrying A or
B antigens are integral parts of the mem-

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens

branes of red cells, epithelial cells, and
endothelial cells (Fig 13-1) and are also
present in soluble form in plasma. Glycoproteins secreted in body fluids such as saliva contain molecules that may, if the person possesses an Se allele, carry A, B, and H
antigens. A and B antigens that are unattached to carrier protein or lipid molecules
are also found in milk and urine as free oligosaccharides.
The transferases encoded by the A, B, H,
and Se alleles add a specific sugar to a precursor carbohydrate chain. The sugar that
is added is referred to as immunodominant
because when it is removed from the structure, the specific blood group activity is
lost. The sugars can be added only in a sequential manner. H structure is made first,
then sugars for A and B antigens are added
to H. The H and Se alleles encode a fucosyltransferase that adds fucose (Fuc) to the
precursor chain; thus, fucose is the immunodominant sugar for H (see Fig 13-2).
The A allele encodes N-acetyl-galactosaminyltransferase that adds N-acetyl-D-

291

galactosamine (or GalNAc) to H to make A
antigen on red cells. The B allele encodes
galactosyltransferase that adds D-galactose
(or Gal) to H to make B antigen. Group AB
individuals have alleles that make transferases to add both GalNAc and Gal to the precursor H antigen. Attachment of the A or B
immunodominant sugars diminishes the serologic detection of H antigen so that the expressions of A or B antigen and of H antigen
are inversely proportional. Rare individuals
who lack both H and Se alleles (genotype hh
and sese) have no H and, therefore, no A or B
antigens on their red cells or in their secretions (see Oh phenotype below). However, H,
A, and B antigens are found in the secretions
of some hh individuals who appear, through
family studies, to possess at least one Se allele
(see para-Bombay phenotype below).
The oligosaccharides to which the A or B
immunodominant sugars are attached may
exist as simple repeats of a few sugar molecules linked in linear fashion, or as part of
more complex structures, with many sugar
residues linked in branching chains. Differ-

Figure 13-1. Schematic representation of the red cell membrane showing antigen-bearing glycosylation of proteins and lipids. GPI = glycophosphatidylinositol. (Courtesy of ME Reid, New York Blood
Center.)

292

AABB Technical Manual

terminal disaccharide; there are at least six
of these types of disaccharide linkages.9
Type 1 chains and Type 2 chains differ in
the linkage of the terminal Gal to GlcNAc
disaccharide (see Fig 13-3). Type 1 A, B, and
H structures are present in secretions, plasma,
and endodermally derived tissues. They are
not synthesized by red cells but are incorporated into the red cell membrane from
the plasma. Type 2 chains are the predominant ABH-carrying oligosaccharides on red
cells and are also found in secretions. Type
3 chains (repetitive form) are found on red
10
cells from group A individuals. They are
synthesized by the addition of Gal to the
terminal GalNAc Type 2 A chains, thus
forming Type 3 H; Type 3 H chains are subsequently converted to Type 3 A by the
addition of GalNAc through the action of
A1-transferase, but not by A2-transferase
(see Fig 13-4).

Figure 13-2. Gal added to the subterminal Gal
confers B activity; GalNAc added to the subterminal Gal confers A activity to the sugar. Unless the
fucose moiety that determines H activity is attached to the number 2 carbon, galactose does
not accept either sugar on the number 3 carbon.

ences between infants and adults in cellular
A, B, and H activity may be related to the
number of branched structures present on
cellular membranes at different ages. The
red cells of infants are thought to carry predominantly linear carbohydrate chains,
which have only one terminus to which the
H (and subsequent A and/or B) sugars can
be added. In contrast, the red cells of adults
carry a high proportion of branched carbohydrate chains, providing additional sites for
conversion to H and then to A and B antigens.
A, B, and H antigens are constructed on
carbohydrate chains that are characterized
by different linkages and composition of the

Figure 13-3. Type 1 and 2 oligosaccharide chains
differ only in the linkage between the GlcNAc and
the terminal Gal.

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens

293

Figure 13-4. Type 3 A antigen structure.

Active alleles H and Se, of the FUT1 and
FUT2 genes, encode fucosyltransferases with
a high degree of homology. The enzyme
produced by H acts primarily on Type 2
chains, which are prevalent on the red cell
membranes. The enzyme produced by Se
prefers, but is not limited to, Type 1 chains
and acts primarily in the secretory glands.

ABO Genes at the Molecular Level
11

Yamamoto et al have shown that A and B
alleles differ from one another by seven
nucleotide differences, four of which resulted in amino acid substitutions at positions 176, 235, 266, and 268 in the protein
sequence of the A and B transferases. Recent crystallography of A- and B-transferases demonstrated the role of these criti12
cal amino acids in substrate recognition.
The initial O allele examined had a single
nucleotide deletion that resulted in a frame
shift and premature stop codon resulting in
the predicted translation of a truncated (ie,
inactive) protein. Subsequently, other O alleles have been identified as well as mutations of A and B alleles that result in weakened expression of A and B antigens (reviewed
elsewhere).13,14 The A2 allele encodes a protein with an additional 21 amino acids.

Antigens
Agglutination tests are used to detect A and
B antigens on red cells. Reagent antibod-

ies frequently produce weaker reactions
with red cells from newborns than with
red cells from adults. Although A and B
antigens can be detected on the red cells
of 5- to 6-week-old embryos, A and B antigens are not fully developed at birth, presumably because the branching carbohydrate structures develop gradually. By 2 to
4 years of age, A and B antigen expression
is fully developed and remains fairly constant throughout life.

Subgroups
ABO subgroups are phenotypes that differ
in the amount of antigen carried on red
cells and, for secretors, soluble antigen
present in the saliva. Subgroups of A are
more commonly encountered than subgroups of B. The two principal subgroups
of A are A1 and A2. Red cells from A1 and A2
persons both react strongly with reagent
anti-A in direct agglutination tests. The
serologic distinction between Al and A2
cells can be determined by testing with
anti-A1 lectin (see anti-A1 below). There is
both a qualitative and quantitative difference between A1 and A2.15 The A1-transferase is more efficient at converting H
substance into A antigen and is capable of
making the repetitive Type 3 A structures.
There are about 10.5 × 105 A antigen sites
5
on adult A1 red cells, and about 2.21 × 10

294

AABB Technical Manual

A antigen sites on adult A2 red cells. Approximately 80% of group A or group AB
individuals have red cells that are agglutinated by anti-A1 and thus are classified as
A1 or A1B. The remaining 20%, whose red
cells are strongly agglutinated by anti-A but
not by anti-A1, are called A2 or A2B. Routine testing with anti-A1 is unnecessary for
donors or recipients.
Subgroups weaker than A2 occur infrequently and, in general, are characterized
by decreasing numbers of A antigen sites
on the red cells and a reciprocal increase in
H antigen activity. Subgroups are most often recognized when there is a discrepancy
between the red cell (forward) and serum
(reverse) grouping. Generally, classification
of weak A subgroups (A3, Ax, Am, Ael) is based
on the:
1.
Degree of red cell agglutination by
anti-A and anti-A1.
2.
Degree of red cell agglutination by human and some monoclonal anti-A,B.
3.
Degree of red cell agglutination by
anti-H (Ulex europaeus).
4.
Presence or absence of anti-A1 in the
serum.
5.
Presence of A and H substances in
the saliva of secretors.
6.
Adsorption/elution studies.
7.
Family (pedigree) studies.
Identification of the various A subgroups
is not routinely done. The serologic classification of A (and B) subgroups was developed using human polyclonal anti-A,
anti-B, and anti-A,B reagents. These reagents have been replaced by murine
monoclonal reagents, and the reactivity is
dependent upon which clone(s) is selected
by the manufacturer. There are, however,
some characteristics that should be noted.
A3 red cells give a characteristic mixed-field
pattern when tested with anti-A from group
B or O donors. Ax red cells are characteristically not agglutinated by human anti-A
from group B persons but are agglutinated

by anti-A,B from group O persons. Ax red
cells may react with some monoclonal anti-A
reagents, depending on which monoclonal
antibody is selected for the reagent. Ael red
cells are not agglutinated by anti-A or
anti-A,B of any origin, and the presence of
A antigen is demonstrable only by adsorption and elution studies. Subgroups of B are
even less common than subgroups of A.
Molecular studies have confirmed that A
and B subgroups are heterogeneous, and the
serologic classification does not consistently
correlate with genomic analysis; multiple
alleles yield the same weakened phenotype,
and, in some instances, more than one
phenotype has the same allele.16

Antibodies to A and B
Ordinarily, individuals possess antibodies
directed toward the A or B antigen absent
from their own red cells (see Table 13-1).
This predictable complementary relationship permits ABO testing of sera as well as
of red cells (see Methods 2.2 and 2.3). One
hypothesis for the development of these
antibodies is based on the fact that the
configurations that confer A and B antigenic determinants also exist in other biologic entities, notably bacteria cell walls.
Bacteria are widespread in the environment, and their presence in intestinal
flora, dust, food, and other widely distributed agents ensures a constant exposure
of all persons to A-like and B-like antigens.
Immunocompetent persons react to the
environmental antigens by producing antibodies to those that are absent from
their own systems. Thus, anti-A is produced by group O and group B persons
and anti-B is produced by group O and
group A persons. Group AB people, having both antigens, make neither antibody.
This “environmental” explanation for the
emergence of anti-A and anti-B remains a
hypothesis that has not been proven.

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens

Time of Appearance
Anti-A and anti-B produced by an infant
can generally be detected in serum after 3
to 6 months of life. Most of the anti-A and
anti-B present in cord blood are of maternal origin, acquired by the placental
transfer of maternal IgG; occasionally, infants can be found who produce these
17(p124)
Thus,
antibodies at the time of birth.
anti-A and anti-B detected in the sera of
newborns or infants younger than 3 to 6
months cannot be considered valid. Antibody production increases, reaching the
adult level at 5 to 10 years of age, and declines later in life. Elderly people usually
have lower anti-A and anti-B levels than
young adults.

Reactivity of Anti-A and Anti-B
IgM is the predominant immunoglobulin
class of anti-A produced by group B individuals and anti-B produced by group A
individuals, although small quantities of
IgG antibody are also present. IgG is the
dominant class of anti-A and anti-B of group
O serum.17(p124) Because IgG readily crosses
the placenta and IgM does not, group A or
B infants of group O mothers are at higher
risk for ABO hemolytic disease of the fetus
and newborn (HDFN) than the infants of
group A or B mothers; but severe HDFN
can also occur in infants of group A and
group B mothers.
Both IgM and IgG anti-A and anti-B preferentially agglutinate red cells at room temperature (20-24 C) or below and efficiently
activate complement at 37 C. The complement-mediated lytic capability of these antibodies becomes apparent if serum testing
includes an incubation phase at 37 C. Sera
from some people will cause hemolysis of
ABO-incompatible red cells at temperatures below 37 C. Hemolysis due to ABO
antibodies should be suspected when the
supernatant fluid of the serum test is pink

295

to red or when the cell button is absent or
reduced in size. Hemolysis must be interpreted as a positive result. Because the
hemolysis is complement-mediated, it will
not occur if plasma is used for testing, or if
reagent red cells are suspended in solutions
that contain EDTA or other agents that prevent complement activation.

Reactivity of Anti-A,B (Group O Serum)
Serum from a group O individual contains
an antibody designated as anti-A,B because
it reacts with both A and B red cells, and
the anti-A and anti-B cannot be separated
by differential adsorption. In other words,
after adsorption of group O serum, an eluate prepared from the group A or group B
adsorbing cells reacts with both A and B
test cells. Saliva from a secretor of either A
or B substance inhibits the activity of
anti-A,B against A or B red cells, respectively.

Anti-A1
Anti-A1 occurs as an alloantibody in the
serum of 1% to 2% of A2 individuals and
15
25% of A 2 B individuals. Sometimes,
Anti-A1 can also be found in the sera of individuals with other weak subgroups of A.
Anti-A1 can cause discrepancies in ABO
testing and incompatibility in crossmatches with A1 or A1B red cells. Anti-A1
usually reacts better or only at temperatures well below 37 C and is considered
clinically insignificant unless there is reactivity at 37 C. When reactive at 37 C, only
A2 or O red cells should be used for transfusion.
In simple adsorption studies, the anti-A
of group B serum appears to contain separable anti-A and anti-A1. Native group B serum agglutinates A1 and A2 red cells; after
adsorption with A2 red cells, group B serum
reacts only with A1 red cells. If further tests
are performed, however, the differences in

296

AABB Technical Manual

A antigen expression between A1 and A2 red
cells appear to be quantitative rather than
qualitative.9
A reliable anti-A1 reagent from the lectin
of Dolichos biflorus is commercially available or may be prepared (see Method 2.10).
The raw plant extract will react with both A1
and A2 red cells, but an appropriately diluted
reagent preparation will not agglutinate A2
cells and thus constitutes an anti-A1.

Routine Testing for ABO
Routine testing for determining the ABO
group consists of testing the red cells with
anti-A and anti-B (cell or forward type)
and testing the serum or plasma with A1
and B red cells (serum or reverse type).
Both red cell and serum testing are required for routine ABO tests on donors and
patients because each serves as a check
on the other.18(pp32,37) The two exceptions to
performing both cell and serum testing
are confirmation testing of the ABO type of
donor units that have already been labeled and testing blood of infants less
than 4 months of age; in both of these instances, only ABO testing of red cells is required.
Anti-A and anti-B typing reagents agglutinate most antigen-positive red cells on direct contact, even without centrifugation.
Anti-A and anti-B in the sera of some patients and donors are too weak to agglutinate red cells without centrifugation or prolonged incubation. Serum tests should be
performed by a method that will adequately detect the antibodies—eg, tube, microplate, or column agglutination techniques.
Procedures for ABO typing by slide, tube,
and microplate tests are described in Methods 2.1, 2.2, and 2.3.
Additional reagents, such as anti-A,B for
red cell tests and A2 and O red cells for serum
tests, are not necessary for routine testing

but are helpful in resolving typing discrepancies (see below). The use of anti-A,B may
not have the same benefit in detecting weak
subgroups when testing with monoclonal
reagents (depending on the clones used) as
when human polyclonal reagents were in
use. Many monoclonal ABO typing reagents have been formulated to detect
some of the weaker subgroups. Manufacturers’ inserts should be consulted for specific reagent characteristics. Special techniques to detect weak subgroups are not
routinely necessary because a typing discrepancy (eg, the absence of expected serum antibodies) usually distinguishes these
specimens from group O specimens.
The A2 red cells are intended to facilitate
the recognition of anti-A1. Because most group
A specimens do not contain anti-A1, routine
use of this reagent is not necessary.

Discrepancies Between Red Cell and
Serum Tests
Table 13-1 shows the results and interpretations of routine red cell and serum tests
for ABO. A discrepancy exists when the
results of red cell tests do not agree with
serum tests. When a discrepancy is encountered, the discrepant results must be
recorded, but interpretation of the ABO
group must be delayed until the discrepancy has been resolved. If the specimen is
from a donor unit, the unit must not be
released for transfusion until the discrepancy has been resolved. When the blood
is from a potential recipient, it may be
necessary to administer group O red cells
of the appropriate Rh type before the investigation has been completed. It is important to obtain sufficient pretransfusion
blood samples from the patient to complete any additional studies that may be
required.

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens

Red cell and serum test results may be
discrepant because of intrinsic problems
with red cells or serum, or technical errors.
Discrepancies may be signaled either because negative results are obtained when
positive results are expected, or positive results are found when tests should have been
negative (see Table 13-2).

297

Specimen-Related Problems in Testing Red
Cells
ABO testing of red cells may give unexpected results for many reasons.
1.
Red cells from individuals with variant A or B alleles may carry poorly
expressed antigens. Antigen expres-

Table 13-2. Possible Causes of ABO Typing Discrepancies
Category

Causes

Red cell weak/
missing reactivity

ABO subgroup
Leukemia/malignancy
Transfusion
Intrauterine fetal transfusion
Transplantation
Excessive soluble blood group substance

Extra red cell
reactivity

Autoagglutinins/excess protein coating red cells
Unwashed red cells: plasma proteins
Unwashed red cells: antibody in patient’s serum to reagent
constituent
Transplantation
Acquired B antigen
B(A) phenomenon
Out-of-group transfusion

Mixed-field red cell
reactivity

Recent transfusion
Transplantation
Fetomaternal hemorrhage
Twin or dispermic (tetragametic) chimerism

Serum weak/missing
reactivity

Age related (<4-6 months old, elderly)
ABO subgroup
Hypogammaglobulinemia
Transplantation

Serum extra reactivity

Cold autoantibody
Cold alloantibody
Serum antibody to reagent constituent
Excess serum protein
Transfusion of plasma components
Transplantation
Infusion of intravenous immune globulin

298

AABB Technical Manual

sion may also be weakened on the red
cells of some persons with leukemia
or other malignancies.
A patient who has received red cell
transfusions or a marrow transplant
may have circulating red cells of more
than one ABO group and constitute
a transfusion or transplantation chimera (see Mixed-Field Agglutination
below).
Exceptionally high concentrations of
A or B blood group substances in the
serum can combine with and neutralize reagent antibodies to produce an
unexpected negative reaction against
serum- or plasma-suspended red cells.
A patient with potent autoagglutinins
may have red cells so heavily coated
with antibody that the red cells agglutinate spontaneously in the presence of diluent, independent of the
specificity of the reagent antibody.
Abnormal concentrations of serum
proteins or the presence in serum of
infused macromolecular solutions may
cause the nonspecific aggregation of
serum-suspended red cells that simulates agglutination.
Serum- or plasma-suspended red cells
may give false-positive results with
monoclonal reagents if the serum or
plasma contains a pH-dependent
autoantibody.19,20 Serum- or plasmasuspended red cells may also give
discrepant results due to an antibody
21
to a reagent dye/constituent or to
proteins causing rouleaux.
Red cells of some group B individuals are agglutinated by a licensed
anti-A reagent that contains a particular murine monoclonal antibody,
MHO4. These group B individuals had
excessively high levels of B allelespecified galactosyltransferase, and
the designation B(A) was given to
this blood group phenotype.22

Red cells of individuals with the
acquired B phenotype typically agglutinate strongly with anti-A and
weakly with anti-B, and the serum
contains strong anti-B. The acquired
B phenotype arises when microbial
deacetylating enzymes modify the A
antigen by altering the A-determining sugar (N-acetylgalactosamine)
so that it resembles the B-determining galactose. The acquired B phenomenon is found most often in individuals with the A1 phenotype.
Inherited or acquired abnormalities
of the red cell membrane can lead to
what is called a polyagglutinable state.
The abnormal red cells can be unexpectedly agglutinated by human reagent anti-A, anti-B, or both because
human reagents will contain antibodies to the so-called cryptantigens
that are exposed in polyagglutinable
states. In general, monoclonal anti-A
and anti-B reagents will not detect
polyagglutination.

Specimen-Related Problems in Testing
Serum or Plasma
ABO serum/plasma tests are also subject
to false results.
1.
Small fibrin clots that may be mistaken for agglutinates may be seen
in ABO tests with plasma or incompletely clotted serum.
2.
Negative or weak results are seen in
serum tests from infants under 4 to 6
months of age. Serum from newborns
is not usually tested because antibodies present are generally passively
transferred from the mother.
3.
Unexpected absence of ABO agglutinins may be due to the presence of
an A or B variant.
4.
Patients who are immunodeficient
due to disease or therapy may have

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens

such depressed immunoglobulin levels
that there is little or no ABO agglutinin activity. Samples from elderly
patients whose antibody levels have
declined with age or from patients
whose antibodies have been greatly
diluted by plasma exchange procedures may also have unexpectedly weak
agglutinins.
If the patient has received a marrow
transplant of a dissimilar ABO group,
serum antibodies will not agree with
red cell antigens. For example, a group
A individual who receives group O
marrow may have circulating group
O red cells and produce only anti-B in
the serum. Refer to Chapter 25 for more
information on the effects of ABOmismatched transplants.
Cold allo- or autoantibodies that react at room temperature can react
with one or both reverse grouping
cells. For example, if the patient has
a room-temperature-reactive anti-M,
it may cause an unexpected reaction
with M-positive A1 and/or B reagent
red cells.
Antibodies to constituents of the diluents used to preserve reagent A1 and
B red cells can agglutinate the cells
independent of ABO antigens and
antibodies.
Abnormal concentrations of proteins,
altered serum protein ratios, or the
presence of high-molecular-weight
plasma expanders can cause nonspecific red cell aggregation or rouleaux that is difficult to distinguish from
true agglutination. Rouleaux formation is easily recognized on microscopic examination if the red cells
assume what has been described as
a “stack of coins” formation.
Recent transfusion with plasma components containing ABO agglutinins
may cause unexpected reactions.

10.

299

Recent infusion of intravenous immune globulin that may contain ABO
isohemagglutinins can cause unexpected reactions.

Mixed-Field Agglutination
Occasional samples are encountered that
contain two distinct, separable populations
of red cells. Usually, this reflects the recent transfusion of group O red cells to a
non-group-O recipient or receipt of a
marrow transplant of an ABO group different from the patient’s own. Red cell
mixtures also occur in a condition called
blood group chimerism, resulting either
from intrauterine exchange of erythropoietic tissue by fraternal twins or from
mosaicism arising through dispermy. In all
such circumstances, ABO red cell tests may
give a mixed-field pattern of agglutination. Mixed-field reactions due to transfusion last only for the life of the transfused
red cells. After hematopoietic transplantation, the mixed-field reaction usually disappears when the patient’s own red cells
are no longer produced. Persistent mixedcell populations do occur in some marrow
recipients. Mixed-field reactions that arise
through blood group chimerism may persist throughout the life of the individual.
For more information regarding the transfusion and evaluation of hematopoietic
transplant patients, refer to Chapters 21
and 25.

Technical Errors
Technical errors leading to ABO discrepancies include:
1.
Specimen mix-up.
2.
Red cell suspensions are too heavy
or too light.
3.
Failure to add reagents.
4.
Missed observation of hemolysis.
5.
Failure to follow manufacturer’s instructions.

300

6.
7.

AABB Technical Manual

Under- or overcentrifugation of tests.
Incorrect interpretation or recording
of test results.

Resolving ABO Discrepancies
The first step in resolving an apparent serologic problem should be to repeat the
tests on the same sample. If initial tests
were performed on red cells suspended in
serum or plasma, the testing should be repeated after washing the red cells several
times with saline. Washing the red cells
can eliminate many problems for red cell
typing that are associated with plasma
proteins. If the discrepancy persists, the
following initial steps can be incorporated
into the investigation.
1.
Obtain the patient’s diagnosis, historical blood group, and history of
previous transfusions, transplantation, and medications.
2.
Review the results of the antibody
detection test against group O red cells
and autologous red cells to detect
possible interference from allo- or
autoantibodies.
3.
Obtain a new blood specimen and test
the new sample if a discrepancy due
to a contaminated specimen is suspected.
In addition to a discrepancy between the
red cell and serum tests, an ABO discrepancy may also exist when the observed reactivity is not in agreement with a previous
type on record. The first step in resolving
this type of discrepancy is to obtain a new
blood specimen.

Resolving Discrepancies Due to Absence of
Expected Antigens
The cause of a discrepancy can sometimes be inferred from the strength of the
reactions obtained in red cell or serum
tests. For example, serum that strongly
agglutinates group B red cells but not A1

cells probably comes from a group A person, even though the red cells are not
agglutinated by anti-A or anti-B. The following procedures can be used to enhance the detection of weakly expressed
antigens.
1.
Incubate washed red cells with anti-A,
anti-B, and anti-A,B for 15 minutes
at room temperature to increase the
association of antibody with antigen.
Incubating the test system for 15 to
30 minutes at 4 C may further enhance antibody attachment. An inert
(eg, 6% albumin) or autologous control for room temperature and 4 C
tests is recommended. The manufacturer’s directions for any reagent
should be consulted for possible
comments or limitations.
2.
Treat the patient’s red cells with a
proteolytic enzyme such as ficin,
papain, or bromelin. Enzyme treatment increases the antigen-antibody
reaction with anti-A or anti-B. In
some instances, reactions between
reagent antibody and red cells expressing antigens will become detectable at room temperature within
30 minutes if enzyme-treated red
cells are employed. Enzyme-treated
group O red cells and an autologous
control must be tested in parallel as
a control for the specificity of the
ABO reaction. The manufacturer’s
directions should be consulted for
possible comments or limitations.
3.
Incubate an aliquot of red cells at
room temperature or at 4 C with
anti-A or anti-B (as appropriate) to
adsorb antibody to the corresponding red cell antigen for subsequent
elution (see Method 2.4). Group A or
B (as appropriate) and O red cells
should be subjected to parallel adsorption and elution with any reagent to serve as positive and nega-

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens

tive controls. Anti-A1 lectin should
not be used for adsorption/elution
studies because in a more concentrated form, such as an eluate, it may
react nonspecifically with red cells.
Test the eluate against group A1, B,
and O cells. Unexpected reactivity in
control eluates invalidates the results obtained with the patient’s red
cells. This indicates that the adsorption/elution procedure was not performed correctly, another antibody is
present (ie, in a polyclonal reagent),
or the specificity of a monoclonal reagent is not distinct enough for the
reagent to be used by this method.
Test the saliva for the presence of H
and A or B substances (see Method
2.5). Saliva tests help resolve ABO
discrepancies only if the person is a
secretor. This may be surmised from
the Lewis phenotype but may not be
known until after the saliva testing is
complete. See the discussion on Lewis
antigens.

Resolving Discrepancies Due to Absence of
Expected Antibodies
1.

Incubate the serum with A1 and B red
cells for 15 to 30 minutes at room
temperature. If there is still no reaction, incubate at 4 C for 15 to 30
minutes. It is recommended to include an autocontrol and group O
red cells for room temperature and 4
C testing to control for reactivity of
common cold autoagglutinins.
Treat the A1 and B reagent cells with
a proteolytic enzyme such as ficin,
papain, or bromelin. Enzyme-treated
group O and autologous red cells
must be tested in parallel as a control for reactivity. The manufacturer’s
directions should be consulted for
possible comments or limitations.

301

Resolving Discrepancies Due to Unexpected
Red Cell Reactions with Anti-A and Anti-B
Red cell ABO tests sometimes give unexpected positive reactions. For example,
reagent anti-A may weakly agglutinate red
cells from a sample in which the serum
gives reactions expected of a normal
group B or O sample. The following paragraphs describe some events that can
cause unexpected reactions in ABO typing
tests and the steps that can be taken to
identify them.
B(A) Phenotype. When cells react weakly
with monoclonal anti-A and strongly with
anti-B, and the serum reacts with A1 red
cells, but not B cells, the B(A) phenotype
should be suspected. Verification that the
anti-A reagent contains the discriminating
MHO4 clone confirms the suspicion. B(A)
red cells can show varying reactivity with
anti-A; the majority of examples react
weakly, and the agglutinates are fragile and
easily dispersed, although some examples
have reacted as strongly as 2+.22 Sera from
these individuals agglutinate both A1 and A2
cells. Except for newborns and immunocompromised patients, serum testing should
distinguish this phenomenon from the AB
phenotype in which a subgroup of A is accompanied by anti-A 1 . Testing with an
anti-A without the MHO4 clone should resolve the discrepancy. The recipient can be
considered a group B.
Acquired B Phenotype. Red cells agglutinated strongly by anti-A and weakly by
anti-B and a serum containing strong
anti-B suggest the acquired B state. The acquired B phenomenon is found most often
in individuals with the A1 phenotype; a few
examples of A2 with acquired B have been
found. Most red cells with acquired B antigens react weakly with anti-B, but occasional examples are agglutinated quite
strongly. Behavior with monoclonal anti-B
reagents varies with the particular clone

302

AABB Technical Manual

used. Acquired B antigens had been observed with increased frequency in tests with
certain FDA-licensed monoclonal anti-B
blood grouping reagents containing the ES-4
clone,23 but the manufacturers have lowered
the pH of the anti-B reagent so the frequency of the detection of acquired B is
similar to polyclonal anti-B or have discontinued the use of that clone. To confirm that
group A red cells carry the acquired B structure:
1.
Check the patient’s diagnosis. Acquired B antigens are usually associated with tissue conditions that allow colonic bacteria to enter the
circulation, but acquired B antigens
have been found on the red cells of
apparently normal blood donors.23
2.
Test the patient’s serum against autologous red cells or known acquired B
cells. The individual’s anti-B will not
agglutinate his or her own red cells
or red cells known to be acquired B.
3.
Test the red cells with monoclonal
anti-B reagents for which the manufacturer’s instructions give a detailed
description. Unlike most human polyclonal antibodies, some monoclonal
antibodies do not react with the acquired B phenotype; this information
may be included in the manufacturer’s directions.
4.
Test the red cells with human anti-B
serum that has been acidified to pH
6.0. Acidified human anti-B no longer reacts with the acquired B antigen.
Antibody-Coated Red Cells. Red cells
from infants with HDFN or from adults suffering from autoimmune or alloimmune
conditions may be so heavily coated with
IgG antibody molecules that they agglutinate spontaneously in the presence of reagent diluents containing high protein concentrations. Usually, this is at the 18% to
22% range found in some anti-D reagents,

but, sometimes, the sensitized red cells also
agglutinate in ABO reagents with protein
concentrations of 6% to 12%. Methods 2.12
or 2.14 may be used to remove much of this
antibody from the red cells so that the cells
can be tested reliably with anti-A and anti-B.
Red cells from a specimen containing coldreactive IgM autoagglutinins may autoagglutinate in saline tests. Incubating the cell
suspension briefly at 37 C and then washing the cells several times with saline
warmed to 37 C can usually remove the antibodies. If the IgM-related agglutination is
not dispersed by this technique, the red cells
can be treated with the sulfhydryl compound dithiothreitol (DTT) (see Method 2.11).

Resolving Discrepancies Due to Unexpected
Serum Reactions
The following paragraphs describe some
events that can cause unexpected or erroneous serum test results and the steps that
can be taken to resolve them.
1.
Reactivity of the A 1 reagent cells
when anti-A is strongly reactive with
the red cells suggests the presence of
anti-A1 in the serum of an A2 or A2B
individual. To demonstrate this as
the cause of the discrepancy:
a.
Test the red cells with anti-A1
lectin to differentiate group A1
from A2 red cells.
b.
Test the serum against several
examples of each of the following: A1, A2, and O red cells. Only
if the antibody agglutinates all
A1 red cells and none of the A2
or O red cell samples can it be
called anti-A1.
2.
Strongly reactive cold autoagglutinins, such as anti-I, anti-IH, anti-IA,
and anti-IB, can agglutinate red cells
of adults, including autologous cells
and reagent red cells, at room temperature (20-24 C). With few excep-

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens

tions, agglutination caused by the cold
autoagglutinin is weaker than that
caused by anti-A and anti-B. The following steps can be performed when
such reactivity interferes to the point
that the interpretation of serum tests
is difficult.
a.
Warm the serum and reagent red
cells to 37 C before mixing and
testing. Incubate at 37 C for 1
hour and perform a “settled”
reading (ie, observe for agglutination without centrifugation).
Rare weakly reactive examples
of IgM anti-A or anti-B may not
be detected by this method.
b.
Remove the cold autoagglutinin
from the serum using a cold
autoadsorption method as described in Method 4.6. The adsorbed serum can then be tested
against A1 and B reagent red cells.
Unexpected alloantibodies that react
at room temperature, such as anti-P1
or anti-M, may agglutinate the red
cells used in serum tests if the cells
carry the corresponding antigen. One
or more of the reagent cells used in
the antibody detection test may also
be agglutinated if the serum and cell
mixture was centrifuged for either a
room temperature or 37 C reading; a
rare serum may react with an antigen of low incidence on the serum
testing cells that is not present on cells
used for antibody detection. Steps to
determine the correct ABO type of sera
containing cold-reactive alloantibodies
include:
a.
Identify the room temperature
alloantibody, as described in
Chapter 19, and test the reagent A1 and B cells to determine
which, if either, carries the corresponding antigen. Obtain A1
and B red cells that lack the an-

303

tigen and use them for serum
testing.
b.
Raise the temperature to 30 to
37 C before mixing the serum
and cells, incubate for 1 hour, and
perform a “settled” reading (ie,
without centrifugation). If the
thermal amplitude of the alloantibody is below the temperature at which anti-A and anti-B
react, this may resolve the discrepancy.
c.
If the antibody detection test is
negative, test the serum against
several examples of A1 and B
red cells. The serum may contain an antibody directed against
an antigen of low incidence,
which will be absent from most
randomly selected A1 and B red
cells.
Sera from patients with abnormal
concentrations of serum proteins, or
with altered serum protein ratios, or
who have received plasma expanders of high molecular weight can aggregate reagent red cells and mimic
agglutination. Some of these samples cause aggregation of the type
described as rouleaux. More often,
the aggregates appear as irregularly
shaped clumps that closely resemble
antibody-mediated agglutinates. The
results of serum tests can often be
corrected by diluting the serum 1:3
in saline to abolish its aggregating
properties or by using a saline replacement technique (see Method
3.4).

The H System
On group O red cells, there is no A or B
antigen, and the membrane expresses
abundant H. Because H is a precursor of A

304

AABB Technical Manual

and B antigens, A and B persons have less
H substance than O persons. The amount
of H antigen detected on red cells with the
anti-H lectin Ulex europaeus is, in order of
diminishing quantity, O>A2>B>A2B>A1>A1B.
Occasionally, group A1, A1B, or (less commonly) B individuals have so little unconverted H antigen on their red cells that they
produce anti-H. This form of anti-H is generally weak, reacts at room temperature or
below, and is not considered clinically significant. Individuals of the rare Oh phenotype (see below), whose red cells lack H,
have a potent and clinically significant
alloanti-H in their serum (in addition to
anti-A and anti-B).

Oh Phenotype
The term Oh or Bombay phenotype has
been used for the very rare individuals
whose red cells and secretions lack H, A,
and B antigens and whose plasma contains potent anti-H, anti-A, and anti-B.6
This phenotype was first discovered in
Bombay, India. The phenotype initially
mimics normal group O but becomes apparent when serum from the Oh individual is tested against group O red cells, and
strong immediate-spin agglutination
and/or hemolysis occurs. The anti-H of
an Oh person reacts over a thermal range
of 4 to 37 C with all red cells except those
of other Oh people. Oh persons must be
transfused only with Oh blood because
their non-red-cell-stimulated antibodies
rapidly destroy cells with A, B, or H antigens. If other examples of Oh red cells are
available, further confirmation can be obtained by demonstrating compatibility of
the serum with Oh red cells. At the genotypic level, the Oh phenotype arises from
the inheritance of hh at the H locus and
sese at the Se locus. Because the Se allele is
necessary for the formation of Leb, Oh red
cells will be Le(a+b–) or Le(a–b–).

Para-Bombay Phenotype
The para-Bombay phenotype designation,
Ah, Bh, and ABh, is classically used for individuals who are H-deficient secretors, ie,
those who have an inactive H-transferase
but have active Se-transferase. The red
cells lack serologically detectable H antigen but carry small amounts of A and/or
B antigen (sometimes detectable only by
adsorption/elution tests), depending on
the individual’s alleles at the ABO locus.
Tests with anti-A or anti-B reagents may
or may not give weak reactions, but the
cells are nonreactive with anti-H lectin or
with anti-H serum from Oh persons. Individuals with the para-Bombay phenotype
have a functional Se allele and thus will
express A, B, and H antigens in their
plasma and secretions. The sera of Ah and
Bh people contain anti-H and/or anti-IH
in addition to the expected anti-A or
anti-B.
H-deficient secretors may also be group
O. These individuals will have traces of H
antigen, but no A or B antigen, on their red
cells and only have H in their secretions.
In 1994, Kelly et al reported the molecular bases for the Bombay and para-Bombay
24
phenotypes. Many mutations at the H locus have subsequently been associated
with H-deficiency.

The Lewis System
a

The Lewis system antigens, Le and Le ,
result from the action of a glycosyltransferase encoded by the Le allele that, like
the A, B, and H glycosyltransferases, adds
a sugar to a precursor chain. Lea is produced when Le is inherited with sese and
Leb is produced when Le is inherited with
at least one Se allele. When the silent or
amorphic allele le is inherited, regardless
of the secretor allele inherited, no Lea or
b
a
b
Le is produced. Thus, Le and Le are not

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens

antithetical antigens produced by alleles;
rather, they result from the interaction of
independently inherited alleles.
The Lewis antigens are not intrinsic to
red cells but are expressed on glycosphingolipid Type 1 chains adsorbed from plasma
onto red cell membranes. Plasma lipids exchange freely with red cell lipids.

Gene Interaction and the Antigens
The synthesis of the Lewis antigens is dependent upon the interaction of two different fucosyltransferases: one from the
Se locus and one from the Lewis locus.
Both enzymes act upon the same precursor Type 1 substrate chains. The fucosyltransferase encoded by the Le allele attaches fucose in α(1→4) linkage to the
subterminal GlcNAc; in the absence of the
transferase from the Se allele, this configa
uration has Le activity. This product canb
not be further glycosylated. Le occurs
when the Type 1 precursor is converted to
Type 1 H by the fucosyltransferase from
the secretor allele, and subsequently acted
upon by the fucosyltransferase from the
Le allele. This Leb configuration has two
9
b
fucose moieties. Thus, Le reflects the
presence of both the Le and Se alleles. Le
without Se results in Lea activity only; Se
with the amorphic allele le will result in

no secretion of Le or Le and the red cells
will have the Le(a–b–) phenotype.
Table 13-3 shows phenotypes of the Lewis
system and their frequencies in the population. Red cells that type as Le(a+b+) are rare
in people of European and African origin
but are relatively common in persons of
Asian origin, due to a fucosyltransferase encoded by a variant secretor allele that competes less efficiently with the Le fuco9
syltransferase.

Lewis Antibodies
Lewis antibodies occur almost exclusively
in the sera of Le(a–b–) individuals, usually
without known red cell stimulus. Those
individuals whose red cell phenotype is
Le(a–b+) do not make anti-Lea because
small amounts of unconverted Le a are
present in their saliva and plasma. It is
most unusual to find anti-Leb in the sera
b
of Le(a+b–) individuals, but anti-Le may
exist along with anti-Lea in the sera of
Le(a–b–) individuals. Lewis antibodies are
often found in the sera of pregnant women
who transiently demonstrate a Le(a–b–)
phenotype. The Lewis antibodies, however,
are almost always IgM and do not cross
the placenta. Because of this and because
Lewis antigens are poorly developed at
birth, the antibodies are not associated

Table 13-3. Phenotypes in the Lewis System and Their Incidence
Reactions with AntiLe
+
0
0
+

Le
0
+
0
+

305

Adult Phenotype Incidence %
Phenotype

Whites

Blacks

Le(a+b−)
Le(a−b+)
Le(a−b−)
Le(a+b+)

22
72
6
Rare

23
55
22
Rare

+ = agglutination; 0 = no agglutination.

306

AABB Technical Manual

with HDFN. Lewis antibodies may bind
complement, and fresh serum that contains anti-Lea (or infrequently anti-Leb)
may hemolyze incompatible red cells in
vitro. Hemolysis is more often seen with
enzyme-treated red cells than with untreated red cells.
Most Lewis antibodies agglutinate saline-suspended red cells of the appropriate
phenotype. The resulting agglutinates are
often fragile and are easily dispersed if red
cell buttons are not resuspended gently after centrifugation. Agglutination sometimes
is seen after incubation at 37 C, but rarely
of the strength seen in tests incubated at
room temperature. Some examples of
anti-Lea, and less commonly anti-Leb, can be
detected in the antiglobulin phase of testing. Sometimes this reflects complement
bound by the antibody if a polyspecific reagent (ie, containing anticomplement) is
used. In other cases, antiglobulin reactivity
results from an IgG component of the antibody.
Sera with anti-Leb activity can be divided
into two categories. The more common type
reacts best with Le(b+) red cells of group O
and A2; these antibodies have been called
bH
anti-Le . Antibodies that react equally well
with the Leb antigen on red cells of all ABO
phenotypes are called anti-LebL.

Transfusion Practice
Lewis antigens readily adsorb to and elute
from red cell membranes. Transfused red
cells shed their Lewis antigens and assume
the Lewis phenotype of the recipient
within a few days of entering the circulation. Lewis antibodies in a recipient’s serum are readily neutralized by Lewis
blood group substance in donor plasma.
For these reasons, it is exceedingly rare for
Lewis antibodies to cause hemolysis of
transfused Le(a+) or Le(b+) red cells. It is
not considered necessary to type donor

blood for the presence of Lewis antigens
before transfusion or when crossmatching for recipients with Lewis antibodies;
red cells that are compatible in tests at 37
C can be expected to survive normally in
vivo.25,26

Lewis Antigens in Children
Red cells from newborn infants usually do
a
not react with either human anti-Le or
b
anti-Le and are considered to be Le(a–b–).
Some can be shown to carry small amounts
of Le a when tested with potent monoa
clonal anti-Le reagents. Among children,
the incidence of Le(a+) red cells is high
and that of Le(b+) red cells low, reflecting
a greater production of the Le allele-specific transferase in infants; the Se allelespecific transferase is produced in lower
levels. The phenotype Le(a+b+) may be
transiently observed in children as the Se
allele transferase levels increase toward
adult levels. Reliable Lewis typing of young
children may not be possible because test
reactions may not reflect the correct phenotype until approximately 2 to 3 years of
age.25

The I/i Antigens and
Antibodies
Cold agglutinins with I specificity are frequently encountered in sera of normal individuals. The antibody is usually not
clinically significant and reacts with all
red cells except cord cells and the rare i
adult phenotype. I and i antigens, however,
are not antithetical but are expressed in a
reciprocal relationship. At birth, infant red
cells are rich in i; I is almost undetectable.
Thus, for practical purposes, cord cells are
considered to be I–, i+. During the first 2
years of life, I antigen gradually increases
at the expense of i. The red cells of most

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens

adults are strongly reactive with anti-I
and react weakly or not at all with anti-i.
Rare adults have red cells that carry high
levels of i and only trace amounts of I;
these red cells have the i adult phenotype.
There is no true I– or i– phenotype.
The I and i antigens on red cells are internal structures carried on the same glycoproteins and glycosphingolipids that carry
H, A, or B antigens and in secretions on the
same glycoproteins that carry H, A, B, Lea,
b
and Le antigens. The I and i antigens are
located closer to the membrane than the
terminal sugars that determine the ABH
antigens. The i structure is a linear chain of
at least two repeating units of N-acetylgalactosamine [Galβ(1→4)GlcNAcβ(1→3)].
On the red cells of adults, many of these
linear chains are modified by the addition
of branched structures consisting of GlcNAc
in a β(1→6) linkage to a galactose residue
internal to the repeating sequence. The
branching configuration confers I specificity.27,28 Different examples of anti-I appear to
recognize different portions of the branched oligosaccharide chain. As branching occurs and as the sugars for H, A, and B antigens are added, access of anti-i and anti-I
may be restricted.25
The I antigen, together with the i antigen, used to comprise the Ii Blood Group
Collection in the ISBT nomenclature. Recent cloning of the gene that encodes the
transferase responsible for converting i active straight chains into I active branched
chains and identification of several mutations responsible for the rare i adult phenotype have caused the I antigen to be assigned to the new I Blood Group System;
29
the i antigen remains in the Ii collection.

Antibodies to I/i
Anti-I is a common, benign autoantibody
found in the serum of many normal healthy
individuals that behaves as a cold aggluti-

307

nin at 4 C with a titer of <64. Agglutination
with adult red cells and weaker or no agglutination with cord cells is the classic
reactivity. Some stronger examples agglutinate cells at room temperature; others
may react only with the strongest I+ red
cells and give inconsistent reactions. Incubating tests in the cold enhances anti-I reactivity and helps to confirm its identity;
albumin and testing enzyme-treated red
cells also enhance anti-I reactivity.
Autoanti-I assumes pathologic significance in cold agglutinin syndrome (CAS),
in which it behaves as a complement-binding antibody with a high titer and high thermal amplitude. The specificity of the autoantibody in CAS may not be apparent when
the undiluted sample is tested (I adult and
cord cells may react to the same strength
even at room temperature); titration studies
and/or thermal amplitude studies may be
necessary to define the specificity (see
Chapter 20). Anti-I is often made by patients with pneumonia due to Mycoplasma
pneumoniae. These patients may experience
transient hemolytic episodes due to the antibody.
Autoanti-i is less often implicated in
symptomatic disease than anti-I. On rare
occasions, anti-i may be seen as a relatively
weak cold autoagglutinin reacting preferentially at 4 C. Anti-i reacts strongest with
cord and i adult red cells, and weakest with
I adult red cells. Patients with infectious
mononucleosis often have transient but
potent anti-i.
Table 13-4 illustrates the serologic behavior of anti-I and anti-i at 4 C and 22 C.
Reaction strengths should be considered
relative; clear-cut differences in reactivity
between the two are seen only with weaker
examples of the antibodies. Titration studies may be needed to differentiate strong
examples of the antibodies.
Serum containing anti-I or anti-i is sometimes reactive at the antiglobulin phase of

308

AABB Technical Manual

Table 13-4. Comparative Serologic Behavior of the I/i Blood Group Antibodies with
Saline Red Cell Suspensions
Temperature

Cell Type

Anti-I

Anti-i

I adult
i cord
i adult
I adult
i cord
i adult

4+
0-2+
0-1+
2+
0
0

0-1+
3+
4+
0
2-3+
3+

22 C

testing when polyspecific antihuman globulin is used. Such reactions rarely indicate
antibody activity at 37 C. Rather, complement components are bound when serum
and cells interact at lower temperatures;
during the 37 C incubation, the antibody
dissociates but the complement remains
bound to the red cells. Thus, the antiglobulin phase reactivity is usually due to
the anticomplement in a polyspecific antiglobulin reagent. Usually, avoiding room
temperature testing and using anti-IgG instead of a polyspecific antihuman globulin
help to eliminate detection of cold autoantibodies. Cold autoadsorption to remove
the autoantibody from the serum may be
necessary for stronger examples; cold autoadsorbed serum or plasma can also be used
in ABO typing (see Method 4.6). The prewarming technique can also be used once
the reactivity has been confirmed as cold
autoantibody (see Method 3.3).

Complex Reactivity
Some antibodies appear to recognize I determinants with attached H, A, or B immunodominant sugars. Anti-IH occurs
quite commonly in the serum of A1 individuals; it reacts stronger with red cells
that have high levels of H as well as I (ie,
group O and A2 red cells) and weaker, if at

all, with group A1 cells of adults or cord
cells of any group. Anti-IH should be suspected when serum from a group A patient causes direct agglutination of all
cells used for antibody detection but is
compatible with all or most group A donor blood. Other examples of complex reactivity include anti-IA, -IP1, -IBH, -ILebH,
and -iH.

The P Blood Group and
Related Antigens
The P blood group has traditionally conk
sisted of the P, P1, and P antigens, and
later Luke (LKE). However, the biochemistry and molecular genetics, although not
yet completely understood, make it clear
that at least two biosynthetic pathways
and genes at different loci are involved in
the development and expression of these
antigens. Thus, in ISBT nomenclature, the
P antigen is assigned to the new globoside
(GLOB) blood group system, the P1 antigen is assigned to the P blood group system,
and Pk and LKE remain in the globoside
29
collection of antigens. For simplicity,
these antigens are often referred to as the
P blood group.

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens

The first antigen of the P blood group
was discovered by Landsteiner and Levine
in 1927, in a series of animal experiments
that led also to the discovery of M and N.
Originally called P, the name of the antigen
was later changed to P1. The designation P
has since been reassigned to an antigen
present on almost all human red cells. The
Pk antigen is also present on almost all human red cells, but it is not readily detected
k
k
unless P is absent, eg, in the rare P1 or P2
phenotypes. The null phenotype, p, is very
rare. LKE antigen is present on almost all
red cells except those of the rare phenotypes
p or Pk and in about 2% of P+ red cells.

Common and Rare Phenotypes
There are two common phenotypes associated with the P blood group, P1 and P2,
k
k
and three rare phenotypes, p, P1 , and P2 ,
as shown in Table 13-5. The P1 phenotype
describes those red cells that react with
anti-P1 and anti-P; red cells that do not react with anti-P1, but do react with anti-P,
are of the P2 phenotype. When red cells
are tested only with anti-P1 and not with
anti-P, the phenotype should be written as
P1+ or P1–.

309

Biochemistry and Genetics
The P blood group antigens, like the ABH
antigens, are sequentially synthesized by
the addition of sugars to precursor chains.
The different oligosaccharide determinants of the P blood group antigens are
shown in Fig 13-5. All the antigens are exclusively expressed on glycolipids on hu30
man red cells, not on glycoproteins. The
precursor of P1 can also be glycosylated to
Type 2H chains, which carry ABH antigens.
There are two distinct pathways for the
synthesis of the P blood group antigens as
shown in Fig 13-6. The common precursor
is lactosylceramide, also known as ceramide dihexose or CDH. One pathway results in the formation of paragloboside and
P1. Paragloboside is also the Type 2 precursor for ABH antigens. The other pathway
results in the formation of the globoside series of antigens: Pk, P, and LKE.
The genes encoding the glycosyltransferases that are responsible for synthesizing
k
P from lactosylceramide and for converting
k
P to P were cloned in 2000. Several mutak
tions that result in the p and P phenotypes
31-33
have been identified.
The genetic relak
tionship between P1, P and P is still not understood. Red cells of the p phenotype are

Table 13-5. Phenotypes of the P Blood Group and Related Antigens
Reactions with Antik

0
+
0

Phenotype Incidence (%)
k

Phenotype

Whites

Blacks

P1k

P1 +P+P

*Usually negative, occasionally weakly positive.

All extremely rare

310

P
P
LKE
P1

AABB Technical Manual

Lactosylceramide (CDH)
Globotriosylceramide (CTH)
Globoside
Sialosylgalactosylgloboside
Paragloboside
Galactosylparagloboside

Galβ(1→4)Glc-Cer
Galα(1→4)Galβ(1→4)Glc-Cer
GalNAcβ(1→3)Galα(1→4)Galβ(1→4)Glc-Cer
NeuAcα(2→3)Galβ(1→3)GalNAcβ(1→3)Galα(1→4)Galβ(1→4)Glc-Cer
Galβ(1→4)GlcnNAcβ(1→3)Galβ(1→4)Glc-Cer
Galα(1→4)Galβ(1→4)GlcnNAcβ(1→3)Galβ(1→4)Glc-Cer

Figure 13-5. Some biochemical structures of P blood group antigens.

P1– in addition to being P– and P –; the P1–
status of p red cells cannot be explained.
The P1 gene is located on chromosome 22
and the P gene is located on chromosome 3.

Anti-P1
The sera of P 1 – individuals commonly
contain anti-P1. If sufficiently sensitive
techniques are applied, it is likely that
anti-P1 would be detected in the serum of
25
virtually every person with P1– red cells.
The antibody reacts optimally at 4 C but
may occasionally be detected at 37 C.
Anti-P1 is nearly always IgM and has not been

reported to cause HDFN. Only rarely has
it been reported to cause hemolysis in
vivo.17(p139),34
The strength of the P1 antigen varies widely
among different red cell samples, and antigen strength has been reported to diminish
when red cells are stored. These characteristics sometimes create difficulties in identifying antibody specificity in serum with a
positive antibody screen. An antibody that
reacts weakly in room temperature testing
can often be shown to have anti-P1 specificity by incubation at lower temperatures or
by the use of enzyme-treated red cells.
Hydatid cyst fluid or P1 substance derived
from pigeon eggs inhibits the activity of
anti-P1. Inhibition may be a useful aid to

Lactosylceramide (CDH)
Lactotriaosylceramide

Paragloboside
(type 2 precursor)

P1 antigen

Pk antigen
Globotriosylceramide (CTH)

P antigen
Globoside

LKE

Figure 13-6. Biosynthesis of P blood group antigens.

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens

antibody identification, especially if anti-P1
is present in a serum with antibodies of
other specificities.

311

fectiosum (Fifth disease). Individuals of p
phenotype who lack globoside are naturally resistant to infection with this patho39
gen.

Rare Antibodies
Alloanti-P, found as a naturally occurring
potent hemolytic antibody in the sera of
P1k and P2k individuals, reacts with all red
cells except those of the rare p and Pk phenotypes. Anti-P can be IgM or a mixture of
IgM and IgG. Anti-PP1Pk, formerly called
a
anti-Tj , is produced by individuals of the
p phenotype without red cell stimulation
and reacts with all red cells except those
of the rare p phenotype. Anti-PP1Pk can be
separated into its components (anti-P,
anti-P1, and anti-Pk) through adsorptions.
These components can be IgM and/or
IgG, react over a broad thermal range, and
can efficiently bind complement, which
make them potent hemolysins. AntiPP1Pk has caused hemolytic transfusion
17(p139)
reactions and, occasionally, HDFN.
There is an association between both
anti-P and anti-PP1Pk and spontaneous
35,36
abortions occurring early in pregnancy
Autoanti-P associated with paroxysmal
cold hemoglobinuria is a cold-reactive IgG
autoantibody that is described as a biphasic
hemolysin.17(pp220-1) The antibody typically
does not react in routine test systems, but is
demonstrable only by the Donath-Landsteiner test (see Chapter 20).

P Antigens as Receptors for Pathogens
The P blood group antigens are receptors
for several pathogens. P, P1, Pk, and LKE
are receptors for uropathogenic Eschek
richia coli; P and P1 are receptors for toxins from enterohemorrhagic E. coli37; and
the meningitis-causing bacterium Streptococcus suis binds to Pk antigen.38 The P
antigen (globoside) has also been shown
to serve as a receptor for erythrovirus (parvovirus) B19, which causes erythema in-

References
1.

7.
8.

9.
10.

11.

12.

13.

14.

Landsteiner K. Zur Kenntnis der antifermentativen, lytischen und agglutinierenden
Wirkungen des Blutserums und der Lymph.
Zbl Balk 1900;27:367.
Landsteiner K. Uber Agglutinationserscherschein ungen normalen menschlichen Blutes.
Wien Klin Wochenschr 1901;14:1132-4.
Garratty G, Dzik W, Issitt PD, et al. Terminology for blood group antigens and genes—historical origins and guidelines in the new millennium. Transfusion 2000;40:477-89.
Von Decastello A, Sturli A. Yber due usiaglutinie
im Serumgesunder und lronker Menschen.
Munchen Med Wochenschr 1902;26:1090-5.
Watkins WM. The ABO blood group system:
Historical background. Transfus Med 2001;
11:243-65.
Oriol R, Candelier JJ, Mollicone R. Molecular
genetics of H. Vox Sang 2000;78(Suppl 2):1058.
Yamamoto F. Molecular genetics of ABO. Vox
Sang 2000;78(Suppl 2):91-103.
Yamamoto F. Molecular genetics of the ABO
histo-blood group system. Vox Sang 1995;69:
1-7.
Daniels G. Human blood groups. 2nd ed. Oxford: Blackwell Science, 2002.
Clausen H, Levery SB, Nudelman E, et al. Repetitive A epitope (type 3 chain A) defined by
group A 1 -specific monoclonal antibody
TH-1: Chemical basis of qualitative A1 and A2
distinction. Proc Natl Acad Sci U S A 1985;82:
1199-203.
Yamamoto F, Clausen H, White T, et al. Molecular genetic basis of the histo-blood group
ABO system. Nature 1990;345:229-33.
Patenaude SI, Seto NOL, Borisova SN, et al.
The structural basis for specificity in human
ABO(H) blood group biosynthesis (letter).
Nat Struct Biol 2002;9:685-90.
Lee AH, Reid ME. ABO blood group system: A
review of molecular aspects. Immunohematology 2000;16:1-6.
Olsson ML, Chester MA. Polymorphism and
recombination events at the ABO locus: A
major challenge for genomic ABO blood
grouping strategies. Transfus Med 2001;11:
295-313.

312

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

AABB Technical Manual

Reid ME, Lomas-Francis C. The blood group
antigen factsbook. 2nd ed. San Diego, CA: Academic Press, 2004.
Olsson ML, Irshaid NM, Hosseini-Maaf B, et
al. Genomic analysis of clinical samples with
serologic ABO blood grouping discrepancies:
Identification of 15 novel A and B subgroup
alleles. Blood 2001;98:1585-93.
Mollison PL, Engelfriet CP, Contreras M.
Blood transfusion in clinical medicine. 10th
ed. Oxford: Blackwell Scientific Publications,
1997.
Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.
Spruell P, Chen J, Cullen K. ABO discrepancies
in the presence of pH-dependent autoagglutinins (abstract). Transfusion 1994;34(Suppl):
22S.
Kennedy MS, Waheed A, Moore J. ABO discrepancy with monoclonal ABO reagents
caused by pH-dependent autoantibody. Immunohematology 1995;11:71-3.
Garratty G. In vitro reactions with red blood
cells that are not due to blood group antibodies: A review. Immunohematology 1998;14:111.
Beck ML, Yates AD, Hardman J, Kowalski MA.
Identification of a subset of group B donors
reactive with monoclonal anti-A reagent. Am
J Clin Pathol 1989;92:625-9.
Beck ML, Kowalski MA, Kirkegaard JR, Korth
JL. Unexpected activity with monoclonal anti-B
reagents (letter). Immunohematology 1992;8:
22.
Kelly RJ, Ernst LK, Larsen RD, et al. Molecular
basis for H blood group deficiency in Bombay
(Oh) and para-Bombay individuals. Proc Natl
Acad Sci U S A 1994;91:5843-7.
Issitt PD, Anstee DJ. Applied blood group serology. 4th ed. Durham, NC: Montgomery
Scientific Publications, 1998.
Waheed A, Kennedy MS, Gerhan S, Senhauser
DA. Success in transfusion with crossmatch-compatible blood. Am J Clin Pathol
1981;76:294-8.
Yu L-C, Twu Y-C, Chang C-Y, Lin M. Molecular basis of the adult I phenotype and the
gene responsible for the expression of the human blood group I antigen. Blood 2001;98:
3840-5.
Yu L-C, Twu Y-C, Chou M-L, et al. The molecular genetics of the human I locus and molecular background explain the partial association of the adult I phenotype with congenital
cataracts. Blood 2003;101:2081-8.
Daniels GL, Cartron JP, Fletcher A, et al. International Society of Blood Transfusion Committee on terminology for red cell surface an-

30.

31.

32.

33.

34.

35.

36.
37.

38.

39.

tigens: Vancouver report. Vox Sang 2003;84:
244-7.
Yang Z, Bergstrom J, Karlsson KA. Glycoproteins with Galα4Gal are absent from human
erythrocyte membranes, indicating that
glycolipids are the sole carriers of blood group
P activities. J Biol Chem 1994;269:14620-4.
Hellberg A, Poole J, Olsson ML. Molecular basis of the globoside-deficient Pk blood group
phenotype. J Biol Chem 2002;277;29455-9.
Furukawa K, Iwamura K, Uchikawa M, et al.
Molecular basis for the p phenotype. J Biol
Chem 2000;275:37752-6.
Koda Y, Soejima M, Sato H, et al. Three-base
deletion and one-base insertion of the α(1,4)
galactosyltransferase gene responsible for the
p phenotype. Transfusion 2002;42:48-51.
Arndt PA, Garratty G, Marfoe RA, Zeger GD.
An acute hemolytic transfusion reaction
caused by an anti-P1 that reacted at 37 C.
Transfusion 1998;38:373-7.
Shirey RS, Ness PM, Kickler TS, et al. The association of anti-P and early abortion. Transfusion 1987;27:189-91.
Cantin G, Lyonnais J. Anti-PP1Pk and early
abortion. Transfusion 1983;23:350-1.
Spitalnik PF, Spitalnik SL. The P blood group
system: Biochemical, serological, and clinical
aspects. Transfus Med Rev 1995;9:110-22.
Haataja S, Tikkanen K, Liukkonen J, et al.
Characterization of a novel bacterial adhesion specificity of Streptococcus suis recognizing blood group P receptor oligosaccharides.
J Biol Chem 1993;268:4311-17.
Brown KE, Hibbs JR, Gallinella G, et al. Resistance to parvovirus B19 infection due to lack
of virus receptor (erythrocyte P antigen). N
Engl J Med 1994;330:1192-6.

Suggested Reading
Chester MA, Olsson ML. The ABO blood group
gene: A locus of considerable genetic diversity.
Transfus Med Rev 2001;15:177-200.
Daniels G. Human blood groups. 2nd ed. Oxford:
Blackwell Science Publications, 2002.
Hanfland P, Kordowicz M, Peter-Katalinic J, et al.
Immunochemistry of the Lewis blood-group system: Isolation and structure of Lewis-c active and
related glycosphingolipids from the plasma of
blood-gro up O L e(a–b–) nonsecretors. Arch
Biochem Biophys 1986;246:655-72.
Issitt PD, Anstee DJ. Applied blood group serology.
4th ed. Durham, NC: Montgomery Scientific Publications, 1998.

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens

Judd WJ. Methods in immunohematology. 2nd ed.
Durham, NC: Montgomery Scientific Publications,
1994.
Lee AH, Reid ME. ABO blood group system: A review of molecular aspects. Immunohematology
2000;16:1-6.
Morgan WTJ, Watkins WM. Unraveling the biochemical basis of blood group ABO and Lewis antigenic specificity. Glycoconj J 2000;17:501-30.
Olsson ML, Chester MA. Polymorphism and recombination events at the ABO locus: A major

313

challenge for genomic ABO blood grouping strategies. Transfus Med 2001;11:295-313.
Palcic MM, Seto NOL, Hindsgual O. Natural and
recombinant A and B gene encoded glycosyltransferases. Transfus Med 2001;11:315-23.
Rydberg L. ABO-incompatibility in solid organ
transplant. Transfus Med 2001;11:325-42.
Watkins WM. The ABO blood group system: Historical background. Transfus Med 2001;11:243-65.
Yamamoto F. Cloning and regulation of the ABO
genes. Transfus Med 2001;11:281-94.

Chapter 14: The Rh System

Chapter 14

The Rh System

HIS CHAPTER USES the DCE nomenclature—a modification of the
nomenclature originally proposed
by Fisher and Race,1 which has been able
to accommodate our present understanding of the genetics and biochemistry of this
complex system. The Rh-Hr terminology
of Wiener is presented only in its historical context, as molecular genetic evidence
does not support Wiener’s one-locus theory.

The D Antigen and Its
Historical Context
Discovery of D
The terms “Rh positive” and “Rh negative”
refer to the presence or absence of the red
cell antigen D. The first human example
of the antibody against the antigen later
called D was reported in 1939 by Levine
and Stetson2; the antibody was found in

the serum of a woman whose fetus had
hemolytic disease of the fetus and newborn (HDFN) and who experienced a
hemolytic reaction after transfusion of
her husband’s blood. In 1940, Landsteiner
3
and Wiener described an antibody obtained by immunizing guinea pigs and
rabbits with the red cells of Rhesus monkeys; it agglutinated the red cells of approximately 85% of humans tested, and
they called the corresponding determinant the Rh factor. In the same year, Le4
vine and Katzin found similar antibodies
in the sera of several recently delivered
women, and at least one of these sera
gave reactions that paralleled those of the
animal anti-Rhesus sera. Also in 1940,
5
Wiener and Peters observed antibodies of
the same specificity in the sera of persons
whose red cells lacked the determinant
and who had received ABO-compatible
transfusions in the past. Later evidence
established that the antigen detected by
315

316

AABB Technical Manual

animal anti-Rhesus and human anti-D
were not identical, but, by that time, the
Rh blood group system had already received its name. Soon after anti-D was
discovered, family studies showed that
the D antigen is genetically determined;
transmission of the trait follows an autosomal dominant pattern.

Clinical Significance
After the A and B antigens, D is the most
important red cell antigen in transfusion
practice. In contrast to A and B, however,
persons whose red cells lack the D antigen do not regularly have anti-D. Formation of anti-D results from exposure,
through transfusion or pregnancy, to red
cells possessing the D antigen. The D antigen has greater immunogenicity than
other red cell antigens; it is estimated that
6,7
30% to 85% of D– persons who receive a
D+ transfusion will develop anti-D. Therefore, in most countries, the blood of all recipients and all donors is routinely tested
for D to ensure that D– recipients are
identified and given D– blood.

Other Rh Antigens
By the mid-1940s, four additional antigens—C, E, c, and e—had been recognized as belonging to what is now called
the Rh system. Subsequent discoveries
have brought the number of Rh-related
antigens to 49 ( Table 14-1), many of
which exhibit both qualitative and quantitative variations. The reader should be
aware that these other antigens exist (see
the suggested reading list), but, in most
transfusion medicine settings, the five
principal antigens (D, C, E, c, e) and their
corresponding antibodies account for the
vast majority of clinical issues involving
the Rh system.

Although Rh antigens are fully expressed
at birth with antigen detection as early as 8
weeks’ gestation,10 they are present on red
cells only and are not detectable on platelets, lymphocytes, monocytes, neutrophils,
or other tissues.11,12

Genetic and Biochemical
Considerations
Attempts to explain the genetic control of
Rh antigen expression were fraught with
controversy. Wiener13 proposed a single
locus with multiple alleles determining
surface molecules that embody numerous
antigens. Fisher and Race14 inferred from
the existence of antithetical antigens the
existence of reciprocal alleles at three individual but closely linked loci. Tippett’s
prediction15 that two closely linked structural loci on chromosome 1 determine
the production of Rh antigens has been
shown to be correct.

RH Genes
Two highly homologous genes on the
short arm of chromosome 1 encode the
nonglycosylated polypeptides that express the Rh antigens (Fig 14-1).16,17 One
gene, designated RHD, determines the
presence of a membrane-spanning protein that confers D activity on the red cell.
In Caucasian D– persons, the RHD gene is
deleted; the D– phenotype in some other
populations (persons of African descent,
Japanese, and Chinese) is associated with
an inactive, mutated, or partial RHD
gene.18 The inactive RHD gene or pseudogene (RHD ) responsible for the D– phenotype in some Africans has been described.19
The RHCE gene determines the C, c, E,
and e antigens; its alleles are RHCe, RHCE,
RHcE, and RHce.20 Evidence derived from

Chapter 14: The Rh System

317

Table 14-1. Antigens of the Rh Blood Group System and Their Incidence
Incidence (%)*
Incidence (%)*
Numeric
Antigen
Numeric
Antigen
White Black Overall Designation Name
White Black Overall
Designation Name
Rh1
Rh2
Rh3
Rh4
Rh5
Rh6
Rh7
Rh8
Rh9
Rh10
Rh11
Rh12
Rh17
Rh18
Rh19
Rh20
Rh21
Rh22
Rh23
Rh26
Rh27
Rh28
Rh29
RH30
Rh31

D
C
E
c
e
f
Ce
Cw
Cx
V
Ew
G
Hr0
Hr
hrs
VS
CG
CE
Dw

85
68
29
80

92
27
22
96
98

65
68
2

92
27
1
<0.01

<0.01
>99.9
>99.9
98
<0.01

32
68
<1
<0.01

80
28

96
22
<0.01
>99.9

total Rh
Goa
hrB

<0.01

Rh32
Rh33
Rh34
Rh35
Rh36
Rh37
Rh39
Rh40
Rh41
Rh42
Rh43
Rh44
Rh45
Rh46
Rh47
Rh48
Rh49
Rh50
Rh51
Rh52
Rh53
Rh54
Rh55
Rh56

Rh32
<0.01
Har
Bastiaan
Rh35
Bea
Evans
C-like
Tar
Ce-like
70
s
Ce
<0.1
Crawford
Nou
Riv
Rh46
Dav
JAL
STEM
<0.01
FPTT
MAR
BARC
JAHK
DAK
LOCR
CENR

1
<0.01
>99.9
<0.01
<0.1
<0.01
>99.9
<0.01
2
<0.01
>99.9
<0.01
>99.9
>99.9
<0.01
6
<0.01
>99.9
<0.01
<0.01
<0.01
<0.01
<0.01

*Incidence in White and Black populations where appropriate.

transfection studies indicates that both
C/c and E/e reside on a single polypeptide
product.

Biochemical and Structural Observations
The predicted products of both RHD and
RHCE are proteins of 417 amino acids

8,9

that, modeling studies suggest, traverse
the red cell membrane 12 times and display only short exterior loops of amino
acids (Fig 14-1). The polypeptides are fatty
acylated and, unlike most blood-groupassociated proteins, carry no carbohydrate residues.

318

AABB Technical Manual

Figure 14-1. Schematic representation of RHD, RHCE, and RHAG genes and RhD, RhCE, and RhAG proteins.
on RhD represents amino acid differences between RhD and RhCE.
on RhCE indicates the
critical amino acids involved in C/c and E/e antigen expression.

Considerable homology exists between
the products of RHD and RHCE; the products of the different alleles of RHCE are
even more similar.18 C and c differ from one
another in only four amino acids, at positions 16, 60, 68, and 103, of which only the
difference between serine and proline at
103 appears to be critical. The presence of
proline or alanine at position 226 distinguishes E from e. The D polypeptide, by
contrast, possesses 32 to 35 amino acids
that will be perceived as foreign by D–
individuals.
Within the red cell membrane, the Rh
polypeptides form a complex with the Rhassociated glycoprotein (RhAG), which has
37% sequence homology with the Rh polypeptides but is encoded by the RHAG gene
on chromosome 6 (Fig 14-1).22
The study of Rhnull red cells, which lack all
Rh antigens, reveals that this complex (Rh
proteins and RhAG) is essential for expression of other membrane proteins. Rhnull cells
lack LW antigens, are negative for Fy5 of the
Duffy system, and have weakened expression of the antigens carried on glycophorin
B (S, s, and U).23 Although Rh/RhAG proteins play a structural role in the red cell
membrane as evidenced by red cell mor-

phology changes in Rhnull syndrome (see
later in this chapter), their function remains
unknown. There is evidence, however, that
the RhAG protein plays a role in ammonium transport.24,25

Rh Terminology
Three systems of nomenclature were developed to convey genetic and serologic
information about the Rh system before
the recent advances in our understanding
of the genetics.

System Terminology
The Rh-Hr terminology derives from
Wiener,13 who believed the RH gene product to be a single entity he called an
agglutinogen. An agglutinogen was characterized by numerous individual specificities, called factors, that were identified
by specific antibodies. This theory was incorrect, but for the designation of phenotype, particularly in conversation, many
serologists use a shorthand system based
on Wiener’s Rh-Hr notation. The phenotype notations convey haplotypes with the
single letters R and r. R is used for haplo-

Chapter 14: The Rh System

types that produce D, r for haplotypes that
do not produce D. Subscripts or, occasionally, superscripts indicate the combinations of other antigens present. For example, R 1 indicates DCe haplotype; R 2
indicates DcE; r indicates dce; R0 indicates
Dce; and so on (Table 14-2).
CDE terminology was introduced by
Fisher and Race,1 who postulated three sets
of closely linked genes (C and c, D and d,
and E and e). Both gene and gene product
have the same letter designation, with italics used for the name of the gene. A modified CDE terminology is now commonly
used to communicate research and sero26
logic findings. Rosenfield and coworkers
proposed a system of nomenclature based
on serologic observations. Symbols were
not intended to convey genetic information, merely to facilitate communication of
phenotypic data. Each antigen is given a
number, generally in the order of its discovery or its assignment to the Rh system. Table 14-1 lists the Rh system antigens by
number designation, name, and incidence.

Determining Phenotype
In clinical practice, five blood typing reagents are readily available: anti-D, -C, -E,

Table 14-2. The Principal RH Gene
Complexes and the Antigens Encoded

Haplotype

R1
R2
Ro
Rz
r′
r″
r
ry

Genes
Present

Antigens
Present

Phenotype

RHD,RHCe
RHD,RHcE
RHD,RHce
RHD,RHCE
RHCe
RHcE
RHce
RHCE

D,C,e
D,c,E
D,c,e
D,C,E
C,e
c,E
c,e
C,E

R1
R2
Ro
Rz
r′
r″
r
ry

319

-c, and -e. Routine pretransfusion studies
include only tests for D. Other reagents
are used principally in the resolution of
antibody problems or in family studies.
The assortment of antigens detected on a
person’s red cells constitutes that person’s
Rh phenotype. Table 14-3 shows reaction
patterns of cells tested with antibodies to
the five antigens and the probable Rh
phenotype in modified Wiener terminology.

Serologic Testing for Rh
Antigen Expression
Expression of D
D– persons either lack RHD, which encodes for the D antigen, or have a nonfunctional RHD gene. Most D– persons
are homozygous for RHce, the gene encoding c and e; less often they may have
RHCe or RHcE, which encode C and e or c
and E, respectively. The RHCE gene,
which produces both C and E, is quite
rare in D– or D+ individuals.
The D genotype of a D+ person cannot
be determined serologically; dosage studies
are not effective in showing whether an individual is homozygous or heterozygous for
RHD. Using serologic tests, RHD genotype
can be assigned only by inference from the
antigens associated with the presence of D.
Molecular techniques, however, allow the
determination of D genotype.19,27,28
Interaction between genes results in socalled “position effect.” If the interaction is
between genes or the product of genes on
the same chromosome, it is called a cis effect. If a gene or its product interacts with
one on the opposite chromosome, it is
called a trans effect. Examples of both effects were first reported in 1950 by Lawler
and Race,29 who noted as a cis effect that the
E antigen produced by DcE is quantitatively

320

AABB Technical Manual

Table 14-3. Determination of Likely Rh Phenotypes from the Results of Tests with
the Five Principal Rh Blood Typing Reagents
Reagent
Anti-D

Anti-C

Anti-E

Anti-c

Anti-e

Antigens
Present

D,C,c,e

R1r

D,C,e

R1R1

D,C,c,E,e

R1R2

D,c,e

R0R0/R0r

D,c,E,e

R2r

D,c,E

R2R2

D,C,E,e

R1Rz

D,C,c,E

R2Rz

D,C,E

RzRz

c,e

C,c,e

r′r

c,E,e

r″r

C,c,E,e

r′r″

weaker than E produced by cE. They noted
as trans effects that both C and E are
weaker when they result from the genotype
DCe/DcE than when the genotypes are
DCe/ce or DcE/ce, respectively.

Expression of C, c, E, e
To determine whether a person has genes
that encode C, c, E, and e, the red cells are
tested with antibody to each of these antigens. If the red cells express both C and c
or both E and e, it can be assumed that
the corresponding genes are present in the
individual. If the red cells carry only C or
c, or only E or e, the person is assumed to
be homozygous for the particular allele.

Ethnic Origin
Ethnic origin influences deductions about
genotype because the incidence of Rh

Probable
Phenotype

genes differs from one geographic group
to another. For example, a White person
with the phenotype Dce would probably
be Dce/ce, but, in a Black person, the genotype could as likely be either Dce/Dce or
Dce/ce. Table 14-4 shows the incidence of
D, C, E, c, and e antigens in White and
Black populations.

Gene Frequency
The phenotype DCcEe (line 3 of Table
14-3) can arise from any of several genotypes. In any population, the most probable genotype is DCe/DcE. Both these
haplotypes encode D; a person with this
phenotype will very likely be homozygous
for the RHD gene, although heterozygous
for the RHCE gene (Ce/cE). Some less
likely genotypes could result if the person
is heterozygous at the D locus (for exam-

Chapter 14: The Rh System

ple, DCe/cE, DcE/Ce, or DCE/ce), but these
are uncommon in all populations. Table
14-4 gives the incidence of the more common genotypes in D+ persons. The figures
given are for Whites and Blacks. The absence of the RHD gene is uncommon in
other ethnic groups.

321

sumptions regarding the most probable genotype rest on the incidence of antigenic
combinations determined from population
studies in different ethnic groups. Inferences about genotype are useful in population studies, paternity tests, and in predicting the Rh genes transmitted by the
husband/partner of a woman with Rh antibodies (Table 14-4).
Molecular techniques are now available
that can determine Rh genotype. Determi-

Determining Genotype
Identifying antigens does not always allow confident deduction of genotype. Pre-

Table 14-4. Incidence of the More Common Genotypes in D+ Persons*
Likelihood of Zygosity for D (%)
Genotype

Incidence (%)

DCE

Mod.
Rh-hr

Whites

Blacks

DCe/ce
DCe/Dce
Dce/Ce

R1r
R1R0
R0r

31.1
3.4
0.2

8.8
15.0
1.8

D,C,e

DCe/DCe
DCe/Ce

R1R1
R1r

17.6
1.7

D,c,E,e

DcE/ce
DcE/Dce

R2r
R2R0

D,c,E

DcE/DcE
DcE/cE

D,C,c,E,e

Antigens
Present
D,C,c,e

D,c,e

Homo- Hetero- Homo- Hetero-

Whites

Blacks

2.9
0.7

10.4
1.1

5.7
9.7

R2R2
R2r

2.0
0.3

1.3
<<0.1

DCe/DcE
DCe/cE

R1R2
R1r

11.8
0.8

3.7
<0.1

DcE/Ce

0.6

0.4

Dce/ce
Dce/Dce

R0r
R0R0

3.0
0.2

22.9
19.4

*For the rare phenotypes and genotypes not shown in this table, consult the Suggested Readings listed at the end of this
chapter.

322

AABB Technical Manual

nations of genotype with polymerase chain
reaction methods can be made using DNA
harvested from white cells or amniocytes19,27,28 or from noncellular fetal DNA in
30
maternal plasma. Rarely, DNA genotype
results will disagree with serologic findings.

Weak D
Most D+ red cells show clear-cut macroscopic agglutination after centrifugation
with reagent anti-D and can be readily
classified as D+. Red cells that are not immediately or directly agglutinated cannot
as easily be classified. For some D+ red
cells, demonstration of the D antigen requires incubation with the anti-D reagent
or addition of antihuman globulin (AHG)
serum after incubation with anti-D [indirect antiglobulin test (IAT)]. These cells are
considered D+, even if an additional step
in testing is required.
In the past, red cells that required additional steps for the demonstration of D
were classified as Du. The term Du is no longer considered appropriate; red cells that
carry weak forms of D are classified as D+
and should be described as “weak D.” Improvement of polyclonal reagents and the
more widespread use of monoclonal anti-D
reagents have resulted in the routine detection of some D+ red cells that would have
been considered weak D when tested with
less sensitive polyclonal reagents. Additionally, monoclonal anti-D may react by direct
agglutination with epitopes of D that had
previously required more sensitive test
methods or, occasionally, may fail to react
with some epitopes of the D antigen. Conversely, some monoclonal anti-D may react
by direct testing with rare D epitopes that
were not detected with polyclonal reagents
(eg, DHAR and Crawford). It is important to
realize that anti-D reagents differ among

manufacturers and to know the characteristics of the product being used.

Quantitative Weak D
In the majority of cases, this form of the
weak D phenotype is due to an RHD gene
encoding an altered RhD protein associated with reduced D antigen expression on
the red cell membrane. The transmembrane or cytoplasmic location of the amino
acid changes in the altered D protein does
not result in the loss of D epitopes; thus,
the production of alloanti-D as in the partial D phenotype (see partial D) would not
be expected.31,32 Weak D expression is fairly
common in Blacks, often occurring as part
of a Dce haplotype. Genes for weak D expression are less common in Whites and
may be seen as part of an unusual DCe or
DcE haplotype.
Red cell samples with a quantitative
weak D antigen either fail to react or react
very weakly in direct agglutination tests with
most anti-D reagents. However, the cells will
react strongly by an IAT.
Red cells from some persons of the genotype Dce/Ce have weakened expression
of D, a suppressive effect exerted by RHC in
the trans position to RHD on the opposite
chromosome. Similar depression of D can
be seen with other RHD haplotypes accompanied by RHCe in trans position. Many of
the weak D phenotypes due to position effect that were reported in the early literature appear as normal D.

Partial D
The concept that the D antigen consists of
multiple constituents arose from observations that some people with D+ red cells
produced alloanti-D that was nonreactive
with their own cells. Most D+ persons who
produce alloanti-D have red cells that react strongly when tested with anti-D. But
some, especially those of the DVI pheno-

Chapter 14: The Rh System

type, give weaker reactions than normal
D+ red cells or react only in the AHG test.
Red cells lacking components of the D antigen have been referred to in the past as
“D mosaic” or “D variant.” Current terminology more appropriately describes these
red cells as “partial D.”
Categorization of partial D phenotypes
was performed by cross-testing red cells
with alloanti-D produced by D+ persons.
The four categories initially described by
Wiener have been expanded considerably
over the years. Tests of many monoclonal
anti-D reagents with red cells of various D
categories suggest that the D antigen comprises numerous epitopes. Partial D phenotypes can be defined in terms of their D
33
epitopes. Tippett et al established at least
10 epitopes but point out that the D antigen is not large enough to accommodate
more than eight distinct epitopes and there
must be considerable overlap between them.
A current model describes 30 epitopes,34
thus demonstrating the dynamic nature of
the D-epitope model as it is revised due to
variation in reagents and techniques. Dogma
regarding the existence of many discrete
epitopes is giving way to a model that is more
topographic in nature, with the fit of antibody to antigen described as a “footprint.”35
Molecular studies have elucidated the
genetic mechanisms behind many of the
partial D phenotypes and have shown that
the phenotypes arise as the result of exchange between the RHCE gene and the RHD
9,18
gene or from single-point mutations.

Significance of Weak/Partial D in Blood
Donors
AABB Standards for Blood Banks and
36(p32)
Transfusion Services
requires donor
blood specimens to be tested for weak expression of D and to be labeled as D+ if
the test is positive. Transfusion of blood
with weak expression of the D antigen to

323

D– recipients is not recommended due to
the fact that some weak or partial D red
cells could elicit an immune response to
D.37 Weak forms of the D antigen, however, seem to be less immunogenic than
normal D+ blood; transfusion of a total of
68 units of blood with weak D to 45 D– recipients failed to stimulate production of
a single example of anti-D.38
Hemolytic transfusion reactions and HDFN
due to weak D red cells were reported in
the early literature, but it is probable that,
with currently available reagents, the responsible cells would have been considered
D+.39

Significance of Weak/Partial D in
Recipients
The transfusion recipient whose red cells
test as weak D is sometimes a topic of debate. Most of these patients can almost always receive D+ blood without risk of immunization, but if the weak D expression
reflects the absence of one or more D epitopes, the possibility exists that transfusion of D+ blood could elicit the production of alloanti-D. This is especially true in
persons of the DVI phenotype. The same
possibility exists, however, for persons
whose partial D red cells react strongly
with anti-D reagents. AABB Standards for
Blood Banks and Transfusion Services36(p48)
only requires recipients’ specimens to be
tested with anti-D by direct agglutination.
The test for weak D is not required. Currently available, licensed anti-D reagents
are sufficiently potent that most patients
with weak D are found to be D+. The few
patients classified as D–, whose D+ status
is only detectable by an IAT, can receive
D – b l o o d w i t h o u t p r o b l e m s. So m e
serologists consider this practice wasteful
of D– blood and prefer to test potential recipients for weak D, then issue D+ blood
when indicated.

324

AABB Technical Manual

If D+ blood is given to recipients of the
weak D phenotype, it is important to safeguard against careless or incorrect interpretation of tests. D– recipients erroneously
classified as D+, possibly due to a positive direct antiglobulin test (DAT) causing a falsepositive test for weak D, run the risk of immunization to D if given D+ blood. Individuals whose weakly expressed D antigen is detectable only by an IAT will be classified as
D– recipients if an IAT is not performed.
However, if they donate blood subsequently,
they will be classified as D+ at the time of
blood donation. Personnel in blood centers
and transfusion services should be prepared
to answer questions from puzzled donors or
their physicians. This can present special
problems in autologous donations, when the
D– patient’s own blood is labeled as D+. In
this case, confirmation of the patient’s D status by the IAT resolves the apparent discrepancy between recipient and donor types.

Other Rh Antigens
Numbers up to 56 have been assigned to
Rh red cell antigens (see Table 14-1); some
of the numbers are now obsolete because
antigens have been rescinded or reassigned. Of the currently included 49, most
beyond D, C, c, Cw, E, and e and their corresponding antibodies are encountered
much less frequently in routine blood
transfusion therapy.

Cis Product Antigens
The membrane components that exhibit
Rh activity have numerous possible antigenic subdivisions. Each gene or gene
complex determines a series of interrelated surface structures, of which some
portions are more likely than others to
elicit an immune response. The polypeptides determined by the genes in the
haplotype DCe express determinants ad-

ditional to those defined as D, C, and e.
These include Ce (rhi), a cis product that
almost always accompanies C and e when
they are encoded by the same haplotype.
The Ce antigen is absent from red cells on
which the C and e were encoded by different haplotypes, for example, in a person
of the genotype DCE/ce. Similar cis product antigens exist for c and e determined
by the same haplotype (the antigen called
ce or f ), for c and E (cE), and for C and E
(CE).
Although antibodies directed at cis product antigens are encountered infrequently,
it would not be correct to consider them
rare. Such antibodies may be present but
unnoticed in serum containing antibodies
of the more obvious Rh specificities; only
adsorption with red cells of selected phenotypes would demonstrate their presence.
Anti-f (ce) may be present, for example, as a
component of some anti-c and anti-e sera,
but its presence would have little practical
significance. The additional antibody
should not confuse the reaction patterns
given by anti-c and anti-e because all red
cells that react with anti-f will express both
c and e. A person with the genotype
DCe/DcE who makes anti-f may receive c–
or e– red cells because cells of either phenotype would also be f–.
Anti-Ce is frequently the true specificity
of the apparent anti-C that a DcE/DcE person produces after immunization with C+
blood. This knowledge can be helpful in establishing an individual’s Rh genotype. If
anti-Ce is the predominant specificity in a
reagent anti-C, the individual whose weak
C antigen resulted from a DCE haplotype
may be mistyped unless test methods and
control red cells are chosen carefully.40

The G Antigen and Cross-Reactions
The G antigen results from serine at position 103 of the Rh polypeptides and is en-

Chapter 14: The Rh System

coded by either RHD or RHCE. As a result, the G antigen is almost invariably
present on red cells possessing either C or
D. Antibodies against G appear superficially to be anti-C+D, but the anti-G activity cannot be separated into anti-C and
anti-D. The fact that G appears to exist as
an entity common to C and D explains the
fact that D– persons immunized by C–D+
red cells sometimes appear to have made
anti-C as well as anti-D, and why D– persons who are exposed to C+D– red cells
develop antibodies appearing to contain
an anti-D component. Differentiation of
anti-D, -C, and -G is not necessary in the
pretransfusion setting because virtually
all D–C– red cells are G–. In obstetric patients, however, some serologists believe
it is essential to distinguish the antibody
specificities to determine the need for Rh
42
immune globulin prophylaxis. Differential adsorption and elution studies to distinguish anti-D, -C, and -G are outlined by
43
Issitt and Anstee.
Rare red cells have been described that
G
possess G but lack D and C. The r phenotype is found mostly in Blacks; generally,
the G antigen is weakly expressed and is associated with the presence of the VS antiG
gen. The r phenotype has been described
G
in Whites but is not the same as r in
Blacks. Red cells also exist that express partial D but lack G entirely, for example, persons of the DIIIb phenotype.39

Variant Antigens
Although red cells from most people give
straightforward reactions with reagent
anti-D, anti-C, anti-E, anti-c, and anti-e
sera, some cells give atypical reactions
and other seemingly normal red cells
stimulate the production of antibodies
that do not react with red cells of common Rh phenotypes. It has been convenient to consider C and c, and E and e as

325

antithetical antigens at specific surface
sites. Antigens that behave as if they have
an antithetical relationship to C/c or E/e
have been found, mainly in Whites. The
w
most common is C , but the relationship
is phenotypic only because Cw and Cx are
antithetical to the high-incidence antigen,
MAR. Variant forms of the e antigen have
been identified, for example, hrS or hrB antigens (Rh19 and Rh31, respectively). PerS
B
sons who are e+ and hr – and/or hr – are
found more frequently in Black populations.9 The absence of hrB is associated in
most cases with the presence of the VS
antigen.44

Rhnull Syndrome and Other
Deletion Types
Rhnull
The literature reports at least 43 persons
in 14 families whose red cells appear to
have no Rh antigens; others are known
but have not been reported. The phenotype, described as Rhnull, is produced by at
least two different genetic mechanisms.
In the more common regulator type of
Rhnull, mutations occur in the RHAG gene
that result in the complete absence of the
core Rh complex (Rh polypeptides and
RhAG) that is necessary for the expression
of Rh antigens.18 Such persons transmit
normal RHD and RHCE genes to their offspring.
The other form of Rhnull, the amorph
type, has a normal RHAG gene; however,
there is a mutation in each RHCE gene together with the common deletion of RHD
(as in D– individuals).18 The amorph type of
Rhnull is considerably rarer than the regulator type. Parents and offspring of this type
of Rhnull are obligate heterozygotes for the
amorph.

326

AABB Technical Manual

Red Cell Abnormalities
Whatever the genetic origin, red cells lacking Rh/RhAG proteins have membrane
abnormalities and a compensated hemolytic anemia. The severity of hemolysis and
resulting anemia varies among affected
persons, but stomatocytosis, shortened
red cell survival, absence of LW and Fy5
antigens, and variably altered activity of S,
s, and U have been consistent features.

Serologic Observations
A few Rhnull individuals were recognized
because their sera contained Rh antibodies. Some came to light, however, when
routine Rh phenotyping of their red cells
revealed the absence of any Rh antigens.
In three cases, the discovery resulted from
deliberate testing for Rh antigens in patients with morphologically abnormal red
cells and hemolytic anemia. Immunized
Rhnull people have produced antibodies
varying in specificity from apparently
straightforward anti-e or anti-C to several
examples that reacted with all red cells
tested except those from other Rhnull people. This antibody, considered to be “antitotal Rh,” has been given the numerical
designation anti-Rh29.

Rhmod
The Rhmod phenotype represents less complete suppression of Rh antigen expression. Unlike Rhnull red cells, those classified as Rhmod do not completely lack Rh
and LW antigens. Rhmod red cells show
much reduced and sometimes varied activity, depending on the Rh system genes
the individual possesses and on the potency and specificity of the antisera used
in testing. Sometimes, the Rh antigens
have sufficiently weakened expression
that only adsorption-elution techniques

will demonstrate their presence. As in
Rhnull, hemolytic anemia is a feature of the
Rhmod condition. It may be appropriate to
think of the two abnormalities as being
essentially similar, differing only in degree. As in the regulator type of Rhnull, a
mutation in RHAG was shown to result in
18
the Rhmod phenotype.

Deleted Phenotypes
Rare RH haplotypes encode D antigen but
fail to encode some or all of the CE antigens. Some examples of deleted phenotypes
include D– – , D••, DCw–, and Dc–. These
rare phenotypes have been shown to arise
from replacement of large portions of RHCE
with RHD.18 Red cells that lack C/c and/or
E/e antigens may show exceptionally
strong D activity, an observation by which
such red cells have sometimes been recognized during routine testing with anti-D.
The D– – phenotype may be identified in
the course of studies to investigate an unexpected antibody. Such persons may have
alloantibody of complex specificity because the person’s red cells lack all the
epitopes expressed on the RhCE polypeptide. Antibody with Rh17 (Hro) specificity is often made by persons of this rare
phenotype, although some sera have been
reported to contain apparently separable
specificities, such as anti-e.
The D•• phenotype is similar to D– –, except that the D antigen is not elevated to
the same degree. D•• red cells may be agglutinated weakly by some examples of sera
from immunized Rh-deletion persons if the
serum sample contains anti-Rh47 in addi39
tion to anti-Rh17. Rh47 is a high-incidence antigen encoded by the RHCE gene
and is absent from other deleted phenotypes (eg, D– –, DCw– , Dc–). Another distinguishing characteristic of D•• red cells is
that they possess the low-incidence antigen
Rh37 (Evans).

Chapter 14: The Rh System

327

Anti-D in D+ Individuals

Rh Antibodies
Most Rh antibodies result from exposure
to human red cells through pregnancy or
transfusion. Occasionally, Rh antibodies
(eg, anti-E, anti-Cw) are naturally-occurring. D is the most potent immunogen,
followed by c and E. Although a few examples of Rh antibodies behave as saline agglutinins, most react best in high-protein,
antiglobulin, or enzyme test systems. Even
sera containing potent saline-reactive
anti-D are usually reactive at higher dilutions in antiglobulin testing. Some workers find enzyme techniques especially useful for detecting weak or developing Rh
antibodies.
Antibody usually persists for many years.
If serum antibody levels fall below detectable thresholds, subsequent exposure to the
antigen characteristically produces a rapid
secondary immune response. With exceedingly rare exceptions, Rh antibodies do not
bind complement, at least to the extent recognizable by techniques currently used. As
a result, primarily extravascular hemolysis,
instead of intravascular hemolysis, occurs
in transfusion reactions involving Rh antibodies.

Dosage Effect
Anti-D seldom shows any difference in reactivity between red cells from individuals homozygous or heterozygous for RHD,
but D expression seems to vary somewhat
with the accompanying alleles of the genotype. For example, red cells from a
DcE/DcE individual carry more D antigen
sites than red cells from a DCe/DCe person and may show higher titration scores
with anti-D. Dosage effects can sometimes be demonstrated with some antibodies directed at the E, c, and e antigens
and, occasionally, at the C antigen.

Alloanti-D may be produced in D+ individuals with partial D phenotype (see Partial D), although not all persons who are
D+ and produce what appears to be anti-D
should be assumed to have epitope-deficient red cells. Weakly reactive anti-LWab
a
or anti-LW may react with D+ cells but
not with D– cells. A D+ person whose antibody is a weakly reactive anti-LWa may
be indistinguishable on initial serologic
testing from an individual with a partial D
antigen who has made anti-D to missing
epitopes. (See the section on the LW system in Chapter 15.) Anti-LW can be differentiated from anti-D by testing the antibody with 0.02M DTT-treated red cells
(Method 3.10); the LW antigen is destroyed by sulfhydryl reagents, whereas D
is unaffected.

Concomitant Antibodies
Some Rh antibodies tend to occur in concert. For example, the DCe/DCe person
manifesting immune anti-E almost certainly has been exposed to c as well as E.
Anti-c may be present in addition to
anti-E, although substantially weaker and
possibly undetectable at the time the
anti-E is found, and transfusion of seemingly compatible E–c+ blood may elicit an
immediate or delayed reaction. Generally,
it is not a sound practice to select donor
blood that is negative for antigens absent
from the recipient’s red cells when antibody has not been detected, but some
practitioners feel that the DCe/DCe recipient with anti-E is a case that merits avoidance of c+ blood as well.45 Anti-E less consistently accompanies anti-c because the
patient can easily have been exposed to c
without being exposed to E. There is little
clinical value in pursuing anti-E in serum
known to contain anti-c because the vast

328

AABB Technical Manual

majority of c– donor blood will be negative for the E antigen.

Rh Typing
Routine Rh typing for donors and patients
involves only the D antigen. Tests for the
other Rh antigens are performed when
identifying unexpected Rh antibodies, obtaining compatible blood for a patient
with an Rh antibody, investigating parentage or other family studies, selecting a
panel of phenotyped cells for antibody
identification, or evaluating whether a
person is likely to be homozygous or heterozygous for RHD.
In finding compatible blood for a recipient with a comparatively weak Rh antibody,
tests with potent blood typing reagents
more reliably confirm the absence of antigen than mere demonstration of a compatible crossmatch. Determination of the patient’s Rh phenotype may help confirm the
antibody specificity and indicate which
other Rh antibodies could also be present.

Routine Testing for D
Until recently, high-protein anti-D reagents of human polyclonal origin that
were suitable for slide, tube, or microplate
tests were used for most routine testing.
More recently, monoclonal anti-D reagents have become widely available.
Tests may employ red cells suspended in
saline, serum, or plasma, but test conditions should be confirmed by reading the
manufacturer’s directions before use. Procedures for microplate tests are similar
to those for tube tests, but very light suspensions of red cells are used.
Slide tests produce optimal results only
when a high concentration of red cells and
protein are combined at a temperature of
37 C. A disadvantage of the slide test is
evaporation of the reaction mixture, which

can cause the red cells to aggregate and be
misinterpreted as agglutination. There are
also greater biohazardous risks associated
with increased potential for spillage of the
specimen during manipulation. Representative procedures for tube, slide, and microplate tests are given in Methods 2.6, 2.7,
and 2.8.
Techniques to demonstrate weak D are
required by AABB Standards only for donor
blood or for testing blood from neonates
born to Rh-negative women to determine
Rh immune globulin candidacy. 36(pp32,48)
When there is an indication to test for weak
D, an IAT should be performed (Method
2.9). A reliable test for weak D expression
cannot be performed on a slide.

High-Protein Reagents
Some anti-D reagents designated for use
in slide, rapid tube, or microplate tests
contain high concentrations of protein
(20-24%) and other macromolecular additives. Such reagents are nearly always prepared from pools of human sera and give
rapid reliable results when used in accordance with manufacturers’ directions.
High-protein levels and macromolecular
additives may cause false-positive reactions. A false-positive result could cause a
D– patient to receive D+ blood and become immunized. An appropriate control
tested according to the manufacturer’s directions must be performed.

Control for High-Protein Reagents
Manufacturers offer their individual diluent formulations for use as control reagents. The nature and concentration of
additives differ significantly among reagents from different manufacturers and
may not produce the same pattern of
false-positive reactions. If red cells exhibit
aggregation in the control test, the results
of the anti-D test cannot be considered

Chapter 14: The Rh System

valid. In most cases the presence or absence of D can be determined with other
reagents, as detailed later in this chapter.

Misleading Results with High-Protein
Reagents
False Positives. The following circumstances can produce false-positive red cell
typing results.
1.
Cellular aggregation resulting from
immunoglobulin coating of the patient’s red cells or serum factors that
induce rouleaux will give positive results in both the test and the control
tubes. Serum factors can be eliminated by thoroughly washing the red
cells (with warm saline if cold agglutinins are present or suspected) and
retesting. If the cells in the control
test remain unagglutinated and the
anti-D test gives a positive result, the
red cells are D+. If agglutination still
occurs in the control tube, the most
likely explanation is immunoglobulin coating of the red cells, which
should then be tested with low-protein reagents.
2.
Red cell aggregation, simulating agglutination, may occur if red cells and
anti-D are incubated too long and
excessive evaporation occurs during
the slide test. It is important to follow the manufacturer’s recommendations to interpret the test within
the recommended period.
False Negatives. The following circumstances can produce false-negative red
cell typing results.
1.
Too heavy a red cell suspension in
the tube test or too weak a suspension in the slide test may weaken agglutination.
2.
Saline-suspended red cells must not
be used for slide testing.

329

Red cells possessing weakly expressed D antigen may not react well
within the 2-minute limit of the slide
test or upon immediate centrifugation in the tube test.

Low-Protein Reagents
The low-protein Rh reagents in current
use are formulated predominantly with
monoclonal antibodies. Immunoglobulin-coated red cells can usually be successfully typed with low-protein Rh reagents that contain saline-agglutinating
antibodies.

Monoclonal Source Anti-D
Monoclonal anti-D reagents are made
predominantly from human IgM antibodies, which require no potentiators and agglutinate most D+ red cells from adults
and infants in a saline system. Monoclonal
anti-D reagents usually promote reactions
stronger than those with polyclonal IgG
reagents, but they may fail to agglutinate
red cells of some partial D categories.
Adding small amounts of IgG anti-D to
the monoclonal IgM antibodies provides
a reagent that will react with weak or partial D red cells in antiglobulin tests. Certain rare D+ red cells (DHAR, DVa, DVc,
Rh43+) may react at immediate spin with
some of these blended reagents, but, with
other reagents, these same cells may be
nonreactive in an IAT or reactive only in
an IAT.
Licensed monoclonal/polyclonal or
monoclonal/monoclonal blends can be
used by all routine typing methods and are
as satisfactory as high-protein reagents in
an IAT for weak D. False-negative findings
can result, however, if tests using monoclonal reagents are incubated in excess of a
manufacturer’s product directions. These reagents, prepared in a low-protein medium,
can be used to test red cells with a positive

330

AABB Technical Manual

DAT, provided those tests are not subjected
to an IAT.

Control for Low-Protein Reagents
Most monoclonal blended reagents have
a total protein concentration approximating that of human serum. False-positive
reactions due to spontaneous aggregation
of immunoglobulin-coated red cells occur
no more often with this kind of reagent
than with other saline-reactive reagents.
False-positive reactions may occur in any
saline-reactive test system if the serum
contains cold autoagglutinins or a protein
imbalance causing rouleaux and the red
cells are tested unwashed. It is seldom
necessary to perform a separate control
test. Absence of spontaneous aggregation
can usually be demonstrated by observing absence of agglutination by anti-A
and/or anti-B in the cell tests for ABO. For
red cell specimens that show agglutination in all tubes (ie, give the reactions of
group AB, D+), a control should be performed as described by the reagent manufacturer; this is not required when donors’ cells are tested. In most cases, a
suitable control is a suspension of the patient’s red cells with autologous serum or
with 6% to 8% bovine albumin, although
exceptions have been noted.46 If the test is
one of several performed concurrently
and in a similar manner, any negative result serves as an adequate control. For example, a separate control tube would be
required only for a red cell specimen that
gives positive reactions with all the Rh
reagents (ie, is typed as D+C+E+c+e+).

Testing for D in Hemolytic Disease of the
Fetus and Newborn
Because red cells from an infant suffering
from HDFN are coated with immunoglobulin, a low protein reagent is usually necessary for Rh testing. Occasionally, the in-

fant’s red cells may be so heavily coated
with antibody that all antigen sites are occupied, leaving none available to react
with a low protein antibody of appropriate specificity. This “blocking” phenomenon should be suspected if the infant’s
cells have a strongly positive DAT and are
not agglutinated by a low protein reagent
of the same specificity as the maternal antibody.
Anti-D is the specificity responsible for
nearly all cases of blocking by maternal antibody. It is usually possible to obtain correct typing results with a low protein anti-D
after 45 C elution of the maternal antibody
from the cord blood red cells. (See Method
2.12.) Elution liberates enough antigen sites
to permit red cell typing, but it must be performed cautiously because overexposure to
heat may denature or destroy Rh antigens.

Tests for Antigens Other than D
Reagents are readily available to test for
the other principal Rh antigens: C, E, c,
and e. These are formulated as either lowprotein (usually monoclonal or monoclonal/polyclonal blends) or high-protein
reagents. High-protein reagents of any
specificity have the same problems with
false-positive results as high-protein anti-D
and require a comparable control test
performed concurrently and under the
same conditions. Observation of a negative result in the control test for anti-D
may not properly control the tests for
other Rh antigens because results with
anti-D are usually obtained after immediate centrifugation; tests for the other Rh
antigens are generally incubated at 37 C
before centrifugation.
Rh reagents may give weak or negative
reactions with red cells possessing variant
antigens. This is especially likely to happen
if anti-e is used to test the red cells from
Blacks, among whom variants of e are rela-

Chapter 14: The Rh System

tively common. It is impossible to obtain
anti-e reagents that react strongly and consistently with the various qualitative and
quantitative variants of e. Variable reactivity
with anti-C reagents may occur if the DCE
or CE haplotypes are responsible for the expression of C on red cells. Variant E and c
antigens have been reported but are considerably less common.
Whatever reagents are used, the manufacturer’s directions must be carefully followed. The IAT must not be used unless the
manufacturer’s instructions state explicitly
that the reagent is suitable for this use. The
pools of human source sera (nonmonoclonal) used to prepare reagents for the
other Rh antigens may contain antiglobulin-reactive, “contaminating” specificities. Positive and negative controls
should be tested; red cells selected for the
positive control should have a single dose
of the antigen or be known to show weak
reactivity with the reagent.

Additional Considerations in Rh Testing
The following limitations are common to
all Rh typing procedures, including those
performed with high-protein reagents.

False-Positive Reactions
The following circumstances can produce
false-positive red cell typing results.
1.
The wrong reagent was inadvertently
used.
2.
An unsuspected antibody of another
specificity was present in the human
source reagent. Antibodies for antigens having an incidence of less than
1% in the population may occasionally be present and cause false-positive reactions, even when the manufacturer’s directions are followed.
For crucial determinations, many
workers routinely perform replicate
tests using reagents from different

331

sources. Replicate testing is not an
absolute safeguard, however, because
reagents from different manufacturers may not be derived from different
sources.
Polyagglutinable red cells may be agglutinated by any reagent containing
human serum. Although antibodies
that agglutinate these surface-altered
red cells are present in most adult
human sera, polyagglutinins in reagents very rarely cause problems.
Aging, dilution, and various steps in
the manufacturing process tend to
eliminate these predominantly IgM
antibodies.
Autoagglutinins and abnormal proteins in the patient’s serum may cause
false-positive reactions when unwashed red cells are tested.
Reagent vials may become contaminated with bacteria, with foreign
substances, or with reagent from another vial. This can be prevented by
the use of careful technique and the
periodic inspection of the vials’ contents. However, bacterial contamination may not cause recognizable turbidity because the refractive index of
bacteria is similar to that of highprotein reagents.

False-Negative Reactions
The following circumstances can produce
false-negative red cell typing results.
1.
The wrong reagent was inadvertently
used.
2.
The reagent was not added to the tube.
It is good practice to add serum to
all the tubes before adding the red
cells and any enhancement medium.
3.
A specific reagent failed to react with
a variant form of the antigen.
4.
A reagent that contains antibody directed predominantly at a cis-prod-

332

5.
6.

AABB Technical Manual

uct Rh antigen failed to give a reliably detectable reaction with red
cells carrying the individual antigens
as separate gene products. This occurs most often with anti-C sera.
The manufacturer’s directions were
not followed.
The red cell button was shaken so
roughly during resuspension that
small agglutinates were dispersed.
Contamination, improper storage,
or outdating cause antibody activity
to deteriorate. Chemically modified
IgG antibody appears to be particularly susceptible to destruction by
proteolytic enzymes produced by certain bacteria.

10.

11.

12.

13.
14.
15.
16.

17.

References
1.

Race RR. The Rh genotypes and Fisher’s theory. Blood 1948; special issue 2:27-42.
Levine P, Stetson RE. An unusual case of
intragroup agglutination. JAMA 1939;113:
126-7.
Landsteiner K, Wiener AS. An agglutinable
factor in human blood recognized by immune sera for rhesus blood. Proc Soc Exp
Biol NY 1940;43:223.
Levine P, Katzin EM. Isoimmunization in
pregnancy and the variety of isoagglutinins
observed. Proc Soc Exp Biol NY 1940;43:
343-6.
Wiener AS, Peters HR. Hemolytic reactions
following transfusions of blood of the homologous group, with three cases in which the
same agglutinogen was responsible. Ann Intern Prn Med 1940;13:2306-22.
Frohn C, Dümbgen L, Brand J-M, et al. Probability of anti-D development in D– patients
receiving D+ RBCs. Transfusion 2003;43:
893-8.
Mollison PL, Engelfriet CP, Contreras M.
Blood transfusion in clinical medicine. 10th
ed. Oxford: Blackwell Scientific Publishers,
1997.
Daniels GL, Fletcher A, Garratty G, et al.
Blood group terminology 2004: From the International Society of Blood Transfusion
committee on terminology for red cell surface antigens. Vox Sang 2004;87:304-16.

18.

19.

20.

21.

22.

23.
24.

25.

Reid ME, Lomas-Francis C. The blood group
antigen factsbook. 2nd ed. London: Academic
Press, 2004.
Gemke RJBJ, Kanhai HHH, Overbeeke MAM,
et al. ABO and Rhesus phenotyping of fetal
erythrocytes in the first trimester of pregnancy. Br J Haematol 1986;64:689-97.
Dunstan RA, Simpson MB, Rosse WF. Erythrocyte antigens on human platelets. Absence of
Rh, Duffy, Kell, Kidd, and Lutheran antigens.
Transfusion 1984;24:243-6.
Dunstan RA. Status of major red cell blood
group antigens on neutrophils, lymphocytes
and monocytes. Br J Haematol 1986;62:301-9.
Wiener AS. Genetic theory of the Rh blood
types. Proc Soc Exp Biol Med 1943;54:316-19.
Fisher RA, Race RR. Rh gene frequencies in
Britain. Nature 1946;157:48-9.
Tippett P. A speculative model for the Rh
blood groups. Ann Hum Genet 1986;50:241-7.
Colin Y, Chérif-Zahar B, Le Van Kim C, et al.
Genetic basis of RhD-positive and RhD-negative blood group polymorphisms as determined by southern analysis. Blood 1991;78:
2747-52.
Arce MA, Thomson ES, Wagner S, et al. Molecular cloning of RhD cDNA derived from a
gene present in RhD-positive, but not RhDnegative individuals. Blood 1993;82:651-5.
Huang CH, Liu PZ, Cheng JG. Molecular biology and genetics of the Rh blood group system. Semin Hematol 2000;37:150-65.
Singleton BK, Green CA, Avent ND, et al. The
presence of an RHD pseudogene containing a
37 base pair duplication and a nonsense mutation in Africans with the Rh D-negative
blood group phenotype. Blood 2000;95:12-8.
Mouro I, Colin Y, Chérif-Zahar B, et al. Molecular genetic basis of the human Rhesus blood
group system. Nat Genet 1993;5:62-5.
Smythe JS, Avent ND, Judson PA, et al. Expression of RHD and RHCE gene products using
retroviral transduction of K562 cells establishes the molecular basis of Rh blood group
antigens. Blood 1996;87:2968-73.
Ridgwell K, Spurr NK, Laguda B, et al. Isolation of cDNA clones for a 50 kDa glycoprotein
of the human erythrocyte membrane associated with Rh (rhesus) blood-group antigen
expression. Biochem J 1992;287:223-8.
Tippett P. Regulator genes affecting red cell
antigens. Transfus Med Rev 1990;4:56-68.
Hemker MB, Goedel C, van Zwieten R, et al.
The Rh complex exports ammonium from
human red blood cells. Br J Haematol 2003;
122:333-40.
Westhoff CM, Seigel D, Burd C, Foskett JK.
Mechanism of genetic complementation of
ammonium transport in yeast by human

Chapter 14: The Rh System

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.
40.

41.

erythrocyte Rh-associated glycoprotein. J
Biol Chem 2003;279:17443-8.
Rosenfield RE, Allen FH Jr, Swisher SN,
Kochwa S. A review of Rh serology and presentation of a new terminology. Transfusion
1962;2:287-312.
Wagner FF, Flegel WA. RHD gene deletion occurred in the Rhesus box. Blood 2000;95:
3662-8.
Chiu RW, Murphy MF, Fidler C, et al. Determination of RhD zygosity: Comparison of a
double amplification refractory mutation
system approach and a multiplex real-time
quantitative PCR approach. Clin Chem 2003;
47:667-72.
Lawler SD, Race RR. Quantitative aspects of
Rh antigens. Proceedings of the International
Society of Hematology 1950:168-70.
Lo YMD, Hjelm NM, Fidler C, et al. Prenatal
diagnosis of fetal RhD status by molecular
analysis of maternal plasma. N Engl J Med
1998;339:1734-8.
Wagner F, Gassner C, Müller T, et al. Molecular basis of weak D phenotypes. Blood 1999;
93:385-93.
Wagner FF, Frohmajer A, Ladewig B, et al.
Weak D alleles express distinct phenotypes.
Blood 2000;95:2699-708.
Tippett P, Lomas-Francis C, Wallace M. The
Rh antigen D: Partial D antigens and associated low incidence antigens. Vox Sang 1996;
70:123-31.
Scott ML. Section 1A: Rh serology. Coordinator’s report. 4th International Workshop on
Monoclonal Antibodies Against Human Red
Cell Surface Antigens, Paris. Transfus Clin Biol
2002;9:23-9.
Chang TY, Siegel DL. Genetic and immunological properties of phage-displayed human
anti-Rh(D) antibodies: Implications for Rh(D)
epitope topology. Blood 1998;91:3066-78.
Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.
Flegel WA, Khull SR, Wagner FF. Primary
anti-D immunization by weak D type 2 RBCs.
Transfusion 2000;40:428-33.
Schmidt PJ, Morrison EG, Shohl J. The antigenicity of the Rho Du blood factor. Blood 1962;
20:196-202.
Daniels G. Human blood groups. 2nd ed. Oxford: Blackwell Scientific Publications, 2002.
Van Loghem JJ. Production of Rh agglutinins
anti-C and anti-E by artificial immunization
of volunteer donors. Br Med J 1947;ii:958-9.
Faas BHW, Beckers EAM, Simsek S, et al. Involvement of Ser103 of the Rh polypeptides
in G epitope formation. Transfusion 1996;36:
506-11.

42.

43.

44.

45.

46.

333

Shirey RS, Mirabella DC, Lumadue JA. Differentiation of anti-D, -C, and -G: Clinical relevance in alloimmunized pregnancies. Transfusion 1997;37:493-6.
Issitt PD, Anstee DJ. Applied blood group serology. 4th ed. Durham, NC: Montgomery
Scientific Press, 1998:350-3.
Reid ME, Storry JR, Issitt PD, et al. Rh haplotypes that make e but not hrB usually make
VS. Vox Sang 1997;72:41-4.
Shirey RS, Edwards RE, Ness PM. The risk of
alloimmunization to c (Rh4) in R1R1 patients
who present with anti-E. Transfusion 1994;34:
756-8.
Rodberg K, Tsuneta R, Garratty G. Discrepant
Rh phenotyping results when testing IgGsensitized rbcs with monoclonal Rh reagents
(abstract). Transfusion 1995;35(Suppl):67S.

Suggested Reading
Agre P, Cartron JP. Molecular biology of the Rh antigens. Blood 1991;78:551-3.
Avent ND, Reid ME. The Rh blood group system: A
review. Blood 2000;95:375-87.
Cartron JP. Defining the Rh blood group antigens.
Blood Rev 1994;8:199-212.
Daniels G. Human blood groups. 2nd ed. Oxford:
Blackwell Scientific Publications, 2002.
Issitt PD. An invited review: The Rh antigen e, its
variants, and some closely related serological observations. Immunohematology 1991;7:29-36.
Issitt PD. The Rh blood group. In: Garratty G, ed.
Immunology of transfusion medicine. New York:
Marcel Dekker, 1994:111-47.
Issitt PD. The Rh blood group system 1988: Eight
new antigens in nine years and some observations
on the biochemistry and genetics of the system.
Transfus Med Rev 1989;3:1-12.
Issitt PD, Anstee DJ. Applied blood group serology.
4th ed. Durham, NC: Montgomery Scientific Press,
1998.
Lomas-Francis C, Reid ME. The Rh blood group
system: The first 60 years of discovery. Immunohematology 2000;16:7-17.
Race RR, Sanger R. Blood groups in man. 6th ed.
Oxford: Blackwell Scientific Publications, 1968.
Reid ME, Ellisor SS, Frank BA. Another potential
source of error in Rh-Hr typing. Transfusion 1975;
15:485-8.

334

AABB Technical Manual

Reid ME, Lomas-Francis C. The blood group antigen factsbook. 2nd ed. London: Academic Press,
2004.

Vengelen-Tyler V, Pierce S, eds. Blood group systems: Rh. Arlington, VA: American Association of
Blood Banks, 1987.

Sonneborn H-H, Voak D, eds. A review of 50 years
of the Rh blood group system. Biotest Bulletin 1997;
5(4):389-528.

White WD, Issitt CH, McGuire D. Evaluation of the
use of albumin controls in Rh typing. Transfusion
1974;14:67-71.

Chapter 15: Other Blood Groups

Chapter 15

Other Blood Groups
15

HERE ARE MANY antigens on red
cells in addition to the ones mentioned in previous chapters. These
antigens are grouped into blood group
systems, collections, and a series of independent antigens, composed mostly of
antigens of low or high incidence. A blood
group system is a group of one or more
antigens governed by a single gene locus
or by a complex of two or more closely
linked homologous genes that have been
shown to be phenotypically and genetically related to each other and genetically
distinct from other blood group systems.
A collection is a group of antigens shown
to have a phenotypic, biochemical, or genetic relationship to each other; however,
there is insufficient information or data that
shows them to be a distinct blood group
system genetically independent from other
blood group systems. Table 10-1 lists the
blood group systems, as defined by the
International Society of Blood Transfusion

(ISBT ) working party on blood group
1-3
terminology, and their gene location.
Additional information on ISBT terminology for all the antigens mentioned in this
chapter can be found in Appendix 6. Table
15-1 shows the serologic behavior and
characteristics of the major blood group
antibodies derived from human sources.
The major systems will be discussed first
in the chapter, followed by the other blood
group systems, collections, and independent high-incidence and low-incidence antigens. In each grouping, the order will
reflect the ISBT number order.

Distribution of Antigens
Antigens present in almost all persons are
known as high-incidence antigens, whereas antigens found in very few persons are
termed very low-incidence antigens. The
frequency of these high- or low-incidence
antigens may also differ by ethnic group.
335

336

AABB Technical Manual

Table 15-1. Serologic Behavior of the Principal Antibodies of Different Blood Group
Systems
Saline
Antibody

In-Vitro
Hemolysis 4 C

Anti-M
Anti-N
Anti-S
Anti-s
Anti-U
Anti-Lua
Anti-Lub
Anti-K
Anti-k
Anti-Kpa
Anti-Kpb
Anti-Jsa
Anti-Jsb
Anti-Lea
Anti-Leb
Anti-Fya
Anti-Fyb
Anti-Jka
Anti-Jkb
Anti-Xga
Anti-Dia
Anti-Dib
Anti-Yta
Anti-Ytb
Anti-Doa
Anti-Dob
Anti-Coa
Anti-Cob
Anti-Sc1
Anti-Sc2

0
0
0
0
0
0
0
0
0
0
0
0
0
Some
Some
0
0
Some
Some
0
0
0
0
0
0
0
0
0
0
0

Most
Most
Few
No
No
Some
Few

Most
Most

Albumin

Papain/Ficin

22 C

37 C

AHG

37 C

AHG

Some
Few
Some
Few
Occ.
Most
Few
Few
Few
Some
Few
Few
0
Most
Most
Rare
Rare
Few
Few
Few
Some

Few
Occ.
Some
Few
Some
Few
Few
Some
Few
Some
Few
Few
0
Some
Some
Rare
Rare
Few
Few
Few
Some

0
0
0
0
See text
See text
Most Most
Few
Few
Few
Few
Some Most
Some Most
Some Most
Some Most
Few
Most
Few
Most
Some Most
Some Most
0
0
0
0
Some Most
Some Most
0
0
Some Some
Some Some
0
Some

0
0

Some

Few
Occ.
Most
Most
Most
Few
Most
Most
Most
Most
Most
Most
Most
Some
Some
Most
Most
Most
Most
Most
Most
Most
Most
All
Some
All
Some
Some
All
Most

Some
All
Some
Some

Most
All
Most
Most

Most

Associated
with
HDFN HTR
Few
Few
Rare No
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Mild Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Rare
No
No
Yes
Yes
Mild Yes
Mild Yes
Mild Yes
No report
Yes
Yes
Yes
Yes
No
Yes
No report
No
Yes
No
Yes
Yes
Yes
Mild Yes
No
No
No
No

AHG = Antihuman globulin; HDFN = Hemolytic disease of the fetus and newborn; HTR = Hemolytic transfusion reaction;
Occ. = Occasionally. The reactivity shown in the table is based on the tube methods in common use. If tests are carried
out by more sensitive test procedures (such as in capillary tubes, in microtiter plates, or by the albumin layering
method), direct agglutination (before the antiglobulin phase) may be observed more often with some antibodies. Blank
spaces indicate a lack of sufficient data for generalization about antibody behavior.

Chapter 15: Other Blood Groups

337

MNS System

molecules or hybrid molecules of the two
proteins. The M, N, S, s, and U antigens
are the most important antigens of the MNS
system with regard to transfusion medicine. They have also been important to
our understanding of biochemistry and
genetics. The M and N antigens are located on glycophorin A (GPA). The S, s,
and U antigens are located on glycophorin
B (GPB). Table 15-2 shows the frequencies
of the common phenotypes of the MNS
system. There is considerable linkage disequilibrium between M,N and S,s due to
the gene location on the chromosome.
For example, the gene complex producing
N with s is much more common than that
producing N with S. The MNSs genes
GYPA and GYPB are in very close proximity on chromosome 4.4 See the section below on Genes Encoding Glycophorins and
Chapters 9 and 10 for more information
about gene interactions.
Red cells that lack S and s may be negative for a high-incidence antigen called U;
persons who lack U may make anti-U when
exposed to U+ red cells.

M, N, S, s, and U Antigens

Low-Incidence Antigens of the MNS System

The MNS system is a complex system of
over 40 antigens carried on two glycophorin

The MNS system includes several low-incidence antigens. Recent biochemical data

Antigens that occur as codominant traits,
such as Jka and Jkb, may have a variable incidence and may differ in ethnic groups.
For an illustration, the Duffy glycoprotein
is known to be a receptor for the parasite
Plasmodium vivax, one of the causative
agents of malaria. In West Africa, where
malaria is endemic, the Fy(a–b–) red cell
phenotype, very rare in Whites, occurs with
an incidence of greater than 80%.
Each of the known antigens described
in this chapter was initially identified
through the detection of its specific antibody in a serum. Tables listing phenotype
frequencies among Whites and Blacks in the
US population are given throughout this
chapter. Frequencies among other groups
in the population are not given because
data are scanty and wide differences between groups of diverse Asian, South American, or Native American origins make generalizations about phenotypes inappropriate.

Table 15-2. Phenotypes and Frequencies in the MNS System
Reactions with AntiM

+
+
0

0
+
+

+
+
0
0
0

0
+
+
0
0

Phenotype Frequency (%)
U

Phenotype

Whites

Blacks

+
+
+
0
(+)

M+N–
M+N+
M–N+
S+s–U+
S+s+U+
S–s+U+
S–s–U–
S–s–U+w

28
50
22
11
44
45
0
0

26
44
30
3
28
69
Less than 1
Rare*

*May not be detected by some antisera and are listed as U–.

338

AABB Technical Manual

attribute the reactivity of various low-incidence determinants to one or more amino
acid substitutions, variation in the extent
or type of glycosylation, or the existence
of a hybrid sialoglycoprotein (SGP).

Genes Encoding Glycophorins
The genes encoding the MNS system antigens are located on chromosome 4 at
4q28-q31. The gene that encodes GPA is
called GYPA and the gene that encodes
GPB is GYPB. The similarities in amino
acid sequences of GPA and GPB suggest
that both genes derive from a common
ancestral gene. GYPA and GYPB consist of
seven and five exons, respectively. The
genes share >95% identity. Although the
genes are highly homologous, GYPB results in a shorter protein because a point
mutation at the 5′ splicing site of the third
intron prevents transcription of exon 3,
called pseudo exon 3. Following the homologous sequences, GYPA and GYPB differ significantly in the 3′ end sequences.

Hybrid Molecules
Pronounced SGP modifications occur in
hybrid molecules that may arise from unequal crossing over or gene conversion
between GYPA and GYPB. Hybrid SGPs
may carry the amino-terminal portion of
GPA and the carboxy-terminal portion of
GPB, or vice versa. Other hybrids appear
as a GPB molecule with a GPA insert or a
GPA molecule with a GPB insert. The lowincidence antigens Hil (MNS20), St a
(MNS15), Dantu (MNS25), and Mur
(MNS10), among others, are associated with
hybrid SGPs. Some variants are found in
specific ethnic groups. For example, the
Dantu antigen occurs predominantly in
Blacks, although the antigen is of low incidence.
Many of the MNS low-incidence antigens were categorized into a subsystem

called the Miltenberger system, based on
reactivity with selected sera. As more antigens have been identified and knowledge of
the genetic events that give rise to these
novel antigens has increased, it is clear that
the Miltenberger subsystem is outdated.
These antigens, such as Mia, Vw, Hil, etc,
should be considered glycophorin variants.

Biochemistry of the MNS System
Antigens of the MNS system are carried
on GPA and GPB, which are single-pass transmembrane glycoproteins. The carboxy (C)
terminal of each glycophorin extends into
the cytoplasm of the red cell, and a hydrophobic segment is embedded within the
lipid bilayer. An amino (N) terminal segment extends into the extracellular environment. The molecules are sensitive to
cleavage at varying positions by certain
proteases (see Fig 15-1).
There are approximately 1,000,000 copies of GPA per red cell. M and N blood
group antigen activity resides on the extracellular segment, a sequence of 72 amino
acids with carbohydrate side chains attached within the first 50 residues of the
amino terminal. When GPA carries M antigen activity (GPAM), the first amino acid residue is serine and the fifth is glycine. When
it carries N antigen activity (GPAN), leucine
and glutamic acid replace serine and glycine at positions one and five, respectively
(see Fig 15-1).
Red cells that lack most or all of GPA are
described as En(a–). These rare En(a–) individuals may produce antibodies (collectively called anti-Ena) that react with various portions of the extracellular part of the
glycoprotein. Some En(a–) persons may
produce an antibody against an antigen
b
called Wr that is part of the Diego blood
b
group system. Wr arises from an interaction between GPA and the anion exchange
molecule, AE-1 (also known as band 3).5

Chapter 15: Other Blood Groups

339

Figure 15-1. Schematic diagram of glycophorin A and glycophorin B. The amino acid sequences that
determine M, N, S, and s are given. indicates an O -linked oligosaccharide side chain,
indicates
an N -linked polysaccharide side chain. Approximate locations of protease cleavage sites are indicated. (Courtesy New York Blood Center.)

GPB is a smaller protein than GPA, and
there are fewer (approximately 200,000)
copies per red cell. GPB carries S, s, and U
antigens. GPB that expresses S activity has
methionine at position 29; GPB with s activity has threonine at that position (see Fig
15-1). The N-terminal 26 amino acids of
N
GPB are identical to the sequence of GPA ,
which accounts for the presence of an N
antigen (known as ‘N’) on all red cells of
normal MNS types. Red cells that lack GPB
altogether lack not only S, s, and U activity
but also ‘N’. Immunized individuals of the
rare M+N–S–s–U– phenotype can produce
a potent anti-N (anti-U/GPB) that reacts
with all red cells of normal MNS types,
whether N-positive or N-negative, and

should be considered clinically significant.
Some S–s– red cells also have a variant GPB.

The Effect of Proteolytic Enzymes on MNS
Antigens
Proteolytic enzymes, such as ficin or papain,
cleave red cell membrane SGPs at welldefined sites. Reactivity with anti-M and
anti-N is abolished by commonly used
enzyme techniques. The effect of different
enzymes on the expression of MNS system antigens reflects the point at which
the particular enzyme cleaves the antigen-bearing SGP and the position of the
antigen relative to the cleavage site (see
Fig 15-1). Sensitivity of the antigens to

340

AABB Technical Manual

proteases may help in the identification
of antibodies to M and N antigens, but the
effects of proteases on tests for the S and s
antigens are more variable. In addition,
the S antigen is sensitive to trace amounts
of chlorine bleach.6

MNS System Antibodies
The antibodies most commonly encountered are directed at the M, N, S, and s antigens.

Anti-M
Anti-M is detected frequently as a saline
agglutinin if testing is done at room temperature. Anti-M is often found in the sera
of persons who have had no exposure to
human red cells. Although M antibodies
are generally thought to be predominantly
IgM, many examples that are partly or
wholly IgG are frequently found. However,
these antibodies are rarely clinically significant. Some examples of anti-M cause
stronger agglutination if the pH of the test
system is reduced to 6.5 and when testing
red cell samples possessing a double-dose
expression of the M antigen. Examples that
react at 37 C or at the antiglobulin phase
of testing should be considered potentially
significant. Compatibility testing performed by a strictly prewarmed method
(see Method 3.3) should eliminate the reactivity of most examples of anti-M. In a
few exceptional cases, anti-M detectable
at the antiglobulin phase has caused
hemolytic disease of the fetus and newborn
(HDFN) or hemolysis of transfused cells.

because these people lack or possess an
altered form of GPB.

Antibodies to S, s, and U
Unlike anti-M and anti-N, antibodies to S,
s, and U usually occur following red cell
immunization. All are capable of causing
hemolytic transfusion reactions (HTRs)
and HDFN. Although a few saline-reactive
examples have been reported, antibodies
to S, s, and U are usually detected in the
antiglobulin phase of testing. Most, but
not all, investigators 7 have found that
papain or ficin destroys the reactivity of
S+ red cells with anti-S. Depending on the
enzyme solution used, the reactivity of
anti-s with s+ cells can be variable.8(p477)
Most examples of anti-U react equally
with untreated and enzyme-treated red
cells, but there have been examples of
broadly reactive anti-U, which detect an
enzyme-sensitive determinant.
Anti-U is rare but should be considered
when serum from a previously transfused
or pregnant Black person contains antibody to a high-incidence antigen.

Antibodies to Low-Incidence Antigens
There are many examples of antibodies to
g
low-incidence antigens, such as anti-M
W
or anti-V , in the MNS blood group system. Many of these antibodies occur as a
saline agglutinin in sera from persons who
have no known exposure to human red
cells. The rarity of these antigens makes it
unlikely that the antibodies will be detected if present.

Anti-N
Anti-N is comparatively rare. Examples are
usually IgM and typically appear as weakly
reactive cold agglutinins. Some powerful and
potentially significant IgG examples have
been observed in a few persons of the rare
phenotypes M+N–S–s–U– and M+N–S–s–U+w

Kell System
Kell System Antigens
Kell system antigens are expressed on the
red cell membrane in low density and are
weakened or destroyed by treatment with

Chapter 15: Other Blood Groups

reducing agents and with acid. The antigens are carried on one protein and encoded by a single gene. For an in-depth
review, see Lee, Russo, and Redman.9

K and k
The K antigen was first identified in 1946
because of an antibody that caused HDFN.
The allele responsible for the K antigen is
present in 9% of Whites and approximately 2% of Blacks. The existence of the
expected allele for k was confirmed when
an antithetical relationship was established between K and the antigen detected by anti-k. Anti-k reacts with the red
cells of over 99% of all individuals.

341

same chromosome. Kp a is an antigen
a
found predominantly in Whites, and Js is
found predominantly in Blacks. The
a
haplotype producing K and Kp has also
not been found. Table 15-3 shows some
phenotypes of the Kell system. The table
also includes K o , a null phenotype in
which the red cells lack all of the antigens
of the Kell system. Several high-incidence
antigens were assigned to the Kell system
because the identifying antibodies were
found to be nonreactive with Ko red cells.
For simplicity, various Kell antigens of
high and low incidence have not been
included in the table.

Phenotypes with Depressed Kell Antigens

Other Kell Blood Group Antigens
Other antithetical antigens of the Kell system include Kpa, Kpb, and Kpc; Jsa and Jsb;
K11 and K17; and K14 and K24. Not all
theoretically possible genotype combinations have been recognized in the Kell
system. For example, Kp a and Js a have
never been found to be produced by the

Kmod is an umbrella term used to describe
phenotypes characterized by weak expression of Kell system antigens. Adsorption/elution tests are often necessary for
their detection. The K mod phenotype is
thought to arise through several different
point mutations of the KEL gene. Red cells
of persons with some Gerbich negative

Table 15-3. Some Phenotypes and Frequencies in the Kell System
Reactions with AntiK

+
+
0

0
+
+

+
+
0

Frequency (%)
Js

0
+
+

+
+
0
0

0
+
+
0

Phenotype

Whites

K+k–
K+k+
K–k+
Kp(a+b–)
Kp(a+b+)
Kp(a–b+)
Js(a+b–)
Js(a+b+)
Js(a–b+)
K0

0.2
Rare
8.8
2
91.0
98
Rare
0
2.3
Rare
97.7
100
0.0
1
Rare
19
100.0
80
Exceedingly rare

Blacks

342

AABB Technical Manual

phenotypes also exhibit depressed Kell
phenotypes (see the Gerbich System).
Persons of the Ge:–2,–3 and Ge:–2,–3,–4
(Leach) phenotypes have depression of at
least some Kell system antigens.
The presence of the Kpa allele weakens
the expression of other Kell antigens when
in cis position. For example, the k antigen
of Kp(a+) red cells reacts more weakly than
expected and, when tested with weaker examples of anti-k, may be interpreted as k–.

Biochemistry of the Kell System
The Kell system antigens are carried on a
93-kD single-pass red cell membrane protein. Kell system antigens are easily inactivated by treating red cells with sulfhydryl reagents such as 2-mercaptoethanol
(2-ME), dithiothreitol (DTT), or 2-aminoethylisothiouronium bromide (AET ).
Such treatment is useful in preparing red
cells that artificially lack Kell system antigens to aid in the identification of Kell-related antibodies. Treatment with sulfhydryl
reagents may impair the reactivity of
other antigens (LWa, Doa, Dob, Yta, and others). Thus, although treatment with these
reagents may be used in antibody problem solving, specificity must be proven by
other means. As expected, Kell system antigens are also destroyed by ZZAP, a mixture of DTT and enzyme (see Methods
3.10, 4.6, and 4.9). This susceptibility to
sulfhydryl reagents suggests that disulfide
bonds are essential to maintain activity of
the Kell system antigens. This hypothesis
has been supported by the biochemical
characterization of Kell proteins deduced
from cloned DNA10; they exhibit a number
of cysteine residues in the extracellular
region. Cysteine readily forms disulfide
bonds, which contribute to the folding of
a protein. Antigens that reflect protein
conformation will be susceptible to any
agent that interferes with its tertiary struc-

ture. Kell antigens are also destroyed by
treatment with EDTA-glycine acid.
The function of the Kell protein is unknown, but it has structural similarities to a
family of zinc-binding neutral endopeptidases. It has most similarity with the
common acute lymphoblastic leukemia
antigen (CALLA or CD10), a neutral endopeptidase on leukocytes.8(pp647-648)

Kx Antigen, the McLeod Phenotype, and
Their Relationship to the Kell System
Although the Kell system locus is on the
long arm of chromosome 7 and the Kx locus (XK) is on the Xp21 region of the X
chromosome, evidence suggests that the
Kell and Kx proteins form a covalently
8
linked complex on normal red cells. On
red cells that carry normal expressions of
Kell antigens, Kx appears to be poorly expressed. It is believed that this finding
represents steric interference by the Kell
glycoprotein in the approach of anti-Kx to
its antigen. Red cells of Ko individuals react strongly with anti-Kx. Similarly, the removal or denaturation of Kell glycoproteins with AET or DTT renders the cells
strongly reactive with anti-Kx.
It is believed that the presence of the
glycoprotein on which Kx is carried is essential for the antigens of the Kell system to
attach to or be expressed normally on red
cells. Therefore, a lack of Kx is associated
with poor expression of Kell system antigens.
Red cells that lack Kx exhibit not only
markedly depressed expression of Kell system antigens but also shortened survival,
reduced deformability, decreased permeability to water, and acanthocytic morphology. This combination or group of red cell
abnormalities is called the McLeod phenotype, after the first person in whom these
observations were made. Persons with McLeod
red cells also have a poorly defined abnormality of the neuromuscular system, char-

Chapter 15: Other Blood Groups

acterized by persistently elevated serum
levels of the enzyme creatine phosphokinase and, in older people, disordered
muscular function. The McLeod phenotype
arises through deletion and mutations of
the XK locus of chromosome X.
In a few instances, the McLeod phenotype has been found in patients with chronic
granulomatous disease (CGD), in which
granulocytes exhibit normal phagocytosis
of microorganisms but an inability to kill
ingested pathogens. The McLeod phenotype associated with CGD appears to result
from deletion of a part of the X chromosome that includes the XK locus as well as
the gene responsible for X-linked CGD.

Kell System Antibodies

Anti-K and Anti-k
Because the K antigen is strongly immunogenic, anti-K is frequently found in sera
from transfused patients. Rare examples
of anti-K have appeared as a saline agglutinin in sera from subjects never exposed
to human red cells. Most examples are of
immune origin and are reactive in antiglobulin testing; some bind complement.
Some workers have observed that examples of anti-K react less well in tests that incorporate low-ionic-strength-saline (LISS)
solutions (notably the Polybrene test) than
in saline tests or tests that include albumin.
Others, however, have not shown differences in antibody reactivity, testing many
examples of anti-K in low ionic systems.
Anti-K has caused HTRs on numerous occasions, both immediate and delayed.
Anti-K can cause severe HDFN and fetal
anemia may be caused by the immune destruction of K+ erythroid progenitor cells by
macrophages in the fetal liver.11
Because over 90% of donors are K–, it is
not difficult to find compatible blood for
patients with anti-K. Anti-k has clinical and

343

serologic characteristics similar to anti-K
but occurs much less frequently. Only about
one person in 500 lacks the k antigen and
finding compatible blood is correspondingly more difficult.

Other Kell System Antibodies
a

Anti-Kp , anti-Kp , anti-Js , and anti-Js are
all much less common than anti-K but
show similar serologic characteristics and
are considered clinically significant. Any of
them may occur following transfusion or
fetomaternal immunization. Antibody frequency is influenced by the immunogenicity of the particular antigen and by the
distribution of the relevant negative phenotypes among transfusion recipients and
positive phenotypes among donors. In
Black patients frequently transfused with
phenotypically matched blood, usually
from other Black donors, anti-Jsa is relatively common. This is due to the approximate 20% incidence of the Jsa antigen in
the Black population (see Table 15-3). Ordinarily, however, these antibodies are rare.
Assistance from a rare donor file is usually
needed to find compatible blood for patients
immunized to the high-incidence antigens
Kpb and Jsb. Anti-Ku is the antibody characteristically seen in immunized Ko persons. It has been reported to cause a fatal
HTR,12 and it appears to be directed at a
single determinant because it has not been
separable into other Kell specificities.
However, antibodies to other Kell system
antigens may be present in serum containing anti-Ku. Some people of the Kmod phenotype have made a Ku-like antibody.

Duffy System
Duffy System Antigens
a

The antigens Fy and Fy are encoded by a
pair of codominant alleles at the Duffy

344

AABB Technical Manual

(FY) locus on chromosome 1. Anti-Fy and
b
anti-Fy define the four phenotypes observed
in this blood group system, namely:
Fy(a+b–), Fy(a+b+), Fy(a–b+), and Fy(a–b–)
(see Table 15-4). In Whites, the first three
phenotypes are common and Fy(a–b–) individuals are extremely rare. However, the
incidence of the Fy(a–b–) phenotype among
Blacks is 68%.
The Duffy gene encodes a glycoprotein
that is expressed in other tissues, including
the brain, kidney, spleen, heart, and lung.
The Fy(a–b–) individual can be the result of
the FyFy genotype or null phenotype. However, in many Black Fy(a–b–) individuals,
the transcription in the marrow is prevented and Duffy protein is absent from the
red cells. These individuals have an allele
that is the same in the structural region as
the Fyb gene that prevents the transcrip8(p439)
tion.
However, the Duffy protein is expressed normally in nonerythroid cells of
these persons.13 Other Fy(a–b–) individuals
either appear to have a total absence or
markedly altered Duffy glycoprotein.8(pp457-458)
This affects other cell lines and tissues, not
only the red cells. Those individuals who
have absent or altered glycoprotein can
make anti-Fy3, which will react with cells
that are Fy(a+) and/or Fy(b+).
A rare inherited form of weak Fyb called
x
Fy has been described and is probably due
x
to a point mutation. The Fy antigen may go

undetected unless potent anti-Fy is used in
testing. The Fy5 antigen appears to be defined by an interaction of the Duffy and Rh
gene products because it is not expressed
on Rhnull red cells. The Fy6 antigen has been
described only by murine monoclonal antibodies and is not present on red cells that
are Fy(a–b–) and Fy:–3,–5.8(p448)

Biochemistry of the Duffy System
In red cells, the Duffy gene encodes a multipass membrane glycoprotein. The antigens
Fya, Fyb, and Fy6 are located on the N-terminal of the Duffy glycoprotein and are
sensitive to denaturation by proteases such
as ficin, papain, and α-chymotrypsin, unlike Fy3 or Fy5. Fy3 has been located on
the last external loop of the Duffy glycoprotein. It is unaffected by protease treatm e n t (reviewed in Pierce and Macpherson).14 The glycoprotein is the receptor for the malarial parasite Plasmodium
vivax, and persons whose red cells lack
Fya and Fyb are resistant to that form of
the disease. In sub-Saharan Africa, notably West Africa, the resistance to P. vivax
malaria conferred by the Fy(a–b–) phenotype may have favored its natural selection, and most individuals are Fy(a–b–).
13
The Duffy gene has been cloned and
the Duffy glycoprotein has been identified
as an erythrocyte receptor for a number of

Table 15-4. Phenotypes and Frequencies in the Duffy System
Reactions with AntiFy
+
+
0
0

Fy
0
+
+
0

Adult Phenotype Frequency (%)
Phenotype

Whites

Blacks

Fy(a+b–)
Fy(a+b+)
Fy(a–b+)
Fy(a–b–)

17
49
34
Very rare

9
1
22
68

Chapter 15: Other Blood Groups

chemokines, notably interleukin-8. Because chemokines are biologically active
molecules, it has been postulated that Duffy
acts as a sponge for excess chemokines,
without ill effect on the red cells.

Duffy System Antibodies
a

Anti-Fy is quite common and may cause
HDFN and HTRs. Anti-Fyb is rare and genb
erally is weakly reactive. Anti-Fy can cause
rare mild HDFN and has been responsible
for mostly mild HTRs. Both antibodies are
usually IgG and react best by antiglobulin
testing. The glycoprotein that expresses
the antigens is cleaved by most proteases
used in serologic tests, so anti-Fya and
b
anti-Fy are usually nonreactive in enzyme test procedures.
a
b
Weak examples of anti-Fy or anti-Fy
may react only with red cells that have a
double dose of the antigen. In Whites, red
cells that express only one of the two antigens are assumed to come from persons
homozygous for the gene and to carry a
double dose of the antigen. In Blacks, such
cells may express the antigen only in single
dose and may not give the expected strong
reaction with antibodies that show dosage.
For example, the patient typing Fy(a+b–)
a
may be Fy Fy.
Anti-Fy3 was first described in the serum
of a White person of the Fy(a–b–) phenotype and is directed at the high-incidence
antigen Fy3. The only cells with which it is
nonreactive are Fy(a–b–). Unlike Fya and
b
Fy , the Fy3 antigen is unaffected by protease treatment, and anti-Fy3 reacts well with
enzyme-treated cells positive for either Fya
b
or Fy . Anti-Fy3 is rare but is sometimes
made by Black Fy(a–b–) patients lacking
Fy3 who have been immunized by multiple
transfusions.
Two other rare antibodies have been described, both reactive with papain-treated
red cells. One example of anti-Fy4 has been

345

reported. It reacted with red cells of the
Fy(a–b–) phenotype and with some Fy(a+b–)
and Fy(a–b+) red cells from Blacks but not
with Fy(a+b+) red cells, suggesting reactivity with a putative product of the Fy gene.
However, different reference laboratories
obtained equivocal results and evidence for
the existence of the Fy4 antigen is weak.
Anti-Fy5 is similar to anti-Fy3, except
that it fails to react with Rhnull red cells that
express Fy3 and is nonreactive with cells
from Fy(a–b–) Blacks. It may react with the
red cells from Fy(a–b–) Whites. This provided a previously unrecognized distinction
between the Fy(a–b–) phenotype so common in Blacks and the one that occurs, but
8(p447)
very rarely, in Whites.
Anti-Fy6 is a murine monoclonal antibody that describes a high-incidence antigen in the same region as Fya and Fyb. The
antibody reacts with all Fy(a+) and/or
Fy(b+) red cells, is nonreactive with Fy(a–b–)
red cells, but, unlike anti-Fy3, is nonreactive with enzyme-treated red cells.

Kidd System
a

Jk and Jk Antigens
a

The Jk and Jk antigens are located on the
urea transporter, encoded by the HUT 11
gene on chromosome 18. Jk(a–b–) red cells,
which lack the JK protein, are more resistant to lysis by 2M urea.16 Red cells of normal Jk phenotype swell and lyse rapidly in
a solution of 2M urea.
The four phenotypes identified in the Kidd
system are shown in Table 15-5. The
Jk(a–b–) phenotype is extremely rare, except in some populations of Pacific Island
origin. Two mechanisms have been shown
17
to produce the Jk(a–b–) phenotype. One is
the homozygous presence of the silent Jk
allele. The other is the action of a dominant
inhibitor gene called In(Jk). The dominant
suppression of Kidd antigens is similar to

346

AABB Technical Manual

Table 15-5. Phenotypes and Frequencies in the Kidd System
Reactions with AntiJk

Phenotype Frequency (%)
Phenotype

Whites

Blacks

Jk(a+b–)

Jk(a+b+)

Jk(a–b+)

Jk(a–b–)

Exceedingly rare

the In(Lu) suppression of the Lutheran system.

Kidd System Antibodies

Anti-Jka and Anti-Jkb
a

Anti-Jk was first recognized in 1951 in the
serum of a woman who had given birth
to a child with HDFN. Two years later,
anti-Jkb was found in the serum of a patient who had suffered a transfusion reaction. Both antibodies react best in antiglobulin testing, but saline reactivity is
sometimes observed in freshly drawn
specimens or when antibodies are newly
forming. Both anti-Jka and anti-Jkb are often weakly reactive, perhaps because,
sometimes, they are detected more readily
through the complement they bind to red
cells. Some examples may become undetectable on storage. Other examples may
react preferentially with red cells from
homozygotes.
Some workers report no difficulties in
detecting anti-Jka and anti-Jkb in low ionic
tests that incorporate anti-IgG. Others find
that an antiglobulin reagent containing an
anticomplement component may be important for the reliable detection of these
inconsistently reactive antibodies. Stronger
reactions may be obtained with the use of
polyethylene glycol (PEG) or enzymetreated red cells in antiglobulin testing.

Kidd system antibodies occasionally
cause HDFN, but it is usually mild. These
antibodies are notorious, however, for their
involvement in severe HTRs, especially delayed hemolytic transfusion reactions
(DHTRs). DHTRs occur when antibody develops so rapidly in an anamnestic response to antigens on transfused red cells
that it destroys the still-circulating red cells.
In many cases, retesting the patient’s pretransfusion serum confirms that the antibody was undetectable in the original tests.

Anti-Jk3
Sera from some rare Jk(a–b–) persons
have been found to contain an antibody
that reacts with all Jk(a+) and Jk(b+) red
cells but not with Jk(a–b–) red cells. Although a minor anti-Jka or anti-Jkb component is sometimes separable, most of
the reactivity has been directed at an antigen called Jk3, which is present on both
Jk(a+) and Jk(b+) red cells.

Other Blood Group Systems
So far, this chapter has been devoted to
blood group systems of red cell antigens
of which the principal antibodies may be
seen fairly frequently in the routine blood
typing laboratory. The other blood group
systems listed in Table 10-1 will be re-

Chapter 15: Other Blood Groups

viewed here briefly; the interested reader
should refer to other texts and reviews for
greater detail.

Lutheran System

Lua and Lub Antigens
The phenotypes of the Lutheran system,
a
b
as defined by anti-Lu and anti-Lu , are
shown in Table 15-6. The Lu(a–b–) phenotype is very rare and may arise from one
of three distinct genetic circumstances
14
(reviewed in Pierce and Macpherson ). In
the first, a presumably amorphic Lutheran gene (Lu) is inherited from both
parents. In the second and most common, the negative phenotype is inherited
as a dominant trait attributed to the independently segregating inhibitor gene,
In(Lu), which prevents the normal expression of Lutheran and certain other blood
group antigens (notably P1, I, AnWj, Ina,
b
and In ). The third Lu(a–b–) phenotype is
due to an X-borne suppressor, recessive in
its effect.

Other Lutheran Blood Group Antigens
A series of high-incidence antigens (Lu4,
Lu5, Lu6, Lu7, Lu8, Lu11, Lu12, Lu13,

Table 15-6. Phenotypes and Frequencies
in the Lutheran System in Whites*
Reactions with
AntiLu

Phenotype

Phenotype
Frequency
(%)

Lu(a+b–)

0.15

Lu(a+b+)

7.5

Lu(a–b+)

92.35

Lu(a–b–)

Very rare

*Insufficient data exist for the reliable calculation of frequencies in Blacks.

347

Lu16, Lu17, and Lu20) has been assigned
to the Lutheran system because the corresponding antibodies do not react with
Lu(a–b–) red cells of any of the three genetic backgrounds. Two low-incidence
antigens, Lu9 and Lu14, have gained admission to the Lutheran system because
of their apparent antithetical relationship
to the high-incidence antigens Lu6 and
Lu8, respectively.
Aua (Lu18), an antigen of relatively high
incidence [90% of all populations are
Au(a+)] and its antithetical partner, Aub
(Lu19), present in 50% of Whites and 68%
of Blacks, have been shown to belong to the
Lutheran system.18,19

Biochemistry of the Lutheran System
Lutheran antigens are carried on a glycoprotein bearing both N-linked and O-linked
oligosaccharides. This protein exists in
two forms and has been shown to have a
role in cell adhesion. The antigens are destroyed by trypsin, α-chymotrypsin, and
sulfhydryl-reducing agents. 20 These results and results of immunoblotting experiments suggest the existence of interchain or intrachain disulfide bonds. Tests
performed with monoclonal anti-Lub sugb
gest that the number of Lu antigen sites
per red cell is low, approximately 6001600 per Lu(a+b+) red cell and 1400-3800
per Lu(a–b+) red cell.21
The Lu and Se (secretor) loci were shown
to be linked in 1951, the first recorded example of autosomal linkage in humans.
The two loci have been assigned to chromosome 19. The gene encoding the Lu
glycoproteins has been cloned.22

Lutheran System Antibodies
a

The first example of anti-Lu (-Lu1) was
found in 1945 in a serum that contained
several other antibodies. Anti-Lu a and
b
anti-Lu are not often encountered. They

348

AABB Technical Manual

are most often produced in response to
pregnancy or transfusion but have occurred in the absence of obvious red cell
stimulation. Lutheran antigens are poorly
developed at birth. It is not surprising that
anti-Lua has not been reported to cause
HDFN; neither has it been associated with
HTRs. Anti-Lub has been reported to shorten the survival of transfused red cells but
causes no, or at most very mild, HDFN.
Most examples of anti-Lua and some antiLub will agglutinate saline-suspended red
cells possessing the relevant antigen, characteristically producing a mixed-field appearance with small to moderately sized,
loosely agglutinated clumps of red cells
interspersed among many unagglutinated
red cells.

Diego System

Diego System Antigens
The Diego system consists of two independent pairs of antithetical antigens,
called Dia/Dib and Wra/Wrb. The system
also contains a large number of low-incidence antigens as seen in Appendix 6.
The antigens are located on AE-1 (band
3), which is encoded by a gene on chromosome 17. The Dia and Dib antigens are
useful as anthropologic markers because
the Dia antigen is almost entirely confined
to populations of Asian origin and Native
North and South Americans, in which the
incidence of Dia can be as high as 54%.8(p583)
a
b
The Wr and Wr antigens are located on
AE-1 in close association with GPA. Wrb expression is dependent upon the presence of
GPA (see MNS Blood Group System).

Diego System Antibodies
a

Anti-Di may cause HDFN or destruction
b
of transfused Di(a+) red cells. Anti-Di is
rare but clinically significant. Anti-Wra is
fairly common and can occur without red
cell alloimmunization. It is a rare cause of

HTR or HDFN. Anti-Wrb is a rarely encountered antibody that may be formed
by rare Wr(a+b–) and some En(a–) individuals. Anti-Wrb may recognize an enzyme-resistant or enzyme-sensitive anti23
gen. It should be considered to have
potential to destroy Wr(b+) red cells.8(p589)

Cartwright System

Cartwright Antigens
The Yt (Cartwright) blood group system
a
b
consists of two antigens, Yt and Yt (see
Table 15-7). A gene on chromosome 7 encodes the antigens. The Yt antigens are located on red cell acetylcholinesterase
24
(AChE), an enzyme important in neural
transmission, but the function of which is
unknown on red cells. Enzymes have a
variable effect on the Yta antigen but 0.2M
DTT appears to destroy the Yta antigen expression.

Cartwright Antibodies
a

Some examples of anti-Yt are benign. A
a
few cases of anti-Yt have shown accelerated destruction of transfused Yt(a+) red
cells. Prediction of the clinical outcome by
the monocyte monolayer assay has proved
successful.25,26 Anti-Yta is not known to cause
b
HDFN. Anti-Yt is rare and has not been
implicated in HTR or HDFN.

Xg System

Xga Antigen
In 1962, an antibody was discovered that
identified an antigen more common among
women than among men. This would be
expected of an X-borne characteristic because females inherit an X chromosome
from each parent, whereas males inherit
X only from their mother. The antigen was
named Xga in recognition of its X-borne
manner of inheritance. Table 15-8 gives
the phenotype frequencies among White

Chapter 15: Other Blood Groups

349

Table 15-7. Phenotype Frequencies in Other Blood Group Systems with
Co-Dominant Antithetical Antigens

System
Yt

Dombrock

Colton

Scianna

Indian

Diego

Reactions with AntiYta
+
+
0
Doa
+
+
0
Coa
+
+
0
0
Sc1
+
+
0
0
Ina
+
+
0
Dia
+
+
0

Ytb
0
+
+
Dob
0
+
+
Cob
0
+
+
0
Sc2
0
+
+
0
Inb
0
+
+
Dib
0
+
+

Phenotype

Phenotype
Frequency in
Whites (%)*

Yt(a+b–)
Yt(a+b+)
Yt(a–b+)

91.9
7.9
0.2

Do(a+b–)
Do(a+b+)
Do(a–b+)

17.2
49.5
33.3

Co(a+b–)
Co(a+b+)
Co(a–b+)
Co(a–b–)

89.3
10.4
0.3
Very rare

Sc:1,–2
Sc:1,2
Sc:–1,2
Sc:–1,–2

99.7
0.3
Very rare
Very rare

In(a+b–)
In(a+b+)
In(a–b+)

Very rare
<1
>99

Di(a+b–)
Di(a+b+)
Di(a–b+)

Rare
Rare
>99.9

*There are insufficient data for the reliable calculation of frequencies in Blacks.

males and females. Enzymes, such as papain and ficin, denature the antigen. The
gene encoding Xga has been cloned.27,28

Scianna System

Xga Antibody
a

linkage with the Xg locus has been demonstrated for few traits to date.

Anti-Xg is an uncommon antibody that
usually reacts only in antiglobulin testing.
Anti-Xga has not been implicated in HDFN
a
or HTRs. Anti-Xg may be useful for tracing the transmission of genetic traits associated with the X chromosome, although

Scianna Antigens
Five antigens—Sc1, Sc2, Sc3, Rd, and
STAR—are recognized as belonging to the
Scianna blood group system. Scianna antigens are expressed by the red cell adhesion protein ERMAP.3 Sc1 is a high-inci-

350

AABB Technical Manual

Table 15-8. Frequencies of the Xg(a+)
and Xg(a–) Phenotypes in White Males
and Females
Phenotype Frequency (%)
Phenotype

Males

Females

Xg(a+)

65.6

88.7

Xg(a–)

34.4

11.3

Frequencies are based on the combined results of testing
nearly 7000 random blood samples from populations of
Northern European origin. There are insufficient data for
reliable calculation of frequencies in Blacks.

dence antigen, whereas Sc2 occurs very
infrequently. Sc1 and Sc2 behave as products of allelic genes (see Table 15-7). Sc3
is thought to be present on the red cells of
any individual who inherits a functional
Sc1or Sc2 gene, analogous to Fy3. Rd and
STAR are low-incidence antigens recently
assigned to the Scianna system. The antigens are resistant to enzymes routinely
used in blood group serology. The gene
encoding the Scianna antigens is located
on chromosome 1.

Scianna Antibodies
The antibodies are rare. Anti-Sc1 has not
been reported to cause HTR or HDFN.
Anti-Sc2 has caused positive DATs in the
neonate but no clinical HDFN.

Dombrock Blood Group System

Dombrock Antigens
Initially, this blood group system consisted
a
b
of Do and Do , with the phenotype frequency as shown in Table 15-7. The discovery that red cells negative for the higha
29
incidence antigen Gy were Do(a–b–) has
led to the recent expansion of the Dombrock
blood group system. Three high-incidence
antigens are Gya, Hy, and Joa. The interrelationship of the phenotypes is shown in
Table 15-9.30 Gy(a–) red cells represent the
null phenotype. The Gy(a+w), Hy–, and
Jo(a–) phenotypes have been found exclusively in Blacks. The Dombrock antigens
are located on a glycoprotein of 46 to 58
kD, the function of which is unknown.

Dombrock Antibodies
a

Anti-Do and anti-Do are uncommon antibodies and are usually found in sera
containing multiple red cell antibodies.
This can make the detection and identification of anti-Doa and anti-Dob difficult.
a
31
Anti-Do has caused HDFN and HTR.
b
HDFN due to anti-Do has not been reported, but examples have caused HTR.
Antibodies to Gya, Hy, and Joa may cause
shortened survival of transfused antigen-positive red cells or mild HDFN. Reactivity of Dombrock antibodies may be
enhanced by papain or ficin treatment of

Table 15-9. Relationship of the Dombrock Blood Group Antigens
a

Phenotype

Normal Do(a+b–)
Normal Do(a+b+)
Normal Do(a–b+)
Gy(a–)
Hy–
Jo(a–)

+
+
0
0
0
(+)

0
+
+
0
(+)
(+)

+
+
+
0
(+)
+

+
+
+
0
0
(+)

+
+
+
0
(+)
0

(+) = weak antigen expression.

Chapter 15: Other Blood Groups

red cells but is weakened or destroyed by
sulfhydryl reagents.

351

Table 15-10. Phenotypes and
Frequencies in the LW System in
Whites

Colton System
Reactions with
Anti-

Colton Antigens

Phenotype

Phenotype
Frequency
(%)

The Colton system consists of Co , a highincidence antigen; Cob, a low-incidence
antigen; and Co3, an antigen (like Fy3)
considered to be the product of either the
Coa or Cob gene. The antigens are products
of a gene on chromosome 7. The phenotype frequencies in Whites are shown in
Table 15-7. The Colton antigens have
been located on membrane protein CHIP
28 (Aquaporin), which functions as the
red cell water transporter.32

Colton Antibodies
a

Anti-Co has been implicated in HTRs and
HDFN.8 Anti-Cob has caused an HTR and
mild HDFN. Enzyme treatment of red
cells enhances reactions with the Colton
antibodies.

LW System

LW(a+b–)

>99%

LW(a+b+)

<1%

LW(a–b+)

Very rare

LW(a–b–)

Very rare

mosome 19 and has been cloned. The
glycoprotein it encodes has homology
33
with cell adhesion molecules.
Red cells from persons of the Rhnull phenotype are LW(a–b–). It appears that the LW
glycoprotein requires an interaction with
Rh proteins for expression, although the
basis of this interaction is not clear. (For a
review of the evolution of the LW system,
see Storry.34)

LW Antibodies

LW Antigens

Table 15-10 shows the LW phenotypes and
their frequencies. The antigens are denatured by sulfhydryl reagents, such as DTT,
and by pronase but are unaffected by
papain or ficin. There are reported cases
of both inherited and acquired LW(a–b–)
individuals.

Association with Rh
a

LW is more strongly expressed on D+
than D– red cells. However, the gene encoding the LW antigens is independent of
the genes encoding the Rh proteins. Genetic independence was originally established through the study of informative
families in which LW has been shown to
segregate independently of the RH genes.
The LW gene has been assigned to chro-

Anti-LW has not been reported to cause
HTRs or HDFN and both D+ and D–
LW(a+) red cells have been successfully
transfused into patients whose sera contained anti-LWa. Reduced expression of
LW antigens can occur in pregnancy and
some hematologic diseases. LW antibodies may occur as an autoantibody or as an
apparent alloantibody in the serum of
ab
such individuals. Anti-LW has been reported in LW(a–b–) individuals as well as
in patients with suppressed LW antigens.19

Chido/Rodgers System

Chido/Rodgers Antigens
The Chido (Ch) and Rodgers (Rg) antigens
are high-incidence antigens present on
the complement component C4. The an-

352

AABB Technical Manual

tigens are not intrinsic to the red cell. In
antigen-positive individuals, the antigens
are adsorbed onto red cells from the
plasma through an attachment mechanism that remains unclear.35 Ch has been
subdivided into six antigens and Rg into
two antigens. A ninth antigen, WH, requires the interaction of Rg1 and Ch6 for
expression. C4 is encoded by two linked
genes, C4A and C4B, on chromosome 6.
Existing sera are poorly classified, but
the phenotype frequency may be considered as in Table 15-11. The antigens are destroyed by papain/ficin treatment but unaffected by DTT/AET treatment.

Chido/Rodgers Antibodies
Antibodies to Ch and Rg are generally benign but may be a great nuisance in serologic investigations. Rapid identification
is possible using red cells coated with C4

or by inhibition with pooled plasma from
antigen-positive individuals. (See Method
3.9.) The antibodies are nonreactive with
enzyme-treated red cells.

Gerbich System

Gerbich Antigens
The Gerbich system includes eight antigens, of which three (Ge2, Ge3, and Ge4)
a
are of high incidence and five (Wb, Ls ,
a
a
An , Dh , and GEIS) are of low incidence.
Several phenotypes that lack one or more
of the high-incidence antigens are shown
in Table 15-12; all are rare. Red cells with
the Gerbich or Leach phenotype have a
weakened expression of some Kell system
a
a
a
antigens. Ge2, Ge4, Wb, Ls , An , Dh , and
GEIS are destroyed by papain and ficin,
but Ge3 resists protease treatment.

Table 15-11. Some Blood Group Antigens with Phenotypic Relationships
Approximate Frequency (%)
Antigens

Phenotypes

Whites

Blacks

Chido (Ch) and Rodgers (Rg)

Ch+,Rg+
Ch–,Rg+
Ch+,Rg–
Ch–,Rg–

95.0
2.0
3.0
Very rare

Cost (Csa)* and York (Yka)

Cs(a+),Yk(a+)
Cs(a+),Yk(a–)
Cs(a–),Yk(a+)
Cs(a–),Yk(a–)

82.5
13.5
2.1
1.9

95.6
3.2
0.6
0.6

Knops-Helgeson (Kna) and McCoy (McCa)

Kn(a+),McC(a+)
Kn(a+),McC(a–)
Kn(a–),McC(a+)
Kn(a–),McC(a–)

97.0
2.0
1.0
Rare

95.0
4.0
1.0
Rare

*Although Csa is not part of the Knops blood group system, there is a phenotypic association between Yk a and Csa.

Chapter 15: Other Blood Groups

353

Table 15-12. Ge– Phenotypes
Phenotype

Antibody Produced

Ge: –2,3,4 (Yus type)

Anti-Ge2

Ge:–2,–3,4 (Gerbich type)

Anti-Ge2 or -Ge3

Ge:–2,–3,–4 (Leach type)

Anti-Ge2, -Ge3, or -Ge4

The antigens of the Gerbich blood group
system are carried on glycophorin C (GPC)
and glycophorin D (GPD). GPC carries Ge3
and Ge4, whereas GPD carries Ge2 and
Ge3. Ana is carried on an altered form of
GPD. Dha and Wb are located on altered
a
forms of GPC. Ls is found on an altered
36
form of GPC and GPD. The proteins are
the product of a single gene, GYPC, on
chromosome 2. GPC is approximately four
times more abundant than GPD. The
mechanism whereby these two proteins are
derived from a single gene involves an alternative initiation site in the gene. GPC
and GPD interact directly with protein
band 4.1 in the membrane skeleton. It is
clear that the interaction is important in
maintaining cell shape because deficiencies of either band 4.1 or GPC/D cause
elliptocytosis.36

incidence antigens and three low-incidence antigens (see Table 15-13). Tca is
antithetical to the low-incidence antigen
Tcb in Blacks and to Tcc in Whites. WESb is
the high-incidence antigen antithetical to
WESa. Cra, Dra, Esa, UMC, GUTI, SERF, and
ZENA are not associated with low-incidence antigens. IFC is absent only in the
null phenotype (Inab).
The antigens are located on the complement regulatory protein called decay-accelerating factor (DAF). The protein is encoded by DAF, one gene of the regulators of
complement activation (RCA) complex, on
chromosome 1. The antigens are on leukocytes, platelets, and trophoblasts of the placenta as well as in soluble form in the serum/plasma and urine.37 The antigens are
not affected by ficin or papain. DTT or AET
may weaken the antigens but do not completely destroy them.37

Gerbich Antibodies
The antibodies that Ge-negative individuals may produce are shown in Table 15-12;
they may be immune or occur without red
cell stimulation. Anti-Ge is usually IgG
but may have an IgM component. The
clinical significance of the antibodies is
variable. Antibodies to the Gerbich antigens may be a rare cause of HDFN.

Cromer System

Cromer Antigens
A total of 13 antigens have been assigned
to the Cromer blood group system: 10 high-

Cromer Antibodies
Antibodies to antigens of the Cromer system are immune-mediated and extremely
a
uncommon. Most examples of anti-Cr ,
b
a
-WES , and -Tc have been found in the
sera of Black individuals. Anti-GUTI was
found in a Canadian of Chilean ancestry.37
The clinical significance of the antibodies
is variable, and some examples cause decreased survival of transfused red cells.
The antibodies will not cause HDFN because the placenta tissue is a rich source
of DAF, which is thought to adsorb the
maternal antibodies.37

354

AABB Technical Manual

Table 15-13. Antigens of High and Low
Incidence in the Cromer Blood Group
System
Antigen

Incidence (%)

Cra
Tca
Tcb
Tcc
Dra
Esa
IFC
WESa
WESb
UMC
GUTI

>99
>99
<1
<1
>99
>99
>99
<1
>99
>99
>99

SERF
ZENA

>99
>99

Knops System

Knops Antigens

that the variable reactivity of anti-CR1-related sera is a direct reflection of the
number of CR1 sites that exhibit both size
and expression polymorphisms and vary
widely among individuals. The antibodies
are of no clinical significance.

Indian System
a

In and In are located on CD44, a protein
of wide tissue distribution with the chara
acteristics of a cell adhesion molecule. In
b
is a low-incidence antigen, and In is of
high incidence (see Table 15-7). Inb shows
reduced expression on Lu(a–b–) red cells
of the In(Lu) type but is normally expressed on Lu(a–b–) red cells from persons homozygous for the amorph or possessing the X-borne suppressor gene. The
antigens are destroyed by papain and ficin
as well as by reducing agents such as 0.2
M DTT. There are few data on the clinical
significance of the corresponding antibodies.

Other Blood Group Systems

Most of the eight Knops system antigens
a
b
a
b
a
a
(Kn , Kn , McC , McC , Sl , Yk , Vil, and Sl3)
have been located on the C3b/C4b receptor (CR1), the primary complement receptor on red cells. A gene on chromoa
a
a
a
some 1 encodes CR1. Kn , McC , Sl , Yk ,
b
and Sl3 are high-incidence antigens. Kn ,
b
McC , and Vil are low-incidence antigens.
Some variation in frequency is observed
between the red cells of Whites and Blacks
(see Table 15-11).
The Knops system antigens are not destroyed by ficin or papain but may be weakened or destroyed by DTT or AET.

Knops Antibodies
The antibodies commonly show variable
weak reactivity in the antiglobulin phase
of testing but may continue to react even
at high dilutions. Moulds et al38 have shown

Ok System
The Ok system consists of a single higha
incidence antigen, Ok . The few rare
Ok(a–) individuals to date have been Japanese. Proteases do not seem to weaken
expression of Oka in routine agglutination
a
tests. Anti-Ok reacts optimally by an indirect antiglobulin test and appears to be
clinically significant in transfusion therapy, causing rapid destruction of Ok(a+)
red cells.

Raph System
The Raph system consists of a single antigen, MER2. Anti-MER2 has been reported
in three Israeli Jews. All three were on renal dialysis, raising the possibility that antibody production may be associated with
kidney disease. The MER2 antigen has

Chapter 15: Other Blood Groups

been detected on the red cells of 92% of
those tested. The MER2 antigen is sensitive to DTT but is not affected by treatment with ficin, papain, or chloroquine.
The antibodies have been IgG and some
have bound complement. To date, there
has been no information on whether these
antibodies are capable of causing HTR or
HDFN.

John Milton Hagen System
The John Milton Hagen (JMH) antigen is
carried on a GPI-linked CD108 glycoprotein. JMH antigen decreases over time. The
JMH– phenotype can be transient. The
JMH– phenotype can be acquired or inherited. The antigen is destroyed or altered by ficin or papain treatment and by
AET or DTT treatment.
Antibodies that react with JMH show
variation in reactivity and are usually weak.
Autoanti-JMH can often be found in older
people, along with an acquired absent or
weak JMH antigen expression. The antibody is not routinely considered capable of
HTR or HDFN.

GIL System
There is one antigen of high frequency, GIL,
in this system. GIL is located on aquaporin
3 (AQP3), which is a glycerol transporter.
The antibody has not been reported to
cause HDFN or HTR.

Blood Group Collections
In addition to the blood group systems,
there are collections of antigens that exhibit shared characteristics but do not as
yet meet the criteria for blood group system status defined by the ISBT. They include Cost (ISBT 205), Er (ISBT 208), and
Vel (ISBT 211).

355

Cost
Csa and Csb are all that remain of this collection after the Knops antigens were
identified on CR1. Cs a occurs in a frequency greater that 98% in most populab
tions, whereas Cs appears in about 34%
of the population.19 There is, however, an
a
unexplained connection between the Yk
a
and Cs antigens, such that red cells negative for one antigen are often weak or negative for the other. (See Table 15-11.) The
antigens are not destroyed by ficin, papain,
a
or DTT. Anti-Cs behaves similarly to antibodies produced to the Knops system antigens and is not considered clinically significant.

Er
The Er collection consists of two antigens,
which give rise to four phenotypes: Er(a+b–),
Er(a+b+), Er(a–b+), and Er(a–b–). Era is a
high-incidence antigen present on the red
cells of >99% of all individuals, but Erb has
a prevalence of less than 1%. The presence
of a silent third allele, Er, is thought to account for the Er(a–b–) phenotype, as demonstrated by family studies. The antigens
are not destroyed by ficin, papain, or DTT
but are destroyed by EDTA-glycine acid.

Vel
The Vel collection was recently created to
include two serologically related antigens
of high incidence, Vel and ABTI.
Vel is a high-incidence antigen that is
unaffected by protease and sulfhydryl treatment. It is well developed at birth, but antigen expression is variable.8 Despite its occurrence after known immunizing stimuli,
anti-Vel is most commonly IgM. It has been
reported to range from causing only a positive DAT in the neonate to causing severe
19
HDFN. It has been implicated in HTRs.
Anti-Vel binds complement, and in-vitro
hemolysis of incompatible red cells is often

356

AABB Technical Manual

seen when testing freshly drawn serum
containing this antibody. Reactivity of
anti-Vel is usually enhanced by enzyme
treatment of red cells expressing the antigen.

High-Incidence Red Cell
Antigens Not Assigned to a
Blood Group System or
Collection
Table 15-14 lists the antigens of high incidence that are independent of a blood
group system or collection. Persons who
make alloantibody to a specific blood
group antigen necessarily have red cells
lacking that antigen. For this reason, antibodies directed at high-incidence antigens
are rarely encountered. The antibodies
corresponding to these antigens usually
react best by antiglobulin testing.

Lan Antigen
Lan is a high-incidence antigen that is resistant to enzyme-treatment and to 0.2 M
DTT treatment. A weak form of the Lan
antigen has been reported.19 Anti-Lan is
characteristically IgG, may bind complement, and may cause HTRs. Cases of
HDFN due to anti-Lan have been mild
even though the Lan antigen is present on
cord red cells.19
a

At Antigen
a

At is a high-incidence antigen that is resistant to enzyme treatment and to 0.2 M
DTT treatment. The At(a–) phenotype has
been found only in Black individuals.19
a
Anti-At is characteristically IgG. The antibody appears to cause only moderate
HTRs and no clinical HDFN.19

Table 15-14. Some Antigens of High
Incidence Not Assigned to a Blood
Group System or Collection
Name

Symbol

August

Ata

Langereis

Lan

Sid

Sda

Duclos
Jra
Emm
AnWj
PEL
MAM

Jr Antigen
a

Jr is a high-incidence antigen that is resistant to enzyme treatment and to 0.2 M
DTT treatment. The Jr(a–) phenotype is more
commonly found in Japanese individuals
but has been found in other populations
as well.8(pp805-806) Anti-Jra has been shown to
cause reduced red cell survival. 8(pp805-806)
a
Other examples of anti-Jr have shown little or no clinical significance in HDFN or
HTR.8(p806),39

AnWj Antigen
AnWj is a high-incidence antigen that is
resistant to enzyme treatment but weakened by 0.2 M DTT treatment.19 The antigen is carried on CD44, which also carries
the Indian blood group system antigens.
The AnWj antigen is weakened on the red
cells from individuals with the In(Lu)
gene (see section on Lutheran System).
Some patients with Hodgkin’s disease
may experience a long-term suppression
of the AnWj antigen.8(pp783-784) The AnWj antigen is the receptor for Haemophilus
influenzae. 8(pp784-785) Anti-AnWj has been

Chapter 15: Other Blood Groups

implicated in severe HTRs but not in HDFN
because the antigen is not present on cord
red cells.
a

Sd Antigen
a

Sd is an antigen of fairly high incidence,
widely distributed in mammalian tissues
and body fluids. The antigen is variably
expressed on the red cells of Sd(a+) india
viduals. Sd expression may diminish during pregnancy and the Sd(a–) phenotype
is observed in 30% to 75% of pregnant
women. The antigen is not present on
cord cells. The strongest expression of Sda
has been observed on polyagglutinable
red cells of the Cad phenotype. The frequency of Sd(a–) blood is considered to
be around 9%, but weakly positive reactions are often difficult to distinguish from
negative ones.
Anti-Sda can be reactive by antiglobulin
testing. Microscopic examination of positive reactions generally shows mixed-field
agglutination, with relatively small, tightly
agglutinated clumps of red cells present
against a background of free red cells.
These agglutinates are refractile and may
have a shiny appearance. Because the majority of the examples of anti-Sda are IgM,
the wide use of anti-IgG means that many
examples of anti-Sda are no longer detected. Anti-Sda is not considered to be clinically significant. However, there have been
reported cases of HTRs with red cells with
strong Sda expression.8(pp816-817)
a
The immunodominant sugar of Sd is
N-acetylgalactosamine (GalNAc), also the
immunodominant sugar of the A blood
group antigen and of the Tamm-Horsfall
glycoprotein, found in human and guinea
pig urine. Anti-Sda activity can be inhibited
by incubation with urine from guinea pigs
or from Sd(a+) humans. See Method 3.11
for the performance of a urine neutralization of anti-Sda.

357

Low-Incidence Red Cell
Antigens Not Assigned to a
Blood Group System or
Collection
Many independent low-incidence red cell
antigens have been recognized in addition
to a growing number that have been assigned to the MNS, Rh, and Diego systems.
Table 15-15 lists those that have been
studied and shown to be inherited in a
dominant manner. Antibodies specific for
these low-incidence antigens react with
so few random blood samples that they
virtually never cause difficulties in selecting blood for transfusion but may be implicated in some rare cases of HDFN. The
antibodies are of interest to the serologist,
however, because of the unexpectedly
high incidence with which they occur,
often without an identifiable antigenic
stimulus.

Table 15-15. Antigens of Low Incidence
Not Assigned to a Blood Group System
or Collection
Batty (By)

Livesay (Lia)

Biles (Bi)

Milne

Box (Bx )

Oldeide (Ola)

Christiansen (Chra)

Peters (Pta)

HJK

Rasmussen (RASM)

HOFM

Reid (Rea)

JFV

REIT

JONES

SARA
a

Jensen (Je )

Torkildsen (Toa)

Katagiri (Kg)
The antigens occur with a frequency of 1 in 500 or less.

358

AABB Technical Manual

Antibodies to Low-Incidence
Antigens
Antibodies to low-incidence antigens have
sometimes been implicated in transfusion
reactions and HDFN. These antibodies
are usually encountered by chance, when
the red cells used for antibody detection
or selected for crossmatching happen to
carry the corresponding antigen.
Antibodies to low-incidence antigens may
also be present as unsuspected contaminants
in blood typing reagents prepared from human serum and may cause false-positive
test results if the red cells tested carry the
antigen. Testing with reagents from different manufacturers may not eliminate this
error because it is not uncommon for a single individual with an uncommon antibody
to provide the serum used for reagent preparation by different manufacturers.
Some antibodies to low-incidence antigens react as saline agglutinins. They can
also occur as IgG antibodies reactive only
by antiglobulin testing, even if there has
been no exposure to red cell immunization.
It is common for several low-incidence antibodies to occur together in a single serum;
multiple specificities are especially likely in
sera from patients with autoimmune conditions.

strengths are observed when a single serum containing “anti-Bg” is tested with
different Bg+ red cells. Reactivity is most
commonly observed in antiglobulin testing, but highly potent anti-Bg sera may directly agglutinate red cells with an unusually strong expression of the Bg antigens.
Confident and precise classification of
reactivity is made difficult by the weak expression of these antigens on some red cells
and by multiple specificities among different examples of the Bg antibodies. These
antibodies may also occur as unsuspected
contaminants in human source blood typing sera, where they may cause false-positive reactions with cells having unusually
strong expression of the corresponding Bg
antigen. Bg-related antigens are denatured
by chloroquine diphosphate or a solution
of glycine-HCl/EDTA.

References
1.

Bg (Bennett-Goodspeed) Antigens
Antibodies directed at certain leukocyte
antigens sometimes cause confusing reactions in serologic tests with red cells. At
least three separate specificities have been
given names as Bg antigens: Bga corresponds
b
to HLA-B7; Bg corresponds to HLA-B17;
c
and Bg corresponds to HLA-A28. A fourth
antibody in some antileukocyte sera reacts with red cells of persons who express
HLA-A10. The so-called Bg antigens are
expressed to variable degrees on red cells,
with the result that reactions of differing

Garratty G, Dzik W, Issitt PD, et al. Terminology for blood group antigens and genes—historical origins and guidelines in the new millennium. Transfusion 2000;40:477-89.
Daniels GL, Anstee DJ, Cartron JP, et al. Terminology for red cell surface antigens. ISBT
Working Party Oslo Report. International Society of Blood Transfusion. Vox Sang 1999;77:
52-7.
Daniels GL, Cartron JP, Fletcher A, et al. International Society of Blood Transfusion Committee on terminology for red cell surface antigens: Vancouver Report. Vox Sang 2003;84:
244-7.
Race RR, Sanger R. Blood groups in man. 6th
ed. Oxford: Blackwell Scientific Publications,
1975.
Bruce LJ, Ring SM, Anstee DJ, et al. Changes
in the blood group Wright antigens are associated with a mutation at amino acid 658 in
human erythrocyte band 3: A site of interaction between band 3 and glycophorin A under certain conditions. Blood 1995;85:299306.
Rygiel SA, Issitt CH, Fruitstone MJ. Destruction of the S antigen by Clorox (abstract).
Transfusion 1983;23:410.
Case J. The behavior of anti-S antibodies with
ficin-treated human red cells. In: Abstracts of

Chapter 15: Other Blood Groups

10.

11.

12.

13.

14.

15.

16.
17.

18.

19.

20.

21.

volunteer papers. 30th Annual Meeting of the
American Association of Blood Banks. Washington, DC: American Association of Blood
Banks, 1977:36.
Issitt PD, Anstee DJ. Applied blood group serology. 4th ed. Durham, NC: Montgomery
Scientific, 1998.
Lee S, Russo D, Redman CM. The Kell blood
group system: Kell and XK membrane proteins. Semin Hematol 2000;37:113-21.
Zelinski T, Coghlan G, Myal Y, et al. Genetic
linkage between the Kell blood group system
and prolactin-inducible protein loci: Provisional assignment of KEL to chromosome 7.
Ann Hum Genet 1991;55:137-40.
Daniels G, Hadley A, Green CA. Causes of fetal anemia in hemolytic disease due to anti-K
(letter). Transfusion 2003;43:115-16.
Lin M, Wang CL, Chen FS, et al. Fatal hemolytic transfusion reaction due to anti-Ku in a
K null patient. Immunohematol 2003;19:1921.
Chaudhuri A, Polyakova J, Zbrezezna V, et al.
Cloning of glycoprotein D cDNA which encodes the major subunit of the Duffy blood
group system and the receptor for the Plasmodium vivax malaria parasite. Proc Natl Acad
Sci U S A 1993;90:10793-7.
Pierce SP, Macpherson CR, eds. Blood group
systems: Duffy, Kidd and Lutheran. Arlington,
VA: American Association of Blood Banks,
1988.
Horuk R, Chitnis C, Darbonne W, et al. A receptor for the malarial parasite Plasmodium
vivax: The erythrocyte chemokine receptor.
Science 1993;261:1182-4.
Heaton DC, McLoughlin K. Jk(a–b–) red blood
cells resist urea lysis. Transfusion 1982;22:70-1.
Mougey R. The Kidd blood group system. In:
Pierce SR, Macpherson CR, eds. Blood group
systems: Duffy, Kidd and Lutheran. Arlington,
VA: American Association of Blood Banks,
1988:53-71.
Zelinski K, Kaita H, Coghlan G, Philipps S. Assignment of the Auberger red cell antigen
polymorphism to the Lutheran blood group
system: Genetic justification. Vox Sang 1991;
61:275-6.
Reid ME, Lomas-Francis C. The blood group
antigen factsbook. 2nd ed. San Diego, CA: Academic Press, 2004.
Daniels G. Effect of enzymes on and chemical
modifications of high-frequency red cell antigens. Immunohematology 1992;8:53-7.
Merry AH, Gardner B, Parsons SF, Anstee DJ.
Estimation of the number of binding sites for
a murine monoclonal anti-Lu b on human
erythrocytes. Vox Sang 1987;53:57-60.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.
35.

36.

37.

38.

359

Crew VK, Green C, Daniels G. Molecular
bases of the antigens of the Lutheren blood
group system. Transfusion 2003;43:1729-37.
Storry JR, Reid ME, Chiofolo JT, et al. A new
Wr(a+b–) Proband with anti-Wrb recognizing
a ficin sensitive antigen (abstract). Transfusion 2001;41(Suppl):23S.
Spring FA. Characterization of blood-groupactive erythrocyte membrane glycoproteins
with human antisera. Transfus Med 1993;3:
167-78.
Eckrich RJ, Mallory DM. Correlation of monocyte monolayer assays and posttransfusion
survival of Yt(a+) red cells in patients with
anti-Yt a (abstract). Transfusion 1993;33
(Suppl):18S.
Garratty G, Arndt P, Nance S. The potential
clinical significance of blood group alloantibodies to high frequency antigens (abstract).
Blood 1997;90(Suppl):473a.
Ellis NA, Ye T-Z, Patton S, et al. Cloning of
PBDX, a MIC2-related gene that spans the
pseudoautosomal boundary on chromosome
Xp. Nat Genet 1994;6:394-9.
Ellis NA, Tippett P, Petty A, et al. PBDX is the
XG blood group gene. Nat Genet 1994;8:28590.
Banks JA, Parker N, Poole J. Evidence to show
that Dombrock antigens reside on the Gya/
Hy glycoprotein. Transfus Med 1992;(Suppl)
1:68.
Scofield TL, Miller JP, Storry JR, et al. Evidence that Hy– RBCs express weak Joa antigen. Transfusion 2004;44:170-2.
Judd WJ, Steiner EA. Multiple hemolytic
transfusion reactions caused by anti-Do a .
Transfusion 1991;31:477-8.
Smith BL, Preston GM, Spring F, et al. Human
red cell aquaporin CHIP. J Clin Invest 1994;94:
1043-9.
Bailly P, Hermand P, Callebaut I, et al. The LW
blood group glycoprotein is homologous to
intercellular adhesion molecules. Proc Natl
Acad Sci U S A 1994;91:5306-10.
Storry JR. Review: The LW blood group system. Immunohematology 1994;8:87-93.
Moulds JM, Laird-Fryer B, eds. Blood groups:
Chido/Rodgers, Knops/McCoy/York and Cromer. Bethesda, MD: American Association of
Blood Banks, 1992.
Reid ME, Spring FA. Molecular basis of glycophorin C variants and their associated blood
group antigens. Transfus Med 1994;4:139-46.
Storry JR, Reid ME, The Cromer blood group
system: A review. Immunohematology 2002;
18:95-101.
Moulds JM, Moulds JJ, Brown M, Atkinson JP.
Antiglobulin testing for CR1-related (Knops/
McCoy/Swain-Langley/York) blood group an-

360

39.

AABB Technical Manual

tigens: Negative and weak reactions are
caused by variable expression of CR1. Vox Sang
1992;62:230-5.
Kwon M, Ammeus M, Blackall D. A Japanese
patient with a Jra antibody: Apparent lack of
clinical significance despite multiple incompatible transfusions (abstract). Transfusion
2001;41(Suppl):58S.

Suggested Reading
Akane A, Mizukami H, Shiono H. Classification of
the standard alleles of the MN blood group system. Vox Sang 2000;79:183-7.
Liu M, Jiang D, Liu S, et al. Frequencies of the major alleles of Diego, Dombrock, Yt, and Ok blood
group systems in the Chinese, Han, Hui, and Ti-

betan nationalities. Immunohematology 2003;19:
22-5.
Lögdberg L, Reid M, Miller J. Cloning and genetic
characterization of blood group carrier molecules
and antigens. Transfus Med Rev 2002;16:1-10.
Pogo AO, Chaudhuri A. The Duffy protein: A malarial and chemokine receptor. Semin Hematol
2000;37:122-9.
Reid ME. The Dombrock blood group system: A review. Transfusion 2003;43:107-14.
Reid ME, Rios M, Yazdanbakhsh K. Applications of
molecular biology techniques to transfusion medicine. Semin Hematol 2000;37:166-76.
Reid ME, Storry JR. Low-incidence MNS antigens
associated with single amino acid changes and their
susceptibility to enzyme treatment. Immunohematology 2001;17:76-81.

Chapter 16: Platelet and Granulocyte Antigens and Antibodies

Chapter 16

Platelet and Granulocyte
Antigens and Antibodies

NTIBODIES REACTIVE WITH antigens expressed on platelets and
leukocytes are assuming increasing importance. Some blood group antigens are shared by red cells, white cells,
and platelets; others are specific to certain cell types. This chapter discusses antibodies directed at antigens expressed on
platelets and neutrophils, with an emphasis on those specific for these cells. HLA
antibodies and antigens are covered more
fully in Chapter 17.

Platelet Antigens
Antigens Shared with Other Tissues
Platelets express a variety of antigenic
markers on their surface. Some of these
antigens are shared with other cell types,
as in the case of ABH antigens and HLA
antigens, which are shared with virtually
all nucleated cells in the body. Others are

observed to be essentially platelet specific.

ABH Antigens
The ABH antigens expressed on platelets
are a combination of structures intrinsic
to the plasma membrane and those adsorbed from the plasma. The amount of
ABH antigen present on platelets is quite
variable from individual to individual,
with from 5% to 10% of non-group-O individuals expressing extremely elevated
amounts of A or B substance on their
platelets. These people appear to have a
“high-expresser” form of glycosyltransferase in their sera. Although platelets are
often transfused without regard to ABO
compatibility, in some cases, ABO antibodies (particularly IgG antibodies of
high titer in group O recipients) may react
with platelets carrying large amounts of A
or B antigen.1 High-expresser platelets are
particularly vulnerable to this type of im361

362

AABB Technical Manual

mune destruction. ABO antibodies in recipients may also cause reduced survival
of ABO-incompatible platelets from normal-expresser phenotype donors, causing
occasional patients to exhibit refractoriness to platelet transfusions on this basis.
a
Other red cell antigens—including Le ,
b
2
Le , Ii, and P as well as the Cromer antigens
associated with decay accelerating factor3—are also found on platelets, but there
is no evidence that antibodies to these antigens significantly reduce platelet survival in
vivo.

HLA Antigens
HLA antigens are found on the surfaces of
both platelets and white cells (see Chapter 17). In fact, platelets are the major
source of Class I HLA antigens in whole
blood. 2 Recent evidence indicates that
most Class I HLA molecules on platelets
are integral membrane proteins, and
smaller amounts may be absorbed from
surrounding plasma.3
HLA alloantibodies do not occur naturally, arising only after sensitization by
pregnancy or blood transfusion. Studies of
HLA alloimmunization in patients transfused with platelets document the development of antibodies within 3 to 4 weeks after
primary exposure and as early as 4 days after secondary exposure in patients previ4
ously transfused or pregnant. The likelihood of HLA alloimmunization by
transfusion in patients not previously sensitized is variable,4,5 and the risk of HLA
alloimmunization appears to be related to
the underlying disease as well as to the
immunosuppressive effects of treatment
regimens. Platelets carry Class I HLA antigens but lack Class II antigens, which are
necessary for primary sensitization. Therefore, exposure to leukocytes expressing
HLA antigens during transfusion is the

principal cause of primary HLA alloimmunization.

Platelet Transfusion Refractoriness
A less-than-expected increase in platelet
count occurs in about 20% to 70% of
multitransfused thrombocytopenic patients,6 and patients treated for malignant
hematopoietic disorders are particularly
likely to become refractory to platelet
transfusions. A widely accepted definition
of refractoriness was used in a randomized controlled clinical trial of platelet
transfusion therapy, sponsored by the National Institutes of Health (NIH). In this
study, two consecutive 1-hour posttransfusion platelet corrected count increments
2
(CCI) of less than 5000 platelets × m body
surface area/µL indicated refractoriness.7
Others have used less stringent criteria
(eg, three platelet transfusions over a
2-week period that yield inadequate posttransfusion platelet counts).8,9 Response is
often determined by calculating either a
CCI or a posttransfusion platelet recovery
(PPR) between 10 and 60 minutes after
transfusion (see Table 16-1). Responses of
7500 platelets × m2 body surface area/µL
or 20% can be considered acceptable from
the CCI or the PPR calculation, respectively.
Alloimmune platelet refractoriness is
most often the result of HLA sensitization
and can be diagnosed by demonstration of
significant levels of HLA antibodies. Patient
serum is tested against a panel of lymphocytes (or synthetic beads bearing Class I antigens) that represent most of the Class I
HLA specificities in the population. A
panel-reactive antibody (PRA) score of 20%
or higher is evidence that HLA sensitization
may be contributing to the platelet refractoriness (see Chapter 17).
Although platelet alloimmunization is
one cause of refractoriness, there are multi-

Chapter 16: Platelet and Granulocyte Antigens and Antibodies

363

Table 16-1. Determination of Response to Transfused Platelets
Calculation of Corrected Count Increment (CCI)
CCI = Body Surface Area (m2) × Platelet Count Increment × 1011
No. of Platelets Transfused
EXAMPLE: If 4 × 10 platelets are transfused to a patient whose body surface area is 1.8 m and
the increase in posttransfusion platelet count is 25,000/µL, then:
11

CCI =

1.8 m 2 × 25,000 / µL × 1011
4 × 10

= 11,250 platelets × m 2 / µL

Calculation of Posttransfusion Platelet Recovery (PPR)
PPR(%) =

Estimated Total Blood Volume* × Platelet Count Increment
No. of Platelets Transfused

*Total blood volume can be estimated in adult patients as 75 mL/kg
EXAMPLE: If 4 × 1011 platelets are transfused to a 70-kg patient and the increase in
posttransfusion platelet count is 25,000/µL, then:
PPR =

70 kg × 70 mL / kg × 25,000 plts / µL × 10 3
4 × 1011 platelets

ple, nonimmune reasons why transfused
platelets may not yield the expected increase in platelet count [eg, sepsis, disseminated intravascular coagulation (DIC), or
the administration of certain drugs]. Some
of the most commonly cited nonimmune
causes of platelet refractoriness are listed in
Table 16-2. A study of patients undergoing
marrow transplant suggested that patient-related variables such as total body irradiation, advanced disease status, and liver
dysfunction are important predictors of
poor platelet count increments as well.10,11
Even when possible immune causes of refractoriness are identified, nonimmune factors are often simultaneously present.
Several strategies may be considered
when selecting platelets for patients with
immune-mediated refractoriness. When
antibodies to HLA antigens are demonstrated, a widely used approach is to supply

= 30.6%

apheresis platelets matched to the patient’s
12
HLA type. A disadvantage is that a pool of
several thousand HLA-typed potential
apheresis donors is necessary to find sufficient HLA-compatible matches.13 Moreover,
donor selection on the basis of HLA type
can lead to the exclusion of donors with
HLA types different from that of the recipient but potentially effective if the recipient
is alloimmunized to other antigenic determinants.14 For patients who are likely to require multiple platelet transfusions, HLA
typing should be performed in advance of a
planned course of treatment.
It is important to understand the degree
of match that may be provided (see Table
16-3). Platelets received following a request
for “HLA-matched” platelets are typically
the closest match obtainable within the
constraints of time and donor availability.
In one study,15 43% of platelets provided as

364

AABB Technical Manual

Table 16-2. Some Nonimmune Causes
of Platelet Refractoriness
Massive bleeding
Fever
Sepsis
Splenomegaly (splenic sequestration)
Disseminated intravascular coagulation
Allogeneic transplantation
Poor storage of platelets before transfusion
Effects of drugs (may include immune
mechanisms)
Intravenous amphotericin B
Thrombotic thrombocytopenic purpura

HLA-matched were relatively poor grade B
or C matches. The most successful responses occur with the subset of grade A
and B1U or B2U HLA matches, but mismatches for some antigens (B44, 45) that
are poorly expressed on platelets can be
useful. According to AABB Standards for
Blood Banks and Transfusion Services,16(p43)
HLA-matched platelets should be irradiated to prevent transfusion-associated
graft-vs-host disease.
A second approach to provide effective
platelets is to use a pretransfusion platelet

crossmatching assay. This approach can be
used to predict and, therefore, avoid subsequent platelet transfusion failures.17 The
solid-phase red cell adherence test (SPRCA)
is the most widely used method for platelet
crossmatching, and test results are reasonably predictive of posttransfusion platelet
counts.18-20 Compared with HLA matching,
crossmatching can prove both more convenient and economically advantageous. It
avoids exclusion of HLA-mismatched but
compatible donors and has the added advantage of selecting platelets when the antibody (-ies) involved is (are) directed at a
platelet-specific antigen. Platelet crossmatching, however, will not always be successful, particularly when patients are
highly alloimmunized (PRA >50%). In these
instances, finding sufficient compatible
units may be problematic, and selection of
HLA-matched platelets may be more practical. Although the incidence of plateletspecific antibodies causing patients to be
refractory to most or all attempted platelet
transfusions is very small, this possibility
should be investigated when most of the attempted crossmatches are positive. If platelet-specific antibodies are present, donors
of known platelet antigen phenotype or

Table 16-3. Degree of Matching for HLA-Matched Platelets
Match
Grade

Description

Examples of Donor
Phenotypes for a Recipient
Who Is A1,3;B8,27

4-antigen match

A1,3;B8,27

B1U

1 antigen unknown or blank

A1,-;B8,27

B1X

1 cross-reactive group

A1,3;B8,7

B2UX

1 antigen blank and 1 cross-reactive

A1,-;B8,7

1 mismatched antigen present

A1,3;B8,35

2 or more mismatched antigens present

A1,32;B8,35

Random

A2,28;B7,35

Chapter 16: Platelet and Granulocyte Antigens and Antibodies

family members, who are more likely to
share the patient’s phenotype, should be
tested.
An alternative approach to supplying
HLA-compatible transfusions is to determine the specificity of the patient’s HLA antibodies and select donors whose platelets
lack the antigens with which the antibodies
react. This is termed the antibody specific8
ity prediction (ASP) method. One study
compared the effectiveness of transfused
platelets selected by the ASP method with
those selected on the basis of HLA matching, platelet crossmatching, or on a random
basis.8 Platelets selected by the ASP method
were equally effective as those selected by
HLA matching or by crossmatching, and
superior to randomly selected platelets. In
addition, from a file of HLA-typed donors,
many more potential donors were identified by the ASP method than were available
using traditional HLA matching criteria,
making the acquisition of compatible
platelets for alloimmunized refractory patients much more feasible.
A further refinement of HLA matching
was proposed by Duquesnoy.21 A computerized algorithm—HLA Matchmaker, available
at http://tpis.upmc.edu/tpis/HLAMatchmaker—
is employed for evaluation of the molecular
similarities and differences between HLA
Class I epitopes. First developed to aid in
locating compatible organs for alloimmunized prospective renal transplant patients, the strategy is based on the concept
that immunogenic epitopes are represented by amino acid triplets on exposed
parts of protein sequences of the Class I
alloantigens that are accessible to alloantibodies. Using this scheme, many Class I
HLA antigens classified as mismatches to a
patient’s HLA type have no incompatible
exposed amino acid triplets and, therefore,
would not be expected to elicit an antibody
response. The pool of potentially compatible HLA-selected donors is thereby greatly

365

expanded. Although this strategy may
prove useful in selecting platelet donors for
refractory patients, it has not yet been evaluated in a clinical trial for this purpose and
remains to be validated for this indication.

Prevention of Platelet Alloimmunization
Once refractoriness resulting from platelet alloimmunization is established, it is
very difficult, if not impossible, to reverse.
Therefore, in addition to developing methods of selecting compatible platelet donors for the refractory patient, several
strategies have been evaluated to prevent
alloimmunization to platelets from occurring in the first place. They include reduction in the number of leukocytes in the
platelet products and ultraviolet B (UVB)
irradiation. The report of the Trial to Reduce Alloimmunization to Platelets (TRAP)
Study Group7 indicated that use of either
leukocyte-filtered or UVB-irradiated blood
components reduced the incidence of HLA
antibody generation from 45% to between
17% and 21%. The incidence of platelet
refractoriness was reduced from 16% to
between 7% and 10%. Although a relationship had been reported between alloimmunization and the number of donor
exposures in one report,22 a second study23
found no relationship between the number of donor exposures and the rate or severity of alloimmunization. The TRAP
study found that leukocyte reduction, not
the number of donor exposures, was significant in modifying the rate of alloimmunization. Thus, leukocyte-reduced,
pooled, whole-blood-derived platelets
appear to be clinically equivalent to
apheresis platelets, at least in terms of reducing primary alloimmunization.24
Antibodies to HLA antigens may be detected by lymphocytotoxicity tests or by
many of the platelet antibody tests discussed below. Lymphocytotoxicity tests de-

366

AABB Technical Manual

tect complement-binding antibodies capable of killing lymphocytes. One strategy for
managing patients who are receiving multiple platelet transfusions and who have developed clinical refractoriness is to test for
the presence of HLA antibodies using a
lymphocytotoxicity antibody screen against
a panel of lymphocytes representing most
of the Class I HLA antigens present in the
donor population. Reactivity to greater
than 20% of the cells in the panel (PRA
>20%) indicates that HLA sensitization may
be at least a contributing cause of the
platelet refractoriness. Newer, more sensitive HLA antibody detection techniques
such as the Flow PRA 25 (One Lambda,
Canoga Park, CA) have been adapted to a
PRA result format, and some HLA testing
laboratories use these methods instead of
the traditional lymphocytotoxicity test.
The laboratory detection of lymphocytotoxic antibodies does not necessarily indicate that the patient will experience reduced survival of transfused platelets.
Moreover, HLA antibodies may disappear
from the patient’s plasma despite continued exposure through transfusions.26 A
fuller discussion of HLA antibody and antigen testing can be found in Chapter 17.

Platelet-Specific Alloantigens
To date, 22 platelet-specific alloantigens
have been characterized as to their localization to platelet surface glycoprotein
structures, quantification of their density
on the platelet surface, and determination
of DNA polymorphisms in genes encod27
ing for them (see Table 16-4). Several
others have been described serologically,
but genetic polymorphisms underlying
them have not yet been determined.28 The
term “platelet specific,” is a misnomer for
some of these markers because they may
be found on other types of cells as well
(especially endothelial cells). However,

their chief clinical importance remains
linked to their presence on platelets. Of
the dozens of recognized platelet membrane glycoproteins, at least five [GPIa, Ib
(alpha and beta), IIb, IIIa, and CD109] are
polymorphic and have been demonstrated to be alloimmunogenic.28 In addition, rare individuals who lack a sixth
membrane glycoprotein, GPIV (CD36),
may become sensitized to this antigen.29
Approximately 3% to 5% of individuals of
Asian or African ethnicity lack GPIV on
their platelets30 and can become immu31
nized by transfusion or pregnancy. Although antibodies to these various membrane glycoproteins may be associated, in
rare instances, with refractoriness to platelet transfusions, alloantibodies to platelet-specific antigens are more often associated with the alloimmune syndromes
posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia
(NAIT).
Several antigen systems on platelets are
now recognized32 (Table 16-4).27,33 The nomenclature adopted by the International
Society of Blood Transfusion classifies the
systems numerically according to the date
of publication and alphabetically to reflect
their frequency in the population.34 As with
red cells, different terminologies for platelet
antigens often coexist. The first recognized
antigen,35 Zwa, is now designated HPA-1a of
the HPA-1 system. The HPA-1a antigen is
often better known as PlA1. HPA-1a is present on the platelets of about 98% of persons
of European ethnicity and anti-HPA-1a
(anti-PlA1) is the most frequently encountered clinically significant platelet-specific
antibody in this population. Its antithetical
antigen, HPA-1b (PlA2), occurs in 27% of this
population.
The HPA-1a and HPA-1b alleles reside on
the platelet membrane glycoprotein GPIIIa.
Patients with Glanzmann’s thrombasthenia
Type I, a disorder of platelet function, lack

Table 16-4. Alloantigenic Polymorphisms of Platelet Glycoproteins that Have Been Implicated in Alloimmune Syndromes*
HPA System
Name

Antigens (Familiar
Names)

Phenotypic
Frequencies

GP Location

Amino Acid
Substitution

Alloimmune
Syndromes

Leu↔Pro33

NAIT, PTP

Arg↔Gln143

NAIT, PTP

POLYMORPHISMS OF GLYCOPROTEIN IIIa
98%

IIIa

HPA-1b, (Pl , Zw )

27%

IIIa

HPA-4a (Pena, Yukb)†

99.9%

IIIa

HPA-4b (Pen , Yuk )

<1%

IIIa

HPA-6

HPA-6bw (Caa, Tua)

<1%

IIIa

Arg↔Gln489

NAIT

HPA-7

HPA-7bw (Mo)

<1%

IIIa

Pro↔Ala407

NAIT

<1%

IIIa

Arg↔Cys636

NAIT

HPA-4

HPA-8
HPA-10
HPA-11
HPA-14
HPA-16

HPA-8bw (Sr )
a

HPA-10bw (La )

<1%

IIIa

Arg↔Gln62

NAIT

<1%

IIIa

Arg↔His633

NAIT

HPA-14bw (Oe )

<1%

IIIa

Lys611 Deleted

NAIT

<1%

IIIa

Ile↔Thr140

NAIT

IIe↔Ser843

NAIT, PTP

Val↔Met837

NAIT

HPA-11bw (Gro )
a

HPA-16bw (Duv )

Chapter 16: Platelet and Granulocyte Antigens and Antibodies

HPA-1a, (PlA1, Zwa)

HPA-1

POLYMORPHISMS OF GLYCOPROTEIN IIb
HPA-3

HPA-3a (Baka, Leka)

85%

IIb

HPA-3b (Bak , Lek )

63%

IIb

HPA-9bw (Maxa)

0.6%

IIb

HPA-9

367

(cont’d)

368

HPA System
Name

Antigens (Familiar
Names)

Phenotypic
Frequencies

GP Location

Amino Acid
Substitution

Alloimmune
Syndromes

glu↔Lys505

NAIT, PTP

Thr↔Met799

NAIT

Thr↔Met145

NAIT

Gly↔Glu15

NAIT

POLYMORPHISMS OF GLYCOPROTEIN Ia
HPA-5

HPA-5a (Brb, Zavb)

99%

HPA-5b (Br , Zav )

20%

HPA-13bw (Sita)

<0.2%

HPA-13

POLYMORPHISMS OF GLYCOPROTEIN Ib
HPA-2

HPA-2a (Kob, Sibb)

99%

Ib alpha

HPA-2b (Ko , Sib )

15%

Ib alpha

HPA-12bw (Iya)

0.3%

Ib beta

HPA-12

OTHER PROBABLE PLATELET ALLOANTIGEN SPECIFICITIES
HPA-15

HPA-15a (Govb)
a

HPA-15b (Gov )

80%

CD109

60%

CD109

*Modified from Kroll.27 GP = glyprotein; NAIT = neonatal alloimmune thrombocytopenia; PTP = posttransfusion purpurpa.

Tyr↔Ser703

NAIT, PTP

AABB Technical Manual

Table 16-4. Alloantigenic Polymorphisms of Platelet Glycoproteins that Have Been Implicated in Alloimmune Syndromes*
(cont’d)

Chapter 16: Platelet and Granulocyte Antigens and Antibodies

this glycoprotein and, therefore, do not express HPA-1 antigens. The HPA-1 polymorphism arises by the substitution of a single
base pair (leucine in HPA-1a and proline in
HPA-1b) at amino acid position 33 of the
protein’s DNA coding sequence. GPIIIa is
also the carrier of HPA-4, -6, -7, -8, -10, -11,
-14, and -16 antigens. Alleles in each of
these systems also arise as a result of single
amino acid substitutions at different positions. The HPA-2 antigen system is situated
on GPIb alpha; the HPA-3 system on GPIIb;
and the HPA-5 system on GPIa.36,37
On the platelet membrane, most of the
glycoproteins that carry these “platelet-specific” antigens are present as heterodimeric
compounds, ie, each consists of two differ38
ent glycoprotein molecules (see Fig 16-1 ).
Therefore, platelet glycoprotein names are

369

often paired (eg, Ia/IIa, IIb/IIIa, or Ib/IX),
referring to the alpha and beta chains in
each complex. GPIb/IX is a leucine-rich
membrane glycoprotein that serves as a receptor for von Willebrand factor on platelets. The Ia/IIa and IIb/IIIa complexes are
members of a broadly distributed family of
adhesion molecules called integrins. Integrins are essential for platelet adhesion and
aggregation because the molecules serve as
receptors for ligands such as fibrinogen
(IIb/IIIa), von Willebrand factor (Ib/IX), and
collagen (Ia/IIa). When present on other
cells, the glycoprotein pairings may differ.
For example, on platelets, GPIIIa is normally paired with GPIIb. On endothelial
cells, fibroblasts, and smooth muscle, however, GPIIIa is paired with a different
glycoprotein. Thus, these cells share the

Figure 16-1. Schematic diagram of platelet glycoprotein complex IIb/IIIa. Dots and letters (Yuk, Oe,
Pl a , Ca, Gro, Sr, Mo, Bak, Max) designate positions and names of recognized allotypic epitopes. The
molecular regions where autoepitopes have been recognized are indicated by brackets. 38

370

AABB Technical Manual

HPA alloantigens found on the GPIIIa molecule, but not those found on the GPIIb
molecule.
CD109 is an exception to the heterodimeric rule, occurring as a monomeric
structure on the platelet membrane. This
GPI-linked protein is found on activated T
cells, cultured endothelial cells, several tumor cell lines, as well as platelets.39 Two
a
alloantigens designated HPA-15wb (Gov )
b
and HPA-15wa (Gov ) have been localized
to platelet CD109.39 Unlike most plateletspecific alloantigens, both alleles are highly
expressed [0.53% (Govb) and 0.47% (Gova)]
in persons of European ethnicity (Table
16-4). Sensitization to Gov alloantigens has
been associated with platelet refractoriness,
NAIT, and PTP, albeit usually together with
other alloantibodies to platelet antigens.40

Clinical Importance of Platelet-Specific
Antigens and Antibodies

Neonatal Alloimmune Thrombocytopenia
Neonatal alloimmune thrombocytopenia
(variously abbreviated NAIT, NATP, etc) is
described in Chapter 23.

Posttransfusion Purpura
Posttransfusion purpura (PTP) is characterized by the development of dramatic,
sudden, and self-limiting thrombocytopenia
5 to 10 days after a blood transfusion in a
patient with a history of sensitization by
pregnancy or transfusion. Coincident with
the thrombocytopenia is the development
of a potent platelet-specific alloantibody
in the patient’s serum, usually anti-HPA1a. Other specificities have been implicated,
almost always associated with antigens
on GPIIb/IIIa.37,41 PTP differs from transfusion reactions caused by red cell antibodies because the patient’s own antigennegative (usually HPA-1a-negative) platelets as well as any transfused antigen-positive platelets (which may be accompa-

nied by clinically severe reactions) are destroyed. Transfusion of antigen-negative
platelets may be of value during the acute
phase of PTP; however, such platelets
have a reduced in-vivo survival.42 Plasmap h e re s i s — o n c e t h e t re a t m e n t o f
choice—has largely been supplanted by
the use of intravenous immune globulin
(IGIV ). The mechanism by which these
treatments are efficacious is unknown.
Platelet antibody assays usually reveal a
serum antibody with HPA-1a specificity.
Typing of the patient’s platelets after recovery will document a HPA-1a-negative
phenotype or analogous typing for other
platelet-specific antigen systems. Following recovery, future transfusions should
be provided using washed antigen-negative RBC units if possible. Washed RBC
units may offer some protection against
recurrence, although at least one case of
PTP caused by antibody to HPA-5b was
precipitated by transfusion of a washed
RBC unit.43

Testing for Platelet-Specific Antigens and
Antibodies
Clinically useful platelet antibody assays
emerged later than serologic assays to diagnose immunologic disorders involving
red cells. This is mainly because it is difficult to separate platelets from whole blood
specimens and to distinguish antibodydependent endpoints from nonspecific
changes that occur in platelets under assay conditions. Three types of platelet antibody detection methods have been developed44 (Table 16-5). The earliest were
Phase I assays that involved mixing patient serum with normal platelets and
used platelet function-dependent endpoints such as alpha granule release, aggregation, or agglutination. Phase II tests
measured either surface or total plateletassociated immunoglobulin on patient

Chapter 16: Platelet and Granulocyte Antigens and Antibodies

371

Table 16-5. Platelet Antibody Assays
Phase I Assays
Platelet aggregation
Inhibition of platelet aggregation
Inhibition of clot retraction
Inhibition of platelet migration
Complement fixation
Platelet factor 3 release
51
Chromium release
14
C-Serotonin release
Phase II Assays
Detection of platelet surface-associated immunoglobulin
■ Platelet suspension immunofluorescence test (PSIFT)
■ Flow cytometry
125
125
■ Radioimmunoassay ( I staphylococcal Protein A, I antihuman immunoglobulin, polyclonal or
monoclonal)
■ Antiglobulin consumption (two-stage assay)
■ Solid-phase red cell adherence
Detection of total platelet-associated immunoglobulin
■ Nephelometry
■ Electroimmunoassay
■ Radial immunodiffusion
Phase III Assays
Monoclonal antibody immobilization of platelet antigens (MAIPA)
Antigen capture ELISA (ACE)
Modified antigen capture ELISA (MACE)
Immunobead assay
Immunoblotting

platelets or on normal platelets after sensitization with patient serum. Phase III
solid-phase assays were developed in
which the binding of antibodies to isolated platelet surface glycoproteins is detected. The test methods are examples of
Phase I, II, and III assays; variations of each
test method are also used. Lymphocytotoxicity tests are discussed in Chapter 17.
Mixed Passive Hemagglutination Assay
(MPHA). A Phase II assay used for the detection of platelet-specific antibodies as
well as for platelet crossmatching is the
MPHA. Shibata et al were the first to use
this method to detect and identify clinically

significant platelet alloantibodies.45 A modification of MPHA, the SPRCA, is widely
used.18 In this assay, intact platelets are immobilized in the round-bottom wells of a
microtiter tray and are sensitized with antibody to be detected. After washing, detector red cells previously coated with an antibody specific for human immunoglobulin
are added. After incubation from several
hours to overnight, the tray is subjected to a
slow centrifugation and examined visually.
If antibody is bound to the immobilized
platelets, the indicator red cells fail to form
a compact button in the center of the well
because they are evenly distributed like a

372

AABB Technical Manual

“carpet” over the antibody-coated platelets.
In a negative reaction, a red cell button forms
in the center of the well. A limitation of the
MPHA assay is that it fails to distinguish
platelet-specific from non-platelet-specific
antibodies. A modification of the MPHA assay, the Capture-P (Imucor Gamma, Norcross,
GA), is available as a commercial kit and is
most often marketed for platelet crossmatching.18 SPRCA testing may be modified
by treatment of target platelets with chloroquine or acid,46,47 which disrupts the Class I
HLA heavy chain-peptide-β2-microglobulin
trimolecular complex. This modifies antigenic epitopes, reducing the binding of
specific antibodies directed against HLA on
platelets. However, strong HLA antibodies
may still bind, giving the impression that
the antibody is directed to non-HLA antigens.
Flow Cytometry. Another example of a
Phase II assay is platelet antibody detection
using immunofluorescence. Originally a
slide-based method,48 the technique now
uses flow cytometry to detect platelet-reactive antibody in patient sera that binds to
intact platelets.49 In the assay, washed platelets are sensitized with patient or control
serum for up to 60 minutes, usually at room
temperature. The platelets are then washed
repeatedly to remove nonspecific immunoglobulins, and platelet-bound antibodies
are detected with a fluorescent-labeled
(usually fluorescein isothiocyanate, FITC)
polyclonal or monoclonal antibody specific
for human immunoglobulin. The platelets
are analyzed in the flow cytometer and results can be expressed as a ratio of the
mean or peak channel fluorescence of normal platelets sensitized with patient serum
over that of normal platelets incubated in
normal serum. In order to prevent nonspecific binding of the immunoglobulin probe
via Fc receptors on the target platelets, the
probe antibodies are enzyme treated to remove the Fc end of the molecule. There-

fore, binding of the labeled probe can be
assumed to be via its F(ab’)2 or antigen-specific end. A second fluorescent label [eg,
phycoerythrin (PE)] can be attached to an
antihuman IgM probe to detect IgM platelet antibodies. Because FITC and PE fluoresce with peak light intensities at different
wavelengths when exposed to the monochromatic argon laser in the flow cytometer
(520-nm green light and 580-nm reddishorange light, respectively), cells labeled
with FITC can be distinguished from those
labeled with PE. Both anti-IgG and antiIgM labeled with different fluorochromes
can be added to the same tube with washed
sensitized platelets for the simultaneous
detection of antiplatelet IgG and IgM.
Flow cytometry has proven to be a very
sensitive method for detection of alloantibodies. The assay is capable of detecting
very small numbers of antibody molecules
bound to platelets as is the case with alloantibodies specific for antigens of the
HPA-5 (Br) system having only 1000 to 2000
sites per platelet. Moreover, some alloantibodies that are specific for labile epitopes
that are unreliably detected in Phase III assays can be detected on intact platelets
using flow cytometry.
Because the target platelets used in the
assay are intact, flow cytometry does not
differentiate between platelet-specific (ie,
platelet glycoprotein directed) and nonplatelet-specific antibodies. Examples of
the latter are HLA and ABO antibodies. This
is an advantage when the method is used to
detect antibodies that will affect the success
of a platelet transfusion, and, for this reason, flow cytometry has been advocated as
a platelet crossmatching method. However,
when used to investigate cases of suspected
NAIT or PTP, the method has a potential
drawback—the more relevant platelet-specific antibodies characteristic in these diseases can be obscured by non-platelet-specific reactivity.

Chapter 16: Platelet and Granulocyte Antigens and Antibodies

Monoclonal Antibody-Specific Immobilization of Platelet Antigen (MAIPA). An example of a Phase III assay is the MAIPA,50-52
perhaps the most widely used assay to detect platelet-specific antibodies. The assay
requires the use of murine monoclonal antibodies (MoAbs) that recognize the target
antigens of interest but do not compete
with the human antibody being detected.
In the assay, target platelets are simultaneously sensitized with patient serum and
a murine MoAb recognizing the desired target molecule on the platelet surface. After
the initial sensitization step, platelets are
washed and solubilized in a nonionic detergent. After centrifugation to remove
cytoskeletal fragments, an aliquot of the
supernatant lysate is added to wells of a
microtiter tray containing immobilized
goat antibody specific for mouse IgG. The
MoAb is thereby captured and the platelet
surface glycoprotein with its bound human
antibody is immobilized. After a wash step,
the human antibody is detected with an
enzyme-labeled goat antihuman immunoglobulin probe.
There are several other versions of Phase
III assays in use today, including the antigen capture enzyme-linked immunosorbent
assay (ACE), the modified antigen capture
ELISA (MACE),53,54 and the commercially
55
available GTI PAKPLUS (GTI, Waukesha,
WI). Each relies on MoAbs to immobilize
only the glycoproteins of interest, thereby
reducing or eliminating interfering reactions
due to non-platelet-specific antibodies, especially anti-HLA, which, if present, is detected only in wells containing pools of immobilized HLA Class I antigens.
Platelet Typing Using Molecular Methods. Molecular typing by polymerase chain
reaction (PCR) is available for many platelet
antigens. Because immunophenotyping is
limited by the shortage of characterized
typing antisera and by low platelet counts,
several DNA-based HPA typing techniques,

373

such as restriction fragment length polymorphism (RFLP) analysis and sequencespecific oligonucleotide hybridization, have
been developed.56,57 All of these techniques
are reliable, but they are also laborious and
time-consuming. For this reason, PCR
genotyping with sequence-specific primers
(SSP) appears to be much more practical to
use.58,59 In a recent workshop, SSP-PCR was
the most common and reliable method of
determining platelet antigens,60 making it
feasible for genotyping HPAs independent
of the patient’s platelet count and of rare
typing sera.61

Autoimmune Platelet Disorders

Idiopathic (Autoimmune)
Thrombocytopenic Purpura
Autoantibodies directed against platelet
antigens may result in thrombocytopenia.
Chronic idiopathic thrombocytopenic
purpura (ITP), most often a disease in
adults, is characterized by an insidious
onset and moderate thrombocytopenia
that may exist for months to years before
diagnosis. Females are twice as likely to
be affected as males. Spontaneous remissions are rare, and treatment is usually
required to raise the platelet count. Firstline therapy consists of steroids, highdose IGIV or Rh immunoglobulin (RhIG),
followed by splenectomy in nonresponders.
Many other therapies have been used in
patients who fail to respond to splenectomy. Results have varied. Chronic autoimmune thrombocytopenia may be idiopathic or associated with other diseases
(eg, HIV infection, malignancy, other autoimmune conditions). Acute ITP is mainly
a childhood disease characterized by abrupt
onset of severe thrombocytopenia and
bleeding symptomatology, often after a
viral infection. The majority of cases resolve spontaneously over a 2- to 6-month

374

AABB Technical Manual

period. If treatment is required, IGIV or
RhIG infusions are usually effective in
raising the platelet count. Steroids are
used less often because of serious side effects in children. Splenectomy, if used, is
reserved for those children whose disease
is severe and lasts longer than 6 months,
similar to chronic ITP in adults.

Testing for Platelet Autoantibodies
Numerous Phase I, II, and III platelet antibody assays have been developed to detect relevant autoantibodies in ITP patients. Although many tests have been
demonstrated to be quite sensitive, particularly in detecting total or cell surface
platelet-associated immunoglobulins (Phase
II assays), 62 none has been sufficiently
specific to be particularly useful in either
the diagnosis or management of ITP. The
American Society of Hematology’s practice guidelines for ITP state that serologic
testing is unnecessary, assuming the clinical findings are compatible with the diagnosis.63 However, platelet antibody tests
may be helpful in the evaluation of patients
suspected of having ITP when other, nonimmune causes may be present.
The goal of serologic testing in ITP is to
detect autoantibody bound to the patient’s
own platelets with or without demonstration of similar reactivity in the patient’s
plasma. Most of the newer assays offered
for evaluation of patients suspected of having ITP are Phase III assays, designed to detect immunoglobulin binding to plateletspecific epitopes found on platelet glycoprotein complexes GPIIb/IIIa, GPIa/IIa,
and/or GPIb/IX.
These solid-phase GP-specific assays appear to have improved specificity in distinguishing ITP from nonimmune thrombocytopenia when compared to Phase II assays,
but this is often balanced by a decrease in
sensitivity.64 Moreover, all of these methods

have limited usefulness in patients who
have very low platelet counts that prevent
adequate numbers of platelets to be collected
for use in the tests.
One commercially available Phase III
test, the GTI PAKAUTO,65 uses eluates prepared from washed patient platelets. The
eluates are tested against a panel of MoAbimmobilized platelet GP complexes, and
antibody binding is detected using an enzyme-linked antihuman immunoglobulin
probe. In the indirect phase of the assay,
patient plasma is tested against the same
glycoprotein panel. In general, plasma antibodies are detected less often than antibodies in the eluates. ITP patients may
have antibodies that are reactive with one
or several GP targets. To date, there is no
correlation between the specificity of autoantibodies in ITP and disease severity.

Drug-Induced Immune Platelet Disorders
Thrombocytopenia associated with specific
drugs is not uncommon. Drugs often implicated include quinidine/quinine, sulfa
drugs, heparin, and colloidal gold. Both
drug-dependent and drug-independent
antibodies may be produced. Drug-independent antibodies, although stimulated
by drugs, do not require the continued
presence of the drug to react with platelets and are serologically indistinguishable from other platelet autoantibodies.
(Unlike typical autoimmune thrombocytopenia, these antibodies are transient except when caused by therapy with gold,
which is excreted very slowly.) Drug-dependent antibodies result when a drug
combines with platelets in such a way as to
create neoantigens to which antibodies are
formed. The drug must be present for the
antibody to react. These antibodies can
cause a thrombocytopenia of sudden and
rapid onset, usually resolving when the
drug is discontinued.

Chapter 16: Platelet and Granulocyte Antigens and Antibodies

Testing for Drug-Dependent PlateletReactive Antibodies
Serology. Virtually any platelet serology
test that is used to detect platelet-bound
immunoglobulin can be modified for use
in the detection of drug-dependent platelet-reactive antibodies. In performing
drug-dependent antibody testing, it is essential to establish the proper positive
and negative controls for the assay. Each
serum or plasma sample suspected of containing drug-dependent antibody must be
tested against normal target platelets in
the presence and absence of drug. Moreover, at least one normal serum should be
tested with and without drug to control
for any possible drug-related platelet effect that does not require specific antibody. Finally, a positive control serum
known to be reactive with the drug being
assayed should be tested with and without drug to complete the evaluation. A
positive result must show that the serum
is positive against normal target platelets
in the presence of drug and not without
drug, and that the drug did not nonspecifically cause a positive result in the
target platelet. Likewise, the positive control must be positive with the drug and
negative without it.
Flow Cytometry. The flow cytometry test
can be readily adapted to detect both IgG
and IgM drug-dependent platelet antibod53,66
ies. In this modification, fluorescence of
normal platelets sensitized with the patient’s serum in the presence of drug can be
compared with that of the patient’s sample
without drug or to a normal serum with
drug to determine relative intensity of labeling. Flow cytometry has proven to have
superior sensitivity to other assays for detection of quinine-quinidine- and sulfonamide-dependent platelet-reactive antibodies.53 Table 16-6 shows other agents for
which drug-dependent platelet-reactive an-

375

tibodies have been detected and confirmed
by flow cytometry in a large platelet immunology reference laboratory.
Flow cytometry has its limitations, as do
other antibody detection methods, in detecting drug-dependent antibodies. For
many drugs, the optimal concentration to
demonstrate in-vitro binding of antibody
has not been determined. Probably the
most extensively studied drugs in this re67
gard are quinine or quinidine. Another
cause of poor sensitivity is the weak binding of drug to platelets, leading to rapidly
declining numbers of drug molecules on
the platelet surface once drug is removed
from the environment of the platelet. It is
therefore important to maintain a critical
concentration of drug in all washing buffers
before addition of probe at the end of an
assay.66 Yet another potential reason for insensitivity is that a patient may not be sensitized to the native drug but, rather, to a
metabolite of the drug. Antibodies dependent on metabolites of acetaminophen and
sulfamethoxazole have been reported.68,69
A number of other assays have been
adopted for the detection of drug-dependent platelet antibodies. Among these are
the SPRCA.70,71 Phase I assays have also been
modified for detection of heparin-dependent platelet antibodies (see below). In some
cases, determination of the specific glycoprotein to which antibody is directed by
Phase III assays may provide useful clinical
information. For example, drug-dependent
antibody to GPIb/IX was associated with a
more acute, but reversible, quinine-induced thrombocytopenia, whereas antibody to GPIIb/IIIa was associated with a more
prolonged course.72

Heparin-Induced Thrombocytopenia
Two types of heparin-induced thrombocytopenia (HIT) have been recognized.
Type I, of nonimmune origin, presents with

376

AABB Technical Manual

Table 16-6. Drugs Confirmed to Elicit Drug-Dependent Platelet-Reactive Antibodies
In Vitro Using Flow Cytometry Testing
abciximab
acetaminophen
carbamazepine
ceftazidime
ceftizoxime
ceftriaxone
ciprofloxacin
eptifibatide
esomeprazole
fentanyl
fexofenadine

heparin
ibuprofen
levofloxacin
loracarbef
naproxen
orbofiban
oxaliplatin
phenytoin
propoxyphene
quinidine
quinine

mild transient thrombocytopenia within
minutes to several days after heparin exposure but generally resolves despite ongoing heparin therapy and is not clinically
important. In contrast, immune, or Type
II, HIT may lead to life- and limb-threatening thrombotic complications and requires
careful evaluation and management of afflicted patients.
The exact incidence of immune HIT is
unknown, but it may develop in up to 3% of
patients treated with unfractionated heparin. Low-molecular-weight heparin is less
likely to be associated with either antibody
production or thrombocytopenia.73 Bovine
heparin appears somewhat more likely to
cause HIT than porcine heparin.74 A reduction in baseline platelet count by at least
50% occurs generally within 5 to 14 days after primary exposure and sooner after secondary exposure to the drug. The platelet
count is often less than 100,000/µL and
usually recovers within 5 to 7 days upon
discontinuation of heparin.
About 30% of patients with HIT, or approximately 0.9% of patients who receive
heparin, develop thrombosis, which can
occur in the arterial, venous, or both systems.75,76 Patients may develop cardiovascular problems, myocardial infarction, limb

ranitidine
rifampin
sulfamethoxazole
sulfisoxazole
suramin
tirofiban
trimethoprim
vancomycin
xemilofiban

ischemia, deep venous thrombosis, or
ischemia of other organs. The thrombotic
complications may force limb amputation
or may prove fatal.
Thrombosis or an unexplained decrease
in platelet count while on heparin therapy
should raise concern about HIT. Heparin,
including heparin flushes and heparincoated catheters, should be discontinued,
and the patient should be evaluated for
laboratory evidence of HIT and signs of
thrombosis. In mild-to-moderate thrombocytopenia, monitoring of platelet counts
and observation may be sufficient, but because of the high risk of thrombosis, treatment with alternative anticoagulants is
generally recommended. Warfarin should
be avoided in the early treatment of HIT
because it does not prevent thrombosis in
this setting and may provoke limb-threatening venous gangrene by reducing levels
of naturally occurring anticoagulants faster
than it reduces activated coagulation factors. However, warfarin can and should be
used after the patient is anticoagulated
with alternative drugs. Suitable anticoagulants approved by the Food and Drug Administration (FDA) include hirudin (a natural thrombin inhibitor) and argatroban.
Selected patients may also benefit from

Chapter 16: Platelet and Granulocyte Antigens and Antibodies

thrombolectomy or thrombolytic therapy.
Platelet transfusion should be avoided,
given that bleeding is a rare complication in
HIT, and administration of platelets may
precipitate thrombosis.77-79
Heparin forms a complex with platelet
factor 4 (PF4), a tetrameric protein released
from platelet alpha granules. Antibodies
(IgG, IgM, and some IgA) form against various epitopes on this complex and attach to
platelet Fcγ IIa receptors, whereby platelets
become activated. The antibody may also
bind to the complexes at other sites, notably on endothelial cells. Thus, HIT might
involve activation and damage not only of
platelets but also of endothelium, causing
increased susceptibility to thrombosis. This
new understanding of the mechanism of
heparin antibodies is exploited by ELISA
tests in which microwells are coated with
the complexes rather than with the platelets themselves.80

Testing for Heparin-Dependent Antibodies
The PF4 ELISA is an example of a Phase
III assay for HIT. Target complexes of PF4
and heparin or heparin-like molecules are
immobilized on a solid phase. To perform
the test, patient serum is added to premade
complexes of PF4 and heparin or heparin-like molecules (eg, polyvinyl sulfate,
PVS) alone and in the presence of highdose (100 U/mL) heparin. Heparin-dependent antibody binds to the complexes
and is detected via enzyme-conjugated
antihuman immunoglobulin. An optical
density value above 0.4 in the PF4-PVS
well that is inhibited by high-dose heparin confirms the presence of a heparindependent antibody in the patient’s sample. Although IgG antibodies are the most
clinically relevant antibodies causing this
81
syndrome, occasional patients with HIT
appear to have only non-IgG (IgM or IgA)
antibodies.82 The PF4 ELISA detects but

377

does not differentiate IgG, IgM, and IgA
antibodies that bind to the PF4-heparin
complex.
The 14C-serotonin release assay (SRA) is
an example of a Phase I assay for detection
of heparin-dependent antibodies.83 Normal,
fresh target platelets are incubated with
14
C-serotonin that is taken up into the
dense granules of the platelets. Then, target
platelets are exposed to patient serum in
the presence of low and high concentrations of heparin. Release of at least 20% of
the radioactive label at the low dose of heparin and inhibition of this release at the
high dose confirms the presence of heparin-dependent antibodies. Other functional
tests used to detect heparin-dependent antibodies include the heparin-induced
platelet aggregation test and the heparininduced platelet activation test.
The PF4 ELISA and the SRA are both
more sensitive and specific than the platelet aggregation test for the detection of heparin-dependent platelet antibodies in patients for whom there is clinical suspicion
of HIT.83-85 However, in asymptomatic patients receiving heparin or in those who
have not yet received the drug, neither test
is sufficiently predictive of HIT to warrant
its use in screening.

Granulocyte Antigens
Analogous to platelet alloantigens, neutrophil alloantigens are implicated in
clinical syndromes including neonatal
alloimmune neutropenia (NAN), transfusion-related acute lung injury (TRALI),
immune neutropenia after marrow transplantation, refractoriness to granulocyte
transfusion, and chronic benign autoimmune neutropenia of infancy. A neutrophil
equivalent of PTP has not been described.
To date, seven neutrophil alloantigens have
been described, including localization to

378

AABB Technical Manual

neutrophil surface glycoprotein structures
and, in some cases, determination of DNA
polymorphisms in genes encoding for them
(see Table 16-7).
The first granulocyte-specific antigen,
NA1 (HNA-1a), was described in 1966 by
Lalezari and Bernard.86 HNA-1a and its antithetical antigen, HNA-1b, are present on
FcγRIIIb. Antibodies to HNA-1a and -1b
have been implicated in TRALI, NAN, and
autoimmune neutropenia of infancy. About
0.1% of individuals of European ethnicity
have neutrophils with no detectable
FcγRIIIb (NAnull). The FcγRIII protein also
carries the neutrophil alloantigen SH
(HNA-1c).87 NB1 (HNA-2a) is found on another granulocyte surface glycoprotein,
CD177, the function of which is still undetermined. HNA-2a has been reported to
have an allele, NB2, but the product of this
gene cannot be reliably identified with
alloantisera, and no MoAb specific for NB2

has been identified; therefore, the existence
of a second allele to HNA-2a (NB1) is unproven.88 HNA-2a has been associated with
TRALI and NAN. The DNA sequence of the
NB1(HNA-2a) gene has been determined as
have the molecular polymorphisms associated with HNA-1a and HNA-1b and HNA-1c
on the gene for FcγRIII. Therefore, genotyping for these specificities can be performed
on genomic DNA using PCR-SSP.89 Reduced
expression of granulocyte antigens occurs in
paroxysmal nocturnal hemoglobinuria,
chronic myelogenous leukemia, and in premature infants.
Additional antigens on granulocytes are
shared with other cells and are not granulocyte-specific. These include 5b (HNA-3a),
MARTa (HNA-4a), and ONDa (HNA-5a). The
HNA-3a is located on a 70- to 95-Kd protein
that has not yet been cloned. HNA-3a is also
expressed on the surface of lymphocytes.
The antibodies directed at this antigen are

Table 16-7. Neutrophil Alloantigens
Antigen Frequency (%)
Antigen
System

Allele

HNA
Designation

White

Black

Glycoprotein
Location

Neutrophil-specific
NA

NA1

HNA-1a

FcγRIIIb (CD16)

NA2

HNA-1b

FcγRIIIb (CD16)

HNA-1c

FcγRIIIb (CD16)

NB1

HNA-2a

CD177

HNA-3a

70-95 kD GP

HNA-4a

CD11b

HNA-5a

CD11a

Neutrophil-nonspecific
5
MART
OND

5b
MART
OND

Chapter 16: Platelet and Granulocyte Antigens and Antibodies

usually agglutinins; they occasionally occur
in women after pregnancy and may be associated with febrile transfusion reactions.
Potent anti-HNA-3a agglutinins in transfused plasma have been responsible for fa90-92
a
a
MART and OND , both hightal TRALI.
incidence antigens, are also present on
monocytes and lymphocytes. MARTa has
been localized to the alpha M chain
(CD11b) of the C3bi receptor (CR3) and results from a single nucleic acid substitua
tion. MART has been recently reported to
93
a
cause NAN. OND is expressed on the alpha L integrin unit, leukocyte function antigen-1 (CD11a) and also results from a single nucleotide substitution. This marker,
found in a chronically transfused aplastic
anemia patient, has not been reported to
be associated with clinical disease.

Clinical Syndromes in Which
Granulocyte-Specific Alloantigens Are
Implicated
Nonhemolytic, febrile transfusion reactions are often associated with granulocyte antibodies. Although such reactions
are more often caused by antibodies to
Class I HLA antibodies directed at Class I
epitopes present on granulocytes, granulocyte-specific antibodies have been associated with clinical syndromes similar to
those seen with antibodies to red cell and
platelet antigens.

Neonatal Alloimmune Neutropenia
NAN is caused by maternal antibodies
against alloantigens of fetal neutrophils;
the most frequent specificities seen are
against NA1, NA2, and NB1 antigens (see
Table 16-7). NAN most often occurs in
women of the neutrophil alloantigen phenotypes NA1/NA1 and NA2/NA2; it may
also occur in women of the rare NAnull
phenotype who lack the FcγRIII protein.
The neutropenia in all these cases can oc-

379

casionally be life-threatening because of
increased susceptibility to infection. Management with antibiotics, IGIV, granulocyte colony-stimulating growth factor,
and/or plasma exchange may be helpful.

TRALI
TRALI is an acute, often life-threatening
reaction characterized by respiratory distress, hypo- or hypertension, and noncardiogenic pulmonary edema that occurs within 6 hours of a transfusion of a
plasma-containing blood component.
TRALI has been reported to be induced by
neutrophil antibodies, although more recent reports are more likely to implicate
antibodies to both Class I and II HLA anti94
gens. In TRALI, the causative antibodies
are most often found in the plasma of the
blood donor (see Chapters 17 and 27). Recent reports have postulated that another
etiology for TRALI is possible. This theory
suggests that two events must coincide
for TRALI to occur: 1) the patient must
have a predisposing clinical condition
that releases cytokines or other factors
that prime neutrophils, causing adherence to endothelium, and 2) the patient
must receive a transfusion of biologically
active lipids (which also stimulate neutrophils) from stored blood components.95 In
one study, neutrophil antibodies were detected in only three of 10 incidents of
TRALI, and none of the donors had HLA
antibodies.
Autoimmune neutropenia, usually occurring in adults, may be idiopathic or may
occur secondary to such diseases as rheumatoid arthritis, systemic lupus erythematosus, or bacterial infections. In autoimmune neutropenia of infancy, usually
occurring in children between the ages of 6
months to 2 years, the autoantibody has a
neutrophil antigen specificity (usually
HNA-1a and -1b) in about half the cases.

380

AABB Technical Manual

The condition is usually self-limiting (with
recovery usually in 7-24 months) and the
condition is relatively benign and manageable with antibiotics.96 Drug-dependent antibodies can also cause neutropenia.

Testing for Granulocyte Autoantibodies
Tests for granulocyte antibodies are not
widely performed, although the implication of neutrophil antibodies as a cause of
TRALI has increased the demand for this
laboratory resource. Agglutination tests
performed in tube, capillary, or microplate formats use heat-inactivated serum
in the presence of EDTA and require fresh
granulocytes. Immunofluorescence tests,
read with either a fluorescence microscope or a flow cytometer, are also used
and are capable of detecting granulocytebound antiglobulin. A combination of
agglutination and immunofluorescence
tests is beneficial.97 Other methods include
chemiluminescence and a MoAb-specific
immobilization of granulocyte antigens
(MAIGA) assay, similar to the MAIPA assay. An advantage of the MAIGA assay is
its ability to differentiate readily between
HLA and granulocyte-specific antibodies.

10.

11.

12.

13.

14.

References
1.

Ogasawara K, Ueki J, Takenaka M, Furihata K.
Study on the expression of ABO antigens on
platelets. Blood 1993;82:993-9.
Bialek J, Bodmer W, Bodmer J, Payne R. Distribution and quantity of leukocyte antigens
in the formed elements of blood. Transfusion
1966;6:193-204.
Feuerstein N, Mono DS, Cooper HL. Phorbolester effect in platelets, lymphocytes, and
leukemic cells (HL-60) is associated with enhanced phosphorylation of Class I HLA antigens. Biochem Biophys Res Commun 1985;
126:206-13.
Howard JA, Perkins HA. The natural history of
alloimmunization to platelets. Transfusion
1978;18:496-503.
Godeau B, Fromont P, Seror T, et al. Platelet
alloimmunization after multiple transfu-

15.

16.

17.

18.

19.

sions: A prospective study of 50 patients. Br J
Haematol 1992;81:395-400.
Dzik WH. Leukoreduced blood components:
Laboratory and clinical aspects. In: Simon
TL, Dzik WH, Snyder EL, et al, eds. Rossi’s
principles of transfusion medicine. 3rd ed.
Philadelphia, PA: Lippincott Williams and
Wilkins, 2002:270-87.
The Trial to Reduce Alloimmunization to
Platelets Study Group. Leukocyte reduction
and ultraviolet B irradiation of platelets to
prevent alloimmunization and refractoriness
to platelet transfusions. N Engl J Med 1997;
337:1861-9.
Petz LD, Garratty G, Calhoun L, et al. Selecting donors of platelets for refractory patients
on the basis of HLA antibody specificity.
Transfusion 2000;40:1446-56.
Koerner TAW, Vo TL, Wacker KE, Strauss RG.
The predictive value of three definitions of
platelet transfusion refractoriness (abstract).
Transfusion 1988;28:33S.
Ishida A, Handa M, Wakui M, et al. Clinical
factors influencing post-transfusion platelet
increment in patients undergoing hematopoietic progenitor cell transplantation—a
prospective analysis. Transfusion 1998;38:
839-47.
Bishop J, McGrath K, Wolf M, et al. Clinical
factors influencing the efficacy of pooled
platelet transfusions. Blood 1988;71:383-7.
Moroff G, Garratty G, Heal JM, et al. Selection
of platelets for refractory patients by HLA
matching and prospective crossmatching.
Transfusion 1992;32:633-40.
Bolgiano DC, Larson EB, Slichter SJ. A model
to determine required pool size for HLAtyped community donor apheresis programs.
Transfusion 1989;29:306-10.
Duquesnoy RJ, Filip DJ, Rodey G, et al. Successful transfusion of platelets “mismatched”
for HLA antigens to alloimmunized thrombocytopenic patients. Am J Hematol 1997;2:
219-26.
Dahlke MB, Weiss KL. Platelet transfusions
from donors mismatched from cross-reactive
HLA antigens. Transfusion 1984;24:299-302.
Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.
Rachel JM, Summers TC, Sinor LT, et al. Use
of a solid phase red blood cell adherence
method for pretransfusion platelet compatibility testing. Am J Clin Pathol 1988;90:63-8.
Rachel JM, Sinor LT, Tawfik OW, et al. A solidphase red cell adherence test for platelet
crossmatching. Med Lab Sci 1985;42:194-5.
O’Connell BA, Schiffer CA. Donor selection
for alloimmunized patients by platelet cross-

Chapter 16: Platelet and Granulocyte Antigens and Antibodies

20.

21.

22.

23.

24.
25.

26.

27.

28.

29.

30.

31.

32.

33.

matching of random-donor platelet concentrates. Transfusion 1990;20:314-7.
Friedberg RC, Donnelly SF, Mintz PD. Independent roles for platelet crossmatching and
HLA in the selection of platelets for alloimmunized patients. Transfusion 1994;34:
215-20.
Duquesnoy RJ. HLA Matchmaker: A molecularly based algorithm for histocompatibility
determination. I. Description of the Algorithm. Hum Immunol 2002;63:339-52.
Gmür J, von Felten A, Osterwaider B, et al.
Delayed alloimmunization using random single donor platelet transfusions: A prospective
study of thrombocytopenic patients with
acute leukemia. Blood 1983;62:473-9.
Dutcher JP, Schiffer CA, Aisner J, Wiernik PH.
Alloimmunization following platelet transfusion: The absence of a dose-response relationship. Blood 1981;57:395-8.
Kruskall MS. The perils of platelet transfusions
(editorial). N Engl J Med 1997;337:1914-5.
Gebel HM, Bray RA. Sensitization and sensitivity: Defining the unsensitized patient.
Transplantation 2000;69:1370-4.
Murphy MF, Metcalfe P, Ord J, et al. Disappearance of HLA and platelet-specific antibodies in acute leukemia patients alloimmunized by multiple transfusions. Br J
Haematol 1987;67:255-60.
Kroll H. Human platelet alloantigens (HPA).
[Available at http://www.uniklinikum-giessen.de/immunologie/hkroll/HPAs.htm (accessed
January 2005).]
Newman PJ, Valetin N. Human platelet
alloantigens: Recent findings, new perspectives. Thromb Haemost 1995;84:234-9
Greenwalt DE, Lipsky RH, Ockenhouse CF, et
al. Membrane glycoprotein CD36: A review of
its roles in adherence, signal transduction, and
transfusion medicine. Blood 1992;80:1105-15.
Curtis BR, Aster RH. Incidence of the Nak(a)negative platelet phenotype in African Americans is similar to that of Asians. Transfusion
1996;36:331-4.
Curtis GR, Ali S, Glazier AM, et al. Isoimmunization against CD36 (glycoprotein IV): Description of four cases of neonatal isoimmune
thrombocytopenia and brief review of the literature. Transfusion 2002;42:1173-9.
Curtis BR, McFarland JG, Gottschall JL. Platelet immunology and alloimmunization. In:
Simon TL, Dzik WH, Snyder EL, et al, eds.
Rossi’s principles of transfusion medicine.
3rd ed. Philadelphia, PA: Lippincott Williams
and Wilkins, 2002:203-18.
McFarland J. Nomenclature update: HLA,
HPA and RBCs. Platelet-specific antigen nomenclature. In: The compendium: A selec-

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

381

tion of short topic presentations. Bethesda,
MD: AABB, 1997.
Metcalfe P, Watkins NA, Ouwehand WH, et al.
Nomenclature of human platelet antigens.
Vox Sang 2003;85:240-5.
van Loghem JJ, Dorfmeijer H, van der Hart M.
Serological and genetical studies on a platelet
antigen (Zw). Vox Sang 1959;4:161-9.
Aster RH. Platelet-specific alloantigen systems: History, clinical significance and molecular biology. In: Nance ST, ed. Alloimmunity: 1993 and beyond. Bethesda, MD:
AABB, 1993:83-116.
von dem Borne AEGKr, Simsek S, van der Schoot
S, et al. Platelet and neutrophil alloantigens:
Their nature and role in immune-mediated
cytopenias. In: Garratty G, ed. Immunobiology
of transfusion medicine. New York: Marcel
Dekker, 1994:149-71.
Müeller-Eckhardt C. Platelet autoimmunity.
In: Silberstein LE, ed. Autoimmune disorders
of blood. Bethesda, MD: AABB, 1996:115-50.
Smith JW, Hayward CP, Horsewood P, et al.
Characterization and localization of the Gova/b
alloantigens to the glycosylphosphatidylinositol-anchored protein CDw109 on human
platelets. Blood 1995;86; 2807-14.
Berry JE, Murphy CM, Smith GA, et al. Detection of Gov system antibodies by MAIPA reveals an immunogenicity similar to the
HPA-5 alloantigens. Br J Haematol 2000;110:
735-42.
McFarland JG. Posttransfusion purpura. In:
Popovsky MA, ed. Transfusion reactions. 2nd
ed. Bethesda, MD: AABB Press, 2001:187-212.
Brecher ME, Moore SB, Letendre L. Posttransfusion purpura: The therapeutic value of
PlA1-negative platelets. Transfusion 1990;30:
433-5.
Christie D, Pulkrabek S, Putnam J, et al. Posttransfusion purpura due to an alloantibody
reactive with glycoprotein Ia/IIa (anti-HPA5b). Blood 1991;77:2785-9.
Sinha RK, Kelton JG. Current controversies
concerning the measurement of platelet associated IgG. Transfus Med Rev 1990;2:121-35.
Shibata Y, Juji T, Nishizawa Y, et al. Detection
of platelet antibodies by a newly developed
mixed passive agglutination with platelets.
Vox Sang 1981;41:25-31.
Neumuller J, Tohidast-Akrad M, Fisher M,
Mayr WR. Influence of chloroquine or acid
treatment of human platelets on the antigenicity of HLA and the “thrombocyte-specific”
glycoproteins Ia/IIa, IIb, and IIb/IIIa. Vox
Sang 1993;65:223-31.
Gouttefangeas C, Diehl M, Keilholz W, et al.
Thrombocyte HLA molecules retain nonrenewable endogenous peptides of mega-

382

48.

49.

50.

51.

52.

53.

54.

55.
56.

57.

58.

59.

60.

61.

AABB Technical Manual

karyocyte lineage and do not stimulate direct
allocytotoxicity in vitro. Blood 2000;95:316875.
von dem Bor ne AEGKr, Ver heugt FWA,
Oosterhof F, et al. A simple immunofluorescence test for the detection of platelet antibodies. Br J Haematol 1978;39:195-207.
Garratty G, Arndt P. Applications of flow
cytofluorometry to transfusion science.
Transfusion 1995;35:157-78.
Kiefel V, Santoso S, Weisheit M, MüellerEckhardt C. Monoclonal antibody-specific
immobilization of platelet antigens (MAIPA):
A new tool for the identification of plateletreactive antibodies. Blood 1987;70:1722-6.
Kiefel V. The MAIPA assay and its applications in immunohematology. Transfus Med
1992;2:181-8.
Morel-Kopp MC, Daviet L, McGregor J, et al.
Drawbacks of the MAIPA technique in characterizing human anti-platelet antibodies.
Blood Coag Fibrinol 1996;7:144-6.
Visentin GP, Wolfmeyer K, Newman PJ, Aster
RH. Detection of drug-dependent, plateletreactive antibodies by antigen-capture ELISA
and flow cytometry. Transfusion 1990;30:694700.
Menitove JE, Pereira J, Hoffman R, et al. Cyclic thrombocytopenia of apparent autoimmune etiology. Blood 1989;73:1561-9.
GTI PAKPLUSTM platelet antibody screening
kit (package insert). Waukesha, WI: GTI, 1996.
McFarland JG, Aster RH, Bussel JB, et al. Prenatal diagnosis of neonatal alloimmune
thrombocytopenia using allele-specific oligonucleotide probes. Blood 1991;78:2276-82.
Simsek S, Faber NM, Bleeker PM, et al. Determination of human platelet antigen frequencies in the Dutch population by immunophenotyping and DNA (allele-specific
restriction enzyme) analysis. Blood 1993;82:
835-40.
Skogen B, Bellissimo D, Hessner M, et al.
Rapid determination of platelet alloantigen
genotypes by polymerase chain reaction using allele-specific primers. Transfusion 1994;
34:955-60.
Kluter H, Fehlau K, Panzer S, et al. Rapid typing for human platelet antigen systems-1, -2,
-3 and -5 by PCR amplification with sequencespecific primers. Vox Sang 1996;71: 121-5.
Panzer S. Report on the Tenth International
Platelet Genotyping and Serology Workshop
on behalf of the International Society of
Blood Transfusion. Vox Sang 2001;80:72-8.
Meyer O, Hildebrandt M, Schulz B, et al. Simultaneous genotyping of human platelet
antigens (HPA) 1 through 6 using new se-

quence-specific primers for HPA-5. Transfusion 1999;39:1256-8.
62.

George JN. Platelet immunoglobulin G: Its
significance for the evaluation of thrombocytopenia and for understanding the origin of
α-granule proteins. Blood 1990;76:859-70.

63.

George JN, Woolf SH, Raskob GE, et al. Idiopathic thrombocytopenic purpura: A practice
guideline developed by explicit methods for
the American Society of Hematology. Blood
1996;88:3-40.

64.

Warner MN, Moore JC, Warkentin TE, et al. A
prospective study of protein-specific assays
used to investigate idiopathic thrombocytopenic purpura. Br J Haematol 1999;104:442-7.

65.

GTI PAKAUTOTM ELISA screening test for autoantibodies to platelet glycoproteins IIb/IIIa,
Ib/IX and Ia/IIa (package insert). Waukesha,
WI: GTI, 2004.

66.

Curtis BR, McFarland JG, Wu GG, et al. Antibodies in sulfonamide-induced immune
thrombocytopenia recognize calcium-dependent epitopes on the glycoprotein IIb/IIIa
complex. Blood 1994;84:176-83.

67.

Christie DJ, Aster RH. Drug-antibody-platelet
interaction in quinine- and quinidine-induced thrombocytopenia. J Clin Immunol
1982;70:989-98.

68.

Eisner EV, Shahidi NT. Immune thrombocytopenia due to a drug metabolite. N Engl J
Med 1972;87:376-81.

69.

Kiefel V, Santoso S, Schmidt S, et al. Metabolite-specific IgG and drug-specific antibodies
IgG, IgM in two cases of trimethoprimsulfamethoxazole-induced immune thrombocytopenia. Transfusion 1987;27:262-5.

70.

Sinor LT, Stone DL, Plapp FV, et al. Detection
of heparin-IgG immune complexes on platelets by solid phase red cell adherence assays.
(Immucorrespondence) Norcross, GA: Immucor,
1990.

71.

Leach MF, Cooper LK, AuBuchon JP. Detection of drug-dependent, platelet-reactive antibodies by solid-phase red cell adherence assays. Br J Haematol 1997;97:755-61.

72.

Nieminen U, Kekomäki R. Quinidine-induced
thrombocytopenia purpura: Clinical presentation in relation to drug-dependent and
drug-independent platelet antibodies. Br J
Haematol 1992;80:77-82.

73.

Warkentin TE, Levine M, Hirsh J, et al. Heparin-induced thrombocytopenia in patients
treated with low molecular weight heparin or
unfractionated heparin. N Engl J Med 1995;
332:1330-5.

74.

Warkentin TE. Pork or beef? Ann Thorac Surg
2003;75:15-6.

Chapter 16: Platelet and Granulocyte Antigens and Antibodies

75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

86.

87.

Nand S, Wong W, Yuen B, et al. Heparin-induced thrombocytopenia with thrombosis.
Am J Hematol 1997;56:12-6.
Warkentin TE, Kelton JG. A 14-year study of
heparin-induced thrombocytopenia. Am J
Med 1996;101:502-7.
Warkentin TE, Chong BA, Greinacher A. Heparin-induced thrombocytopenia: Towards
consensus. Thromb Haemost 1998;79:1-7.
Cancio LC, Cohen DJ. Heparin-induced
thrombocytopenia thrombosis. J Am Coll
Surg 1998;186:76-91.
Gupta AK, Kovacs MJ, Sauder DN. Heparininduced thrombocytopenia. Ann Pharmacother
1998;32:55-9.
GTI-HATTM for the detection of heparin-associated antibodies (package insert). Waukesha,
WI: GTI, 1997.
Suh JS, Malik MI, Aster RH, Visentin GP. Characterization of the humoral immune response in heparin induced thrombocytopenia. Am J Hematol 1997;54:196-201.
Amiral J, Wolf M, Fischer A, et al. Pathogenicity of IgA and/or IgM antibodies to heparin-PF4 complexes in patients with heparin
induced thrombocytopenia. Br J Haematol
1996;92:954-9.
Sheridan D, Carter C, Kelton JG. A diagnostic
test for heparin-induced thrombocytopenia.
Blood 1986;67:27-30.
Pouplard C, Amiral J, Borg JY, et al. Decision
analysis for use of platelet aggregation test,
carbon 14-serotonin release assay, and heparin platelet factor 4 enzyme-linked immunosorbent assay for diagnosis of heparin-induced thrombocytopenia. Am J Clin Pathol
1999;111:700-6.
Lindhoff-Last E, Gerdsen F, Ackermann H, et
al. Determination of heparin platelet factor 4
IgG antibodies improves diagnosis of heparin-induced thrombocytopenia. Br J Haematol 2001;113:886-90.
Lalezari P, Bernard JE. An isologous antigenantibody reaction with human neutrophils
related to neonatal neutropenia. J Clin Invest
1966;45:1741-50.
Steffensen R, Gulen T, Varming K, Jersild C.
FcγRIIIb polymorphism: Evidence that NA1/

88.
89.

90.

91.

92.

93.

94.

95.

96.

97.

383

NA2 and SH are located in two closely linked
loci and that the SH allele is linked to the NA1
allele in the Danish population. Transfusion
1999;39:593-8.
Stroncek D. Neutrophil alloantigens. Transfus
Med Rev 2002;16:67-75.
Hessner MJ, Curtis BR, Endean DJ, Aster RH.
Determination of neutrophil antigen gene
frequencies in five ethnic groups by polymerase chain reaction with sequence-specific
primers. Transfusion 1996;36:895-9.
Nordhagen R, Conradi M, Promtord SM. Pulmonary reaction associated with transfusion
of plasma containing anti-5b. Vox Sang 1986;
51:102-8.
Davoren A, Curtis RBR, Shulman IA, et al.
TRALI due to granulocyte-agglutinating human neutrophil antigen-3a (5b) alloantibodies in donor plasma: A report of 2 fatalities. Transfusion 2003;43:641-5.
Kopko PM, Marshall CS, MacKenzie MR, et al.
Transfusion-related acute lung injury: Report
of a clinical look-back investigation. JAMA
2002;287:1968-71.
Fung UL, Willett JE, Pitcher LA, et al. Confirming an alloimmune neonatal neutropenia
due to anti-HLA-4a (MART) by DNA characterization. Presented at the 7th European
symposium on platelet, granulocyte and red
cell immunobiology, Lago Maggiore, Italy,
April 11-14, 2002.
Flesch BK, Neppert J. Transfusion-related
acute lung injury caused by human leukocyte
antigen Class II antibody. Br J Haematol
2002;116:673-6.
Silliman R, Paterson AJ, Dickey WO, et al. The
association of biologically active lipids with
the development of transfusion-related acute
lung injury: A retrospective study. Transfusion 1997;37:719-26.
Bux J, Behrens G, Jaeger G, et al. Diagnosis
and clinical course of autoimmune neutropenia in infancy: Analysis of 240 cases. Blood
1998;91:81-6.
Bux J, Chapman J. Report on the second international granulocyte serology workshop.
Transfusion 1997;37:977-83.

Chapter 17: The HLA System

Chapter 17

The HLA System

HE HLA SYSTEM includes a complex array of genes and their protein products. HLA antigens contribute to the recognition of self and
nonself, to the immune responses to antigenic stimuli, and to the coordination of
cellular and humoral immunity. The HLA
genes, which are located in the major
histocompatibility complex (MHC) on the
short arm of chromosome 6, code for
glycoprotein molecules found on cell surface membranes. Class I molecules are
found on the surface of platelets and of all
nucleated cells of the body. Mature red
cells usually lack HLA antigens demonstrable by conventional methods, but nucleated immature erythroid cells express
them. MHC Class II antigens are restricted
to a few cell types; the most important are
B lymphocytes, macrophages, and dendritic cells. Other terms that have been
applied to antigens of the HLA system are:
major histocompatibility locus antigens,
transplantation antigens, and tissue antigens.

The HLA antigen molecules play a key
role in antigen presentation. Immunologic
recognition of differences in HLA antigens
is probably the first step in the rejection of
transplanted tissue. The HLA system is second in importance only to the ABO antigens in influencing the long-term survival
of transplanted solid organs and is of paramount significance in hematopoietic progenitor cell (HPC) transplantation. HLA antigens and antibodies are also important in
such complications of transfusion therapy
as platelet refractoriness, febrile nonhemolytic transfusion reactions (FNHTRs),
transfusion-related acute lung injury (TRALI),
and posttransplant and posttransfusion
graft-vs-host disease (GVHD).
Studies correlating HLA polymorphisms
with susceptibility and disease resistance
began soon after serologic techniques for
HLA Class I typing were developed. Historically, HLA antigen typing has been of value
in parentage testing and in forensic investigations. Molecular analysis of the HLA region permits selection of more closely
385

386

AABB Technical Manual

matched donors for HPC transplantation,
for investigation of disease associations,
and for anthropologic population studies.
Because of the polymorphic nature of the
HLA genes, a complex nomenclature has
been developed to refer to the unique allele
sequences based on the relationship of the
allele to the serologic specificity of the corresponding antigen.1

Genetics of the Major
Histocompatibility Complex
Class I and II HLA antigens are cell surface glycoproteins that are products of
closely linked genes mapped to the p21.3
band on the short arm of chromosome 6
(Fig 17-1). This genomic region is called
the MHC and is usually inherited en bloc
as a haplotype. Each of the several loci
has multiple alleles with codominant expression of the products from each chromosome. The HLA system is the most

polymorphic system of genes described in
humans.
The HLA-A, HLA-B, and HLA-C genes
encode the corresponding Class I antigens
A, B, and C. The HLA-DR, HLA-DQ, and
HLA-DP gene cluster codes for the synthesis of correspondingly named Class II antigens. Located between the Class I and Class
II genes is a group of non-HLA genes that
code for molecules that include the complement proteins C2, Bf, C4A, C4B; a steroid enzyme (21-hydroxylase); and a
cytokine (tumor necrosis factor). This region is referred to as MHC Class III.

Organization of HLA Genetic Regions
The HLA Class I region contains, in addition to the classical genes HLA-A, HLA-B,
and HLA-C, other gene loci designated
HLA-E, HLA-F, HLA-G, HFE, HLA-J, HLA-K,
HLA-L, MICA, and MICAB. These latter
genes encode the nonclassical or Class Ib
HLA proteins, characterized by limited
polymorphism and low levels of expres-

Figure 17-1. (A) The major histocompatibility complex located on the short arm of chromosome 6. The
centromere is to the left. The key Class I, II, and III genetic loci are shown. The Class III region contains complement system genes (C2, Bf, C4A, C4B), the 21-hydroxylase gene (21OH), and the gene for
tumor necrosis factor (TNF). (B) Greater detail of the Class II region.

Chapter 17: The HLA System

sion. Some Class I genes express nonfunctional proteins or are not able to express a protein. Genes unable to express a
functional protein product are termed
pseudogenes and presumably represent
an evolutionary dead end. HLA-E regulates natural killer cells. HLA-G is expressed by the trophoblast and may be involved in the development of maternal
immune tolerance of the fetus. Hereditary
hemochromatosis (HH), an iron overload
disorder with a 10% carrier frequency in
Northern Europeans, is associated with
two missense mutations in a Class I-like
gene.3 The gene conferring HH was initially named HLA-H; however, the HLA-H
designation had already been assigned to
an HLA Class I pseudogene by the World
Health Organization (WHO) Nomenclature Committee.4 The gene conferring HH
is now called HFE. Class I molecules are
also located outside the MHC, such as
CD1, which can present nonprotein antigens (such as lipids) to T cells.
The genomic organization of the MHC
Class II region (HLA-D region) is more
complex. An MHC Class II molecule consists of a noncovalent complex of two structurally similar chains, the α-chain and the
β-chain. Both of these chains are encoded
within the MHC. The polymorphism of
HLA Class II molecules results from differences in both the α-chain and the β-chain;
this depends on the Class II isoform. For
example, with HLA-DR, the α-chain is
monomorphic, but the β-chain is very polymorphic. Multiple loci code for either alpha
or beta chains of the Class II MHC proteins.
Different haplotypes have different numbers of Class II genes and pseudogenes.
The proteins coded by the DRA gene and
the DRB1 gene result in HLA-DR1 through
HLA-DR18. The products of the A gene and
the B3 gene (if present) express HLA-DR52;
those of the A gene and the B4 gene (if present) express HLA-DR53; and those of the A

387

gene and B5 gene (if present) express
HLA-DR51. The HLA-DQ1 through DQ9
antigens are expressed on the glycoproteins
coded by the DQA1 and DQB1 genes in the
DQ gene cluster. Many of the other genes of
the DQ cluster are probably pseudogenes. A
similar organization is found in the HLADP gene cluster.
The MHC Class III region contains four
complement genes, whose alleles are generally inherited together as a unit, termed a
complotype. There are more than 10 different complotypes inherited in humans. Two
of the Class III genes, C4A and C4B, code
for variants of the C4 molecule. These variants have distinct protein structure and
function; the C4A molecule (if present) carries the Rodgers antigen and the C4B molecule (if present) carries the Chido antigen,
both of which are adsorbed onto the red
cells of individuals who possess the gene.

Patterns of Inheritance
Although the organization of the MHC is
complicated, its inheritance follows the
established principles of genetics. Every
person has two different copies of chromosome 6 and, thus, possesses two HLA
haplotypes, one from each parent. An individual’s haplotype is typically determined
by typing multiple family members from
different generations and observing which
alleles are inherited together. The expressed gene products constitute the phenotype, which can be determined for an
individual by typing for the HLA antigens.
Because the HLA genes are autosomal
and codominant, the phenotype represents the combined expression of both
haplotypes. Figure 17-2 illustrates inheritance of haplotypes.

Finding HLA-Identical Siblings
Each child inherits one copy of chromosome 6 from each parent; hence, one MHC

388

AABB Technical Manual

Figure 17-2. The linked genes on each chromosome constitute a haplotype. To identify which haplotypes a person possesses, one must know the antigens present and also the inheritance pattern in the
specific kindred. The observed typing results of the father in this family are interpreted into the following phenotype: A1,3;B7,8;Cw7,-;DR15,17. The observed results plus the family study reveal the
haplotypes of the father to be: a = A1,Cw7,B8,DR17 and b = A3,Cw7,B7,DR15. Offspring of a single
mating pair must have one of only four possible combinations of haplotypes, assuming there has been
no crossing-over.

haplotype is inherited from each parent.
Because each parent has two different
copies of chromosome 6, four different
combinations of haplotypes are possible
in the offspring (assuming no recombination). This inheritance pattern is important in predicting whether family members
will be compatible donors for transplantation. The chance that two siblings will
be HLA-identical is 25%. The chance that
any one patient with “n” siblings will have
at least one HLA-identical sibling is 1-(3/4)n.

Having two siblings provides a 44% chance
and three siblings a 58% chance that one
sibling will be HLA-identical. No matter
how many siblings are available for typing
(aside from identical twins), the probability will never be 100% for finding an HLAidentical sibling.

Absence of Antigens
Usually, both copies of the genes within
the MHC are expressed as antigens; how-

Chapter 17: The HLA System

ever, in certain individuals, only one antigen can be identified. This may occur if
the individual is homozygous for the allele, or if appropriate antisera are not
available to type the individual’s antigen
(referred to as a blank allele). Very rarely,
the absence of an antigen can result from
a null allele. A null allele is characterized
by substitutions within the coding region
of the gene that prevent the expression of
a functional protein at the cell surface. Such
inactivation of a gene may be caused by
nucleotide substitutions, deletions, or insertions, which lead to a premature cessation in the antigen’s synthesis. When referring to phenotypes, a blank is often
written as “x” (for A locus), “y” (for B locus), or “–” (for any locus) (eg, A1,x;B7,40
or A1,–;B7,40). Family studies must be performed to determine the correct genotype.

Crossing-Over
The genes of the HLA region occasionally
demonstrate chromosome crossover, in
which segments containing linked genetic
material are exchanged between the two
chromosomes during meiosis or gametogenesis (see Fig 10-6). These recombinants
are then transmitted as new haplotypes to
the offspring. Crossover frequency is in
part related to the physical distance between genes. For example, the HLA-A,
HLA-B, and HLA-DR loci are close together, with 0.8% crossover between the A
and B loci and 0.5% between the B and
DR loci. In family studies and in parentage testing, the possibility of recombination must be considered.

Linkage Disequilibrium
The MHC system is so polymorphic that,
theoretically, the number of possible unique HLA phenotypes is greater than the
global human population. Moreover, new
HLA alleles are constantly being discov-

389

ered and characterized. As of 2004, there
were 309 HLA-A, 563 HLA-B, and 368
DRB1 alleles. In reality, many HLA haplotypes are overrepresented compared with
what would be expected if the distribution
of HLA genes were random. The phenomenon of linkage disequilibrium accounts
for the discrepancy between expected and
observed HLA haplotype frequencies.
Expected frequencies for HLA haplotypes are derived by multiplication of the
frequencies of each allele. For example, in
individuals of European ancestry, the overall frequency of the gene coding for HLA-A1
is 0.15 and that for HLA-B8 is 0.10; therefore, 1.5% (0.15 × 0.10) of all HLA haplotypes in this population would be expected
to contain genes coding for both HLA-A1
and HLA-B8 if they were randomly distributed. The actual frequency of the A1 and B8
combination, however, is 7% to 8% in this
population. Certain allelic combinations
occur with increased frequency in different
racial groups and constitute common haplotypes in those populations. These are called
ancestral haplotypes because they appear
to be inherited from a single common ancestor. The most common ancestral haplotype in Northern Europeans, the A1, B8,
DR3, DQ2 haplotype, includes both Class I
and Class II regions. It is unclear whether
ancestral haplotypes represent relatively
young haplotypes that have not had sufficient time to undergo recombination, or
whether they are old haplotypes that are resistant to recombination because of selection. Linkage disequilibrium in the HLA
system is important in studies of parentage because haplotype frequencies in the
relevant population make the transmission of certain gene combinations more
likely than others. Linkage disequilibrium
also affects the likelihood of finding suitable unrelated donors for HLA-matched
platelet transfusions and for HPC transplantation.

390

AABB Technical Manual

Biochemistry, Tissue
Distribution, and Structure
The HLA antigens are cell surface glycoproteins. HLA Class I molecules contain
one copy of two polypeptides: a heavy
chain, which is attached to the membrane, and a light chain, which is called
β2-microglobulin. The HLA Class II molecules are composed of one copy each of
an α-chain and a β-chain, both of which
are attached to the cell surface. HLA antigens are divided into Class I and Class II
based on their function, tissue distribution, and biochemistry.

Characteristics of Class I and Class II
Antigens
Class I antigens (HLA-A, -B, and -C) have
a molecular weight around 57,000 daltons

and consist of two chains: a glycoprotein
heavy chain (45,000 daltons) encoded on
the short arm of chromosome 6 and, as a
light chain, the β2-microglobulin molecule
(12,000 daltons) encoded by a gene on
chromosome 15. The heavy chain penetrates the cell membrane. β2-microglobulin is not attached to the cell membrane;
it associates with the heavy chain via the
latter’s nonvariable (α3) domain but is not
covalently bound to it (see Fig 17-3). The
external portion of the heavy chain consists of three amino acid domains (α1, α2,
and α3), of which the outermost α1 and
α2 domains contain the polymorphic regions conferring the HLA antigen specificity.
Class I molecules are found on platelets
and on most nucleated cells in the body,
with some exceptions such as neurons, cor-

Figure 17-3. Stylized diagram of Class I and Class II MHC molecules showing α and β polypeptide
chains, their structural domains, and attached carbohydrate units.

Chapter 17: The HLA System

neal epithelium, trophoblast, and germinal
cells. Only vestigial amounts remain on
mature red cells, with certain allotypes
better expressed than others. These Class I
polymorphisms were independently recognized as red cell alloantigens by serologists
and were designated as Bennett-Goodspeed (Bg) antigens. The specificities called
a
b
c
Bg , Bg , and Bg are identified as HLA-B7,
HLA-B17, and HLA-A28, respectively. Platelets express primarily HLA-A and HLA-B
antigens. HLA-C antigens are present at
very low levels and Class II antigens are
generally not expressed at all on platelets.
Class II antigens (HLA-DR, -DQ, and
-DP) have a molecular weight of approximately 63,000 daltons and consist of two
structurally similar glycoprotein chains (α
and β), both of which traverse the membrane (see Fig 17-3). The extramembranous
portion of each chain has two amino acid
domains, of which the outermost domain
contains the variable regions of the Class II
alleles. The expression of Class II antigens
is more restricted than that of Class I. Class
II antigens are expressed constitutively on
B lymphocytes, monocytes, and cells derived from monocytes such as macrophages and dendritic cells, intestinal epithelium, and early hematopoietic cells.
There is also constitutive expression of
Class II antigens on some endothelial cells,
especially those lining the microvasculature.
However, in general, endothelium, particularly that of larger blood vessels, is negative
for Class II antigen expression, although its
presence can be induced (for instance, by
interferon-gamma during immune activation). T lymphocytes are negative for Class
II antigen expression but become positive
when activated. Class II antigens are expressed abnormally in autoimmune disease and on some tumor cells.
Soluble HLA Class I and Class II antigens
shed from cells are found in blood and
body fluids and may play a role in modulat-

391

ing immune reactivity.5 Levels of soluble
HLA increase with infection [including human immunodeficiency virus (HIV)], inflammatory disease, and transplant rejection, and decline with progression of
malignancy. Levels of soluble HLA in blood
components are proportionate to the number of residual donor leukocytes and to the
length of storage.6 Soluble HLA in blood
components may be involved in the immunomodulatory effect of blood transfusion.

Configuration
A representative three-dimensional structure of these molecules can be obtained
by X-ray crystallographic analysis of purified HLA antigens. The outer domains,
which contain the regions of amino acid
variability and the antigenic epitopes of
the molecules, form a structure known as
the “peptide-binding groove.” Alleles defined by polymorphisms in the HLA gene
sequences have unique amino acid sequences and, therefore, form unique
binding grooves, each able to bind different classes of peptides. The peptide-binding groove is critical for the functional aspects of HLA molecules (see section on
Biologic Function).

Nomenclature
An international committee sponsored by
the World Health Organization establishes the nomenclature of the HLA system. It is updated regularly to incorporate
new HLA alleles. HLA antigens are designated by a number following the letter
that denotes the HLA series (eg, HLA-A1
or HLA-B8). Previously, antigenic specificities that were not fully confirmed carried the prefix “w” (eg, HLA-Aw33). When
identification of the antigen became definitive, the WHO Nomenclature Commit-

392

AABB Technical Manual

tee dropped the “w” from the designation.
The Committee meets regularly to update
nomenclature by recognizing new specificities or genetic loci. The “w” prefix is no
longer applied in this manner and is now
used only for the following: 1) Bw4 and
Bw6, to distinguish these “public” antigens
from other B locus alleles; 2) all C locus
specificities, to avoid confusion with
members of the complement system; and
3) Dw and DP specificities that were defined by mixed lymphocyte reactions and
primed lymphocyte typing. The numeric
designations for the HLA-A and HLA-B
specificities were assigned based on the
order of their discovery.

Splits
Refinement of serologic methods permitted antigens previously believed to represent a single specificity to be “split” into
specificities that were serologically (and,
later, genetically) distinct. The designation for an individual antigen that is a
split of an earlier recognized antigen often includes the number of the parent antigen in parentheses, eg, HLA-B44 (12).

Shared Determinants
As the specificity of HLA typing sera improved, it was found that some epitopes,
the part of the antigen that binds antibody, were common to several antigens.
Antibodies to these epitopes identified
antigens that constituted a group, of which
the individual members could be identified by antisera with more restricted activity.

Cross-Reactive Groups
In addition to “splits,” HLA antigens and
antigen groups may have other epitopes
in common. Antibodies that react with
these shared determinants often cause
cross-reactions in serologic testing. The

collective term for a group of HLA antigens that exhibit such cross-reactivity is
cross-reactive group (CREG).

“Public” Antigens
In addition to splits and CREGs, HLA proteins have reactivity that is common to
many different HLA specificities. Called
“public” antigens, these common amino
acid sequences appear to represent the
less variable portion of the HLA molecule.
Two well-characterized “public” antigens,
HLA-Bw4 and HLA-Bw6, are found in the
HLA-B series. The Bw4 antigen is also
found on some A locus molecules. “Public” antigens are clinically important because patients exposed to foreign HLA
antigens via pregnancy, transfusion, or
transplantation can make antibodies to
them. A single antibody, when directed
against a “public” antigen, can resemble
the sum of multiple discrete alloantibodies.

Nomenclature for HLA Alleles
As nucleotide sequencing is used to investigate the HLA system, increasing
numbers of HLA alleles are being identified, many of which share a common serologic phenotype. The minimum requirement for designation of a new allele
is the sequence of exons two and three for
HLA Class I and exon two for HLA Class II
(DRB1). These exons encode the variable
amino acids that confer HLA antigen specificity. A uniform nomenclature has been
adopted that takes into account the locus,
the major serologic specificity, and the allele determined by molecular typing techniques. For example, isoelectric focusing,
amino acid sequencing, and nucleotide
sequencing have identified several unique
variants of HLA-DR4. The first HLA-DR4
variant is designated DRB1*0401, indicat-

Chapter 17: The HLA System

ing the locus (DR), the protein (β1 chain),
an asterisk to represent that an allele
name follows, the major serologic specificity (04 for HLA-DR4), and the allele
number (variant 01). For Class I alleles, the
name of the locus, for example HLA-B, is
followed by an asterisk and then a number of digits. The first two digits correspond to the serologic specificity of the
antigen. The third and fourth digits are
used to list the subtypes, numbers being
assigned in the order in which the DNA
sequences have been determined. Therefore, B*2704 represents the HLA-B locus,
with a serologic specificity of B27, and
was the fourth allele described in this
family (see Table 17-1). Finally, the nomenclature can accommodate alleles
with silent mutations, ie, those that have
different DNA sequences but identical
amino acid sequences and null alleles.

Biologic Function
The essential function of the HLA system
is self/nonself discrimination. Discrimination of self from nonself is accomplished
by the interaction of T lymphocytes with

393

peptide antigens. T lymphocytes interact
with peptide antigen only when the T-cell
receptor (TCR) for antigen engages both
an HLA molecule and the antigenic peptide contained with its peptide-binding
groove. This limitation is referred to as
“MHC restriction.”7
In the thymus, T lymphocytes whose
TCRs bind to a self HLA molecule are selected (positive selection), with the exception of those whose TCRs also bind to a
peptide derived from a self antigen, in
which case they are deleted (negative selection). Some self-reactive T cells escape negative selection, however. If not functionally
inactivated, for instance, by the mechanism
of anergy, these self-reactive T cells may become involved in an autoimmune process.
(See Chapter 11.)

Role of Class I
Class I molecules are synthesized, and
peptide antigens are inserted into the
peptide-binding groove, in the endoplasmic reticulum. Peptide antigens that
fit into the Class I peptide-binding groove
are typically eight or nine amino acids in
length and are derived from proteins that

Table 17-1. HLA Nomenclature
Genetic
Locus

HLA-A
HLA-B
HLA-C
DRA
DRB1
DQA1
DQB1
DPA1
DPB1

Antigenic
Specificity
A1 to A80
B7 to B81
Cw1 to Cw10
DR1 to DR18
DQ1 to DQ9
DPw1 to DPw6

Allele

Number of
Identified
Alleles

Polypeptide
Location

A*0101 to *8001
B*0702 to *8301
Bw*0102 to *1802
DRA*0101 to *0102
DRB1*0101 to *1608
DQA1*0101 to *0601
DQB1*0501 to *0402
DPA1*0103 to *0401
DPB1*0101 to *8901

207
412
100
2
271
20
45
19
93

a
a
a
a
β1
a
β1
a1
β1

394

AABB Technical Manual

are made by the cell (endogenous proteins). These endogenous proteins, which
may be normal self proteins, altered self
proteins such as those found in cancer
cells, or viral proteins such as those found
in virus-infected cells, are degraded in the
cytosol by a large multifunctional protease (LMP) and transported to the endoplasmic reticulum by a transporter associated with antigen processing (TAP). The
LMP and TAP genes are both localized to
the MHC.
Class I molecules are transported to the
cell surface where they are available to interact with CD8-positive T lymphocytes. If
the TCR of a T lymphocyte can bind the antigenic peptide in the context of the specific
Class I molecule displaying it, then this
binding activates the cytotoxic properties of
the T cell, which will then attack the cell,
characteristically eliciting an inflammatory
response. The presentation of antigen by
Class I molecules is especially important in
a host’s defense against viral pathogens and
against malignant transformation. Tumor
cells that do not express Class I escape this
immune surveillance.

by endocytosis (exogenous proteins). Exogenous proteins, which may be normal
self proteins or proteins derived from
pathogens such as bacteria, are degraded
to peptides by enzymes in the endosomal
pathway. Class II molecules are then
transported to the cell surface where they
are available to interact with CD4-positive
T lymphocytes, which secrete immunostimulatory cytokines in response. This
mechanism is especially important for the
production of antibodies.

Detection of HLA Antigens
and Alleles
Methods for the detection of HLA antigens
and alleles fall into three groups: molecular (DNA-based), serologic, and cellular
assays. Detailed procedures of commonly
used assays are provided in the current
edition of the American Society for Histocompatibility and Immunogenetics Laboratory Manual. Depending on the clinical
situation, a particular HLA antigen detection or typing method may be preferable
(Table 17-2).

Role of Class II
Class II molecules, like Class I molecules,
are synthesized in the endoplasmic reticulum, but peptide antigens are not inserted into the peptide-binding groove
here. Instead, an invariant chain (Ii) is inserted. The Class II-invariant chain molecule is transported to an endosome where
the invariant chain is removed by a specialized Class II molecule called DM
(whose locus is also localized to the MHC).
A Class II antigenic peptide is then inserted into the peptide-binding groove.
Peptide antigens that fit into the Class II
peptide-binding groove are typically 12 to
25 amino acids in length and are derived
from proteins that are taken up by the cell

DNA-Based Assays
DNA-based typing has several advantages
over serologic and cellular assays: high
sensitivity and specificity; small sample
volumes; decreased turnaround time, for
some methods, as short as a few hours;
and absence of the need for cell surface
antigen expression or cell viability. Although serologic methods can readily distinguish only about 21 serologic specificities, high-resolution DNA-based methods
can detect up to 368 alleles.

Polymerase Chain Reaction Testing
Polymerase chain reaction (PCR) technology allows amplification of large quanti-

Chapter 17: The HLA System

395

Table 17-2. HLA Typing Methods and Appropriate Applications
Method

Clinical Application

Resolution

SSP (PCR)

Solid organ, related and unrelated HPC
transplantation

Serologic to allele level,
higher resolution with
large number of primers

DNA sequencing

Unrelated HPC transplantation, resolution of typing problems with other
methods, characterization of new
alleles

Allele level

Forward SSOP
hybridization

Solid organ and HPC transplantation
(can accommodate high-volume
testing)

Serologic to allele level

Reverse SSOP
hybridization

Solid organ, related and unrelated HPC
transplantation

Serologic, higher resolution
with larger number of
probes

Microlymphocytotoxicity

Solid organ transplantation, evaluation
of platelet refractoriness, HLA typing
(Class I only) of platelet recipients
and platelet donors

Serologic specificity

ties of a particular target segment of genomic DNA. Low- to intermediate-resolution
typing detects the HLA serologic equivalents with great accuracy; eg, it distinguishes DR15 from DR16, whereas highresolution typing distinguishes individual
alleles, eg, DRB1*0101 from DRB1*0102.
Several PCR-based methods have been
developed, of which two general approaches
are described below.
Oligonucleotide Probes. The first technique uses sequence-specific oligonucleotide probes (SSOPs) and is known as PCRSSO, PCR-SSOP, or allele-specific oligonucleotide (ASO) hybridization.8 A PCR
product amplified from genomic DNA is
applied to a membrane or filter to which
the labeled SSOPs are hybridized. These
short DNA probes will hybridize with the
complementary sequences and identify
groups of alleles or individual alleles. Advantages are that all Class II loci can be

typed and highly specific information obtained. Disadvantages include potential
difficulty in interpretation of results and the
need to use multiple filters and perform
multiple amplifications and subsequent
hybridizations. A variation of this technique, the reverse line or dot blot, eliminates the need for multiple filters and hybridizations by incorporation of a label
(such as biotin) into the PCR product during its amplification from genomic DNA.
The PCR product is then hybridized to a
membrane containing all the relevant
SSOPs and its pattern of hybridization with
the SSOPs revealed by detection of the incorporated label. In the past few years, a
new method for establishing HLA genotypes that features arrays of oligonucleotide
probes on a solid phase has begun to appear in HLA typing laboratories. The
microbead array assay is an SSOP method
for HLA-A, -B, and -DR antigen level typing.

396

AABB Technical Manual

This technique also has the ability for lowto-intermediate resolution DNA-based tissue typing, with a reduction in sample processing time.
Sequence-Specific Primers. A second
major technique uses sequence-specific
primer pairs (SSPs) that target and amplify
a particular DNA sequence.9 This method
requires the performance of multiple PCR
reactions in which each reaction is specific
for a particular allele or group of alleles. Direct visualization of the amplified alleles is
seen after agarose gel electrophoresis. Because SSPs have such specific targets, presence of the amplified material indicates
presence of the corresponding allele(s). The
pattern of positive and negative PCR amplifications is examined to determine the HLA
alleles present. Primer pair sets are available that can determine the full HLA-A, -B,
-C, -DR, -DQ, and -DP type.

Sequence-Based Typing
High-resolution nucleic acid sequencing
of HLA alleles generates allele-level sequences that are used to characterize new
allele(s). With the ever-increasing availability and ease of use of automated sequencers, sequence-based typing has become a routine HLA typing method in
some HLA laboratories.

Serologic Assays

Lymphocytotoxicity
The microlymphocytotoxicity test can be
used to detect HLA-A, -B, -C, -DR, and
-DQ antigens. Lymphocytes are used for
testing because they are readily obtained
from anticoagulated peripheral blood
and, unlike granulocytes, give reproducible results. Lymphocytes obtained from
lymph nodes or spleen may also be used.
HLA typing sera are obtained primarily
from multiparous women. Some mouse
monoclonal antisera are also available.

HLA sera of known specificities are
placed in wells of a microdroplet test plate.
A suspension of lymphocytes is added to
each well. Rabbit complement is then
added and, if sufficient antibody has bound
to the lymphocyte membranes, the complement cascade will be activated through
the membrane attack complex, leading to
lymphocytotoxicity. Damage to the cell
membrane can be detected by the addition
of dye: cells that have no attached antibody,
no activated complement, and no damage
to the membrane keep the vital dyes from
penetrating; cells with damaged membranes allow the dye to enter. The cells are
examined for dye exclusion or uptake under phase contrast microscopy. If a fluorescent microscope is available, fluorescent vital dyes can also be used.
Because HLA-DR and HLA-DQ antigens
are expressed on B cells and not on resting
T cells, typing for these antigens usually requires that the initial lymphocyte preparation be manipulated before testing to yield
an enriched B-cell population. This is typically accomplished by the use of magnetic
beads, to which monoclonal antibodies to
B cells have been bound.
The interpretation of serologic reactions
requires skill and experience. Control wells
of known reactivity and careful quality control of reagents are required, especially for
the activity of the complement used to induce lymphocytotoxicity. In addition, antigen assignments can be made only on the
basis of results obtained with multiple antisera because few reagent antisera have sufficient monospecific reliability to be used
alone. The extreme polymorphism of the
HLA system, the variation in antigen frequencies among different racial groups, the
reliance on biologic antisera and living target cells, and the complexities introduced
by splits, CREGs, and “public” antigens all
contribute to difficulties in accurate serologic HLA typing.

Chapter 17: The HLA System

Antibodies in Patients
Microlymphocytotoxicity testing can be
used to test serum specimens against selected target cells. This is routinely done
in HLA crossmatching, which consists of
testing serum from a potential recipient
against unfractionated lymphocytes (or
fractionated T and B lymphocytes) from a
potential donor. A variation of the microlymphocytotoxicity test, which uses an antiglobulin reagent, is one of the methods
used to increase sensitivity. Flow cytometry is also used as an independent
method to increase the sensitivity of the
crossmatch.
Testing the patient’s serum against a
panel of 30 to 60 or more different target
cells can assess the extent of HLA alloimmunization. The percent of the panel
cells to which the recipient has formed
cytotoxic antibodies is referred to as the
panel reactive antibody (PRA) level. Determination of PRA can be useful in the investigation of FNHTRs, in the workup of
platelet refractoriness, and in following patients who are awaiting cadaver solid organ
transplants. This “HLA antibody screen” not
only detects the presence of HLA antibodies but also may allow their specificity to be
determined. The presence of HLA antibodies can also be demonstrated by using an
enzyme-linked immunosorbent assay with
solid-phase HLA antigens or by flow cytometric analysis using antigen-coated beads.

Cellular Assays
Historically, the mixed lymphocyte culture (MLC) (also called mixed leukocyte
culture, mixed lymphocyte reaction, or
MLR) was used to detect genetic differences in the Class II region. In the MLR,
lymphocytes from different individuals
are cultured together and have the opportunity to recognize foreign HLA-D region
antigens and to respond by proliferating.

397

The HLA System and
Transfusion
HLA system antigens and antibodies play
important roles in a number of transfusion-related events. They include alloimmunization and platelet refractoriness,
FNHTR, TRALI, and posttransfusion GVHD.
HLA antigens are highly immunogenic. In
response to pregnancy, transfusion, or
transplantation, immunologically normal
individuals are more likely to form antibodies to HLA antigens than to any other
antigen system.

Platelet Refractoriness
The incidence of HLA alloimmunization
and platelet refractoriness among patients receiving repeated transfusions of
cellular components is 20% to 71%.10 The
refractory state exists when transfusion of
suitably preserved platelets fails to increase the recipient’s platelet count. Platelet refractoriness may be due to clinical
factors such as sepsis, high fever, disseminated intravascular coagulopathy, medications, hypersplenism, complement-mediated destruction, or a combination of
these, or it may have an immune basis.
(See Chapter 16 for more information
about platelets.)

Antibody Development
Antibodies against HLA antigens usually
cause immune-mediated platelet refractoriness, but antibodies to platelet-specific or ABH antigens may also be involved. HLA alloimmunization can follow
pregnancy, transfusion, or organ transplantation because the foreign antigens
are the donor MHC antigens themselves.
A common example of this is the development of HLA antibodies directed against
Class I antigens that occurs with transfusion of platelets, which express only Class

398

AABB Technical Manual

I antigens. The presence, in the transfused component, of leukocytes bearing
Class I and II antigens elicits alloimmunization. The likelihood of immunization
can be lessened with leukocyte-reduced
blood components, or by treatment with
ultraviolet light, which alters the costimulatory molecules or impairs antigen-presenting cell activity. The threshold
level of leukocytes required to provoke a
primary HLA alloimmune response is unclear and probably varies among different
recipients. Some studies have suggested
that 5 × 106 leukocytes per transfusion
may represent an immunizing dose. In
patients who have been previously sensitized by pregnancy or transfusion, exposure to even lower numbers of allogeneic
cells is likely to provoke an anamnestic
antibody response.

Finding Compatible Donors
The HLA antibody response of transfused
individuals may be directed against individual specificities present on donor cells
or against “public” alloantigens. Precise
characterization may be difficult. An overall assessment of the degree of HLA alloimmunization can be obtained by measuring the PRA of the recipient’s serum.
Platelet-refractory patients with a high
PRA are broadly alloimmunized and may
be difficult to support with platelet transfusions. HLA-matched platelets, obtained
by plateletpheresis, benefit some, but not
all, of these refractory patients. Because
donors with a four-antigen match for an
immunized recipient are hard to find, strategies for obtaining HLA-matched platelets
vary. Selection of partially mismatched
donors, based on serologic cross-reactive
groups, has been emphasized, but such
donors may fail to provide an adequate
transfusion response in vivo. An alternative approach to the selection of donors is

based on matching for “public” specificities rather than cross-reactive private
antigens. Obtaining an adequate number
of readily available HLA-typed donors can
prove difficult; it has been estimated that
a pool of 1000 to 3000 or more donors
would be needed to provide the transfusion requirements of most HLA-alloimmunized patients.11 Use of single antigen beads to identify HLA antibody
specificities precisely can allow a better
selection of donors who have acceptable
mismatched antigens.12
In the past, it was recommended that
patients who were at risk of becoming
alloimmunized and refractory be serologically Class I HLA-typed early in the course
of their illness, when enough lymphocytes
were present in the peripheral blood to obtain a reliable HLA type. Intensive chemotherapy makes it very difficult to obtain
enough cells for such typing. More recently,
with the advent of molecular typing techniques, a patient’s HLA alleles can be determined using genomic DNA isolated from
very small numbers of white cells or even
nonblood tissue (eg, buccal swabs).
HLA-alloimmunized patients often respond to crossmatch-compatible platelets
selected using patient serum and samples
of apheresis platelets in a platelet antibody
assay. Crossmatching techniques may assess compatibility for both HLA and platelet-specific antibodies.13 These histocompatible platelet components are further
discussed in Chapter 21.

Febrile Nonhemolytic Transfusion
Reactions
HLA antibodies, as well as granulocyte
and platelet-specific antibodies, have
been implicated in the pathogenesis of
FNHTRs. The recipient’s antibodies, reacting with transfused antigens, elicit the
release of cytokines (eg, interleukin-1) ca-

Chapter 17: The HLA System

pable of causing fever. Serologic investigation, if undertaken, may require multiple techniques and target cells from a
number of different donors (see Chapter
27).

Transfusion-Related Acute Lung Injury
In TRALI, a transfusion reaction that is
being recognized with increasing frequency, acute noncardiogenic pulmonary
edema develops in response to transfusion.
Pathogenesis appears to reflect the presence of HLA antibodies in donor blood,
which react with and fix complement to
granulocytes of the recipient, leading to
severe capillary leakage and pulmonary
edema. Rarely, HLA antibodies of the recipient react with transfused leukocytes
from the donor (see Chapter 27). Cases of
TRALI have been reported that appear to
be caused by donor antibodies against
Class II antigens in recipients. Because
Class II antigens are not expressed on
neutrophils, an alternate explanation for
activation of neutrophils in these instances
is required. One hypothesis is that Class II
antigens on the recipient’s pulmonary
macrophages are targeted by these complement-activating antibodies. Subsequent release of cytokines and chemokines
results in the recruitment and activation
of neutrophils in the lungs.14

Chimerism and Posttransfusion
Graft-vs-Host Disease
Chimerism refers to the presence of donor cells in the recipient. Persistent chimerism after blood transfusion may lead
to the development of GVHD in the recipient. The development of posttransfusion
GVHD depends on several factors: the degree to which the recipient is immunocompromised; the number and viability
of lymphocytes in the transfused component; and the degree of HLA similarity be-

399

tween donor and recipient. The observation of posttransfusion GVHD with the use
of fresh blood components from blood
relatives has highlighted the role of the
HLA system in GVHD.
Figure 17-4 illustrates the conditions for
increased risk of GVHD. The parents have
one HLA haplotype in common. Each child,
therefore, has a one in four chance of inheriting the same haplotype from each parent,
and Child #1 is homozygous for the shared
parental HLA haplotype. Transfusion of
blood from this person to an unrelated recipient who did not have this haplotype
would have no untoward consequences. If,
however, Child #1 were a directed donor for
the relatives heterozygous for that haplotype (both parents and Child #3), the recipient would not recognize any foreign antigens on the transfused lymphocytes and
would not eliminate them. The donor cells,
however, would recognize the recipient’s
foreign HLA antigens, would become activated, proliferate, and attack the host. To
avoid this situation, it is recommended that
all cellular components known to be from
blood relatives be irradiated before transfusion. Other specially chosen donor units,
such as HLA-matched platelets, may also
present an increased risk of posttransfusion
GVHD. Rarely, transfusion-associated GVHD
has occurred after the transfusion of blood
from an unrelated donor.15
Chimerism is also proposed to be responsible for the maintenance of tolerance
16
in some organ transplant recipients and
for the maintenance of HLA sensitization.17
It has been postulated that scleroderma is a
form of GVHD resulting from chimeric cells
derived from fetal cells transferred across
the placenta during pregnancy.18

Hemolytic Transfusion Reactions
HLA incompatibility has rarely been implicated as a cause of shortened red cell

400

AABB Technical Manual

Figure 17-4. HLA haplotypes in a family at risk for transfusion-associated GVHD. In contrast to the family shown in Fig 17-2, each parent shares a common HLA haplotype, HLA-A1,B8,DR17. Child 1 is homozygous for the haplotype shared by the parents and by child 3. The lymphocytes of child 1 are capable of producing posttransfusion GVHD if transfused to either parent or to child 3.

survival in patients with antibodies to HLA
a
b
antigens such as Bg (B7), Bg (B17), and
c
Bg (A28) that are expressed, although
weakly, on red cells (see Chapter 15). Such
an incompatibility may not be detected
with conventional pretransfusion testing.

HLA Testing and
Transplantation
HLA testing is an integral part of organ
transplantation. The extent of testing differs for different types of transplants. Organ transplantation is discussed in greater detail in Chapter 26.

Hematopoietic Progenitor Cell Transplants
It has long been recognized that disparity
within the HLA system represents an important barrier to successful HPC transplantation.19 HLA similarity and compatibility between the donor and the recipient
are required for engraftment and to prevent GVHD, but some degree of rejection
or GVHD remain common problems for
recipients of allogeneic HPCs, despite
immunosuppressive conditioning.
Candidate donors and recipients are
typed for their HLA-A, -B, -C, -DR and -DQ
alleles. The goal is to match, as closely as
possible, the alleles of the prospective donor and recipient at the HLA-A, -B, and
-DRB1 loci, with the optimal match being
an allele-level match.20 Some transplant

Chapter 17: The HLA System

programs additionally match for HLA-C
and -DQ alleles. Molecular HLA typing is
performed on samples from both the donor
and recipient for optimal assessment of
Class I and II region compatibility. Although HLA-identical sibling donors remain the best choice for HPC transplantation, there is increasing use of unrelated
donors identified by searching the file of 5
million HPC donors listed in the National
Marrow Donor Program’s registry of volunteer donors. The use of umbilical cord
blood stem cells and hematopoietic stem
cell grafts that have undergone T-cell depletion may allow greater donor-recipient mis21,22
matches.

Kidney and Pancreas Transplants
ABO compatibility is the most important
factor determining the immediate survival
of kidney transplants. Because ABH antigens are expressed in varying amounts on
all cells of the body, transplanted ABO-incompatible tissue comes into continuous
contact with the recipient’s ABO antibodies. Of particular importance is the expression of ABH antigens on vascular endothelial cells because the vascular supply
in the transplant is a common site for rejection.
Both the recipient and the donor are ordinarily tested for ABO, HLA-A, -B, and -DR
antigens. HLA-C and -DQ testing is also
usually performed. Before surgery, a major
crossmatch of recipient serum against donor lymphocytes is required. ASHI Standards for Histocompatibility Testing23 require that the crossmatch be performed
using a method more sensitive than routine
microlymphocytotoxicity testing, such as
prolonged incubation, washing, augmentation with antihuman globulin reagents, or
flow cytometry. Flow cytometry is the most
sensitive method and is especially useful
because it can best predict early acute re-

401

jection and delayed graft function, both of
which are strong predictors of chronic rejection and long-term allograft survival.24 In
patients undergoing cadaveric kidney retransplantation, the 7-year graft survival
rate using the T-cell flow crossmatch to select the donor kidney was comparable to
that of patients undergoing primary cadaveric transplantation (68% vs 72%) and was
significantly better than that of regraft patients for whom only the antiglobulin
lymphocytotoxicity crossmatch was used
25
(45%). Because HLA antibody responses
are dynamic, the serum used for the crossmatch is often obtained within 48 hours of
surgery and is retained in the frozen state
for any required subsequent testing. An incompatible crossmatch with unfractionated
or T lymphocytes is a contraindication to
kidney transplantation. The significance of
a positive B-cell crossmatch is unclear.
Serum from a patient awaiting cadaverdonor kidney transplant surgery is tested at
regular intervals for the degree of alloimmunization by determining the PRA. In addition, many laboratories identify the
specificities of HLA alloantibodies formed.
If an antibody with a defined HLA specificity is identified in a recipient, it is a common practice to avoid the corresponding
antigen when allocating a deceased donor
allograft. The serum samples used for periodic PRA testing are usually frozen. The
samples with the highest PRA are often
used, in addition to the preoperative sample, for pretransplant crossmatching. The
necessity of a prospective crossmatch for
recipients with no evidence of HLA sensitization has been questioned. Prompt transplantation with reduced cold ischemia time
for the renal allograft may provide greater
benefit to the patient than prospective
crossmatching, provided 1) a very sensitive
method for antibody detection, such as
flow cytometry, has been used26,27 and 2) it is
absolutely certain that the patient has had

402

AABB Technical Manual

no additional sensitizing event (ie, immunization or transfusions within 2 weeks before or any time since that serum was
screened).
The approach to kidney transplants using living donors is different. In the past,
when several prospective living donors
were being considered, MLC testing between the recipient and the donors was
sometimes performed, but it is rarely performed today. HLA matching of recipients
with kidney donors (both living and cadaveric donor) contributes to long-term allograft survival by decreasing the likelihood
of chronic rejection. In 1996, the projected
20-year allograft survival rates were 57% for
two-haplotype-matched sibling donors,
30% for one-haplotype-matched parental
donors, and 18% for cadaver donors.28 For
cadaver donors with no mismatches with
the recipient for HLA-A, -B, and -DR, the
projected 20-year allograft survival was
40%. Surprisingly, projected survival for
allografts from living, unrelated donors is
similar to those from parental donors.29 Recently, the 1-year survival rates for grafts
from living and cadaver renal donors were
93.9% and 87.7% respectively, and the halflives of living-donor and cadaver-donor renal allografts were 21.6 and 13.8 years, respectively.30

Other Solid Organ Transplants
For liver, heart, lung, and heart/lung transplants, ABO compatibility remains the
primary immunologic system for donor
selection, and determining pretransplant
ABO compatibility between donor and recipient is mandatory. HLA-A, -B, and -DR
testing of potential recipients is required,
and the transplant crossmatch must be
available before transplantation when the
recipient has demonstrated presensitization, except for emergency situations.
Levels of HLA compatibility do correlate

with graft survival after heart transplantation, but prospective HLA matching has
been difficult to implement.31

Parentage and Other
Forensic Testing
HLA typing (particularly DNA-based HLA
typing) has proven useful in forensic testing. In parentage testing, HLA typing
alone can exclude about 90% of falsely accused males. With the addition of red cell
antigen typing, the exclusion rate rises to
95% and exceeds 99% when typing for red
cell enzymes and serum proteins is included. Haplotype frequencies, rather
than gene frequencies, are used in these
calculations because linkage disequilibrium is so common in the HLA system. It
is important, however, to keep in mind
the racial differences that exist in HLA
haplotype frequencies; recombination
events must also be considered.
Other useful DNA-based assays for forensic testing are detection of alleles with
variable numbers of tandem repeats and alleles with variation in the number of short
tandem repeats, which assess other polymorphic, non-HLA genetic regions. DNAbased assays allow identification of individuals on the basis of extremely small samples of fluid or tissue, such as hairs, epithelial cells, or semen.

HLA and Disease
For some conditions, especially those believed to have an autoimmune etiology,
an association exists between HLA phenotype and occurrence of clinical disease
32-34
HLA-associated dis(see Table 17-3).
eases have several features in common.
They are known or suspected to be inherited, display a clinical course with acute

Chapter 17: The HLA System

403

Table 17-3. HLA-Associated Diseases
32-37

Disease

HLA

Celiac disease

DQ2

>250

Ankylosing spondylitis

B27

>150

Narcolepsy

DQ6

>38

Subacute thyroiditis

B35

Type I diabetes

DQ8

Multiple sclerosis

DR15, DQ6

Rheumatoid arthritis

DR4

Juvenile rheumatoid arthritis

DR8

Grave’s disease

DR17

RR = relative risk.

exacerbations and remissions, usually have
characteristics of autoimmune disorders,
and the exact cause is unknown. Evidence
has been accumulating that implicates
the HLA molecules themselves in disease
susceptibility. For instance, resistance to
cerebral malaria results from a strong
cytotoxic T-cell response to particular malarial peptides that are restricted by (fit
into the peptide-binding grooves of ) two
specific HLA molecules.35 Another mechanism that could lead to the association of
HLA phenotype and disease is the presence of a Class I or II heterodimer encoded by a specific allele that preferentially presents autoantigens to the T-cell
receptor.
The ancestral haplotype A1, B8, DR3,
DQ2 discussed previously (under Linkage
Disequilibrium) is associated with susceptibility to Type 1 diabetes, lupus, celiac disease, common variable immunodeficiency
and IgA deficiency, myasthenia gravis, and
also with an accelerated course of HIV infection, likely due to the presence of multi36
ple genes. However, HLA typing has only
limited value in assessing risk for most diseases because the association is incom-

plete, often giving false-negative and
false-positive results. The association of
HLA-B27 and ankylosing spondylitis in
those of European ancestry is instructive.
The test is highly sensitive; more than 90%
of such patients with ankylosing spondylitis
possess the HLA-B27 antigen. On the other
hand, specificity is low; only 20% of individuals with the B27 antigen will develop ankylosing spondylitis. A second condition,
narcolepsy, is strongly associated with the
HLA allele DQB1*0602.37 As with the case of
HLA-B27 and ankylosing spondylitis, over
90% of individuals with narcolepsy are positive for HLA-DQB1*0602, but only a minority of individuals with this marker develop
the disease.
The degree of association between a
given HLA type and a disease is often described in terms of relative risk (RR), which
is a measure of how much more frequently
a disease occurs in individuals with a specific HLA type when compared to individuals not having that HLA type. Calculation of
RR is usually based on the cross-product
ratio of a 2 × 2 contingency table. However,
because the HLA system is so highly polymorphic, there is an increased possibility of

404

AABB Technical Manual

finding an association between an HLA antigen and a disease by chance alone. Therefore, calculation of RR for HLA disease associations is more complex and is typically
done by Haldane’s modification of Woolf’s
formula.38-39 The RR values for some diseases associated with HLA are shown in Table 17-3.

10.

12.

13.

14.

References
1.

11.

Schreuder GMTh, Hurley CK, Marsh SGE, et
al. The HLA dictionary 2001: A summary of
HLA-A, -B, -C, -DRB1/3/4/5, -DQB1 alleles and
their association with serologically defined
HLA-A, -B, -C, -DR, and -DQ antigens. Hum
Immunol 2001;62:826-49.
Braud VM, Allan DSJ, McMichael AJ. Functions of nonclassical MHC and non-MHC-encoded class I molecules. Curr Opin Immunol
1999;11:100-8.
Feder JN, Gnirke A, Thomas W, et al. A novel
MHC class I-like gene is mutated in patients
with hereditary haemochromatosis. Nat Genet
1996;13:399-408.
Bodmer JG, Parham P, Albert ED, Marsh SG.
Putting a hold on “HLA-H.” Nat Genet 1997;
15:234-5.
McDonald JC, Adamashvili I. Soluble HLA: A
review of the literature. Hum Immunol 1998;
59:387-403.
Ghio M, Contini P, Mazzei C, et al. Soluble
HLA class 1, HLA class II, and Fas ligand in
blood components: A possible key to explain
the immunomodulatory effects of allogeneic
blood transfusion. Blood 1999;93:1770-7.
Zinkernagel RM, Doherty PC. The discovery
of MHC restriction. Immunol Today 1997;18:
14-7.
Cao K, Chopek M, Fernandez-Vina MA. High
and intermediate resolution DNA typing systems for class I HLA-A, -B, -C genes by hybridization with sequence-specific oligonucleotide probes (SSOP). Rev Immunogenet
1999;1:177-208.
Welsh K, Bunce M. Molecular typing for the
MHC with PCR-SSP. Rev Immunogenet 1999;
1:157-76.
Dzik WH. Leukoreduced blood components:
Laboratory and clinical aspects. In: Simon
TL, Dzik WH, Snyder EL, et al, eds. Rossi’s
principles of transfusion medicine. 3rd ed.
Baltimore, MD: Lippincott Williams and
Wilkins, 2002:270-87.

15.

16.

17.

18.

19.
20.

21.

22.

23.

24.

25.

Bolgiano DC, Larson EB, Slichter SJ. A model
to determine required pool size for HLAtyped community donor apheresis programs.
Transfusion 1989;29:306-10.
Pei R, Lee JH, Shih NJ, et al. Single human
leukocyte antigen flow cytometry beads for
accurate identification of human leukocyte
antigen antibody specificities. Transplantation 2003;75:43-9.
Friedberg RC. Independent roles for platelet
crossmatching and HLA in the selection of
platelets for alloimmunized patients. Transfusion 1994;34:215-20.
Kopko PM, Popovsky MA, MacKenzie MR, et
al. HLA class II antibodies in transfusion-related acute lung injury. Transfusion 2001;41:
1244-8.
Gorman TE, Julius CJ, Barth RF, et al. Transfusion-associated graft-vs-host disease. A fatal
case caused by blood from an unrelated HLA
homozygous donor. Am J Clin Pathol 2000;
113:732-7.
Starzl TE, Demetris AJ, Murase N, et al.
Chimerism after organ transplantation. Curr
Opin Nephrol Hypertens 1997;6:292-8.
Sivasai KSR, Jendrisak M, Duffy BF, et al.
Chimerism in peripheral blood of sensitized
patients waiting for renal transplantation.
Transplantation 2000;69:538-44.
Artlett CM, Smith JB, Jimenez SA. Identification of fetal DNA and cells in skin lesions
from women with system sclerosis. N Engl J
Med 1998;338:1186-91.
Thomas ED. Bone marrow transplantation: A
review. Semin Hematol 1999;36:95-103.
Mickelson EM, Petersdorf E, Anasetti PM, et
al. HLA matching in hematopoietic cell transplantation. Hum Immunol 2000;61:92-100.
Kurtzberg J, Laughlin M, Graham ML, et al.
Placental blood as a source of hematopoietic
stem cells for transplantation into unrelated
recipients. N Engl J Med 1996;335:157.
Aversa F, Tabilio A, Velardi A. Treatment of
high-risk acute leukemia with T cell depleted
stem cells from related donors with one fully
mismatched HLA haplotype. N Engl J Med
1998;339:1186-93.
Standards for histocompatibility testing. Mt.
Laurel, NJ: American Society for Histocompatibility and Immunogenetics, 1998.
Utzig MJ, Blumke M, Wolff-Vorbeck G, et al.
Flow cytometry cross-match: A method for
predicting graft rejection. Transplantation
1997;63:551-4.
Bryan CF, Baier KA, Nelson PW, et al. Longterm graft survival is improved in cadaveric
renal retransplantation by flow cytometric
crossmatching. Transplantation 2000;66:
1827-32.

Chapter 17: The HLA System

26.

27.

28.

29.

30.

31.

32.

33.
34.

35.

Taylor CJ, Smith SI, Morgan CH, et al. Selective omission of the donor crossmatch before
renal transplantation: Efficacy, safety, and effects of cold storage time. Transplantation
2000;69:719-23.
Gebel HM, Bray RA. Sensitization and sensitivity: Defining the unsensitized patient.
Transplantation 2000;69:1370-4.
Terasaki PI, Cho Y, Takemoto S, et al. Twentyyear follow-up on the effect of HLA matching
on kidney transplant survival and prediction
of future twenty-year survival. Transplant
Proc 1996;28:1144-5.
Terasaki PI, Cecka JM, Gjertson DW, Takemoto
S. High survival rates of kidney transplants
from spousal and living unrelated donors. N
Engl J Med 1995;333:333-6.
Hariharan S, Johnson CP, Bresnahan BA, et al.
Improved graft survival after renal transplantation in the United States, 1988 to 1996. N
Engl J Med 2000;342:605-12.
Ketheesan N, Tay GK, Witt CS, et al. The significance of HLA matching in cardiac transplantation. J Heart Lung Transplant 1999;18:
226-30.
Thorsby E. Invited anniversary review: HLA
associated diseases. Hum Immunol 1997;53:
1-11.
Pile KS. HLA and disease associations. Pathology 1999;31:202-12.
Howell WM, Jones DB. The role of human
leukocyte antigen genes in the development
of malignant disease. J Clin Pathol Mol Pathol
1995;48:M302-6.
Hill AV. The immunogenetics of resistance to
malaria. Proc Assoc Am Physicians 1999;111:
272-7.

36.

37.

38.

39.

405

Price P, Witt C, Allcock R, et al. The genetic
basis for the association of the 8.1 ancestral
haplotype (A1, B8, DR3) with multiple immunopathological diseases. Immunol Rev
1999;167:257-74.
Pelin Z, Guilleminault C, Risch N, et al. HLADQB1*0602 homozygosity increases relative
risk for narcolepsy but not for disease severity in two ethnic groups. US Modafinil in
Narcolepsy Multicenter Study Group. Tissue
Antigens 1998;51:96-100.
Haldane JBS. The estimation and significance
of the logarithm of a ratio of frequencies. Ann
Hum Genet 1955;20:309-11.
Woolf B. On estimating the relation between
blood groups and disease. Ann Hum Genet
1955;19:251-3.

Suggested Reading
ASHI Clinical Affairs Committee. Guidelines for
clinical histocompatibility practice. Mt. Laurel,
NJ: American Society for Histocompatibility and
Immunogenetics, 1999.
Phelan DL, Mickelson EM, Noreen HS, et al. ASHI
laboratory manual. 4th ed. Mt. Laurel, NJ: American Society for Histocompatibility and Immunogenetics, 2001.
Standards for histocompatibility testing. Mt. Laurel, NJ: American Society for Histocompatibility
and Immunogenetics, 1998.

Chapter 18: Pretransfusion Testing

Chapter 18

Pretransfusion Testing

HE PURPOSE OF pretransfusion
testing is to select blood components that will not cause harm to
the recipient and will have acceptable
survival when transfused. If performed
properly, pretransfusion tests will confirm
ABO compatibility between the component and the recipient and detect most
clinically significant unexpected antibodies.
The AABB Standards for Blood Banks
and Transfusion Services1 requires that the
following procedures be performed before
blood components are issued for transfusion:
■
Positive identification of the recipient and the recipient’s blood sample.
■
ABO group and Rh typing of the recipient’s blood.
■
Red cell antibody detection tests for
clinically significant antibodies using the recipient’s serum or plasma.
■
Comparison of current findings on
the recipient’s sample with records
of previous results.

■
■
■

■
■

Confirmation of the ABO group of
red cell components.
Confirmation of the Rh type of Rhnegative red cell components.
Selection of components of ABO
group and Rh type appropriate for
the recipient.
Performance of a serologic or computer crossmatch.
Labeling of products with the recipient’s identifying information.

Transfusion Requests
Requests for transfusion may be submitted electronically or on paper and must
contain sufficient information for positive
recipient identification. Standards1(p36) requires two independent identifiers to
identify the patient. These identifiers
could be the patient’s first and last names,
an identification number unique to that
individual, a birth date, or other identifying system. Other information necessary
407

408

AABB Technical Manual

to process the request includes identification of the component needed, the quantity, any special requests such as irradiation, gender and age of the recipient (42
CFR 493.1241), and the name of the responsible physician. The diagnosis and
the recipient’s history of transfusion and
pregnancy may be helpful in problemsolving. Each facility should have a written policy defining request acceptance
criteria. Blood requests that lack the required information, are inaccurate, or are
illegible should not be accepted.1(p36) Telephoned requests are acceptable in urgent
situations but should be documented, for
example, in a telephone log; a subsequent
request as a written authorization is to be
made within 30 days.2

Patient Identification
Collection of a properly labeled blood
sample from the intended recipient is
critical to safe blood transfusion. Most
hemolytic transfusion reactions result
from errors in sample or patient identification.3,4 The person drawing the blood
sample must identify the intended recipient in a positive manner. Each facility
must develop and implement policies and
procedures for patient identification and
specimen collection.
Most hospitals identify patients with an
identification wristband. Ideally, this wristband is placed on the patient before specimen collection and remains on the patient
until discharge. The same identifying information on the specimen tube submitted for
testing will be used to label blood components and this information will be compared against the patient’s wristband at the
time of transfusion. Some hospitals use an
internally generated or commercially available identification band with a substitute or
additional “blood bank number” as the
unique patient identifier. Commercial sys-

tems vary in design: color-coded numbers
on wristbands, tubes, and units; a wristband
with an embosser for label printing; and a system for barcoding that provides positive sample and patient identification.
Hospital policies generally require phlebotomists to collect blood specimens only
from patients who have an attached patient
identification wristband. However, in some
circumstances, it may not be possible for
the patient to wear an identification wristband, and an alternative means of positive
patient identification may be needed. The
use of wristbands is difficult when the patient has total body burns, when the patient
is an extremely premature infant, or when
the wristband is inaccessible during surgery. Some facilities allow identifying information to be placed on a patient’s ankle or
forehead. Intraoperative patient identification procedures may allow the use of an alternative identification process in lieu of an
inaccessible wristband. It is important to
remind clinical personnel that transfusions
should not be administered to a patient
who lacks positive identification.
When the patient’s identity is unknown,
an emergency identification method may
be used. This patient identification must be
attached to the patient and affixed or reproduced on blood samples. This identification must be cross-referenced with the patient’s name and hospital identification
number or code when they become known.
When hospitals allow the use of confidential or alias names, the facility must have
policies and procedures that govern their
use.
Outpatients may be identified with the
use of a patient wristband for the purpose
of blood sample collection. Alternative
methods of positive patient identification
include a driver’s license or other photographic identification. Whenever possible,
the patient should be asked to state his or
her name and to provide confirmation of

Chapter 18: Pretransfusion Testing

birth date and address. If a discrepancy is
noted, the sample must not be collected
until the patient’s identity has been clarified.
The identification of specimens used for
preadmission testing must meet the same
requirements as those used for inpatient
transfusion—there must be no doubt about
the identity of the specimen and the patient. With the advent of patient admission
on the same day as surgery, hospitals have
devised several mechanisms to identify patients when specimens have been collected
several days or weeks before surgery. One
option is to require that the patient wear an
identification wristband. Other facilities use
a unique number on specimens and on a
patient identification form that the patient
must provide on the day of surgery in order
for the preadmission specimen to be valid
for transfusion.5 An alternative procedure is
to place on the patient’s medical record the
wristband used during specimen collection. This wristband would be attached to
the patient upon arrival on the day of surgery after proper identification of the patient.6
Regardless of the system used, it must be
well known to all those who collect blood
specimens and be followed routinely.
Ideally, patient identification procedures
should be used as a matter of course to
identify patients for all treatment methods,
not solely transfusions.

Sample Labeling
Before leaving the patient, the phlebotomist must label the blood sample tubes
with two independent patient identifiers
and the date of collection. Either handwritten or imprinted labels may be used
as long as the information on the label is
identical to that on the wristband and
request. There must be a mechanism to
identify the phlebotomist1(p37); this certifi-

409

cation may be placed on the label of the
tube, placed on the requisition, or documented in a computer system.

Confirming Sample Identity in the
Laboratory
When a sample is received in the laboratory, a trained member of the staff must
confirm that the information on the label
and on the transfusion request is identical. If there is any doubt about the identity of the patient, a new sample must be
1(p37)
It is unacceptable for anyobtained.
one to correct identifying information on
an incorrectly labeled sample. Each laboratory should establish policies and procedures that define identifying information
and describe how to document receipt of
mislabeled specimens.

Blood Sample
Pretransfusion testing may be performed
on either serum or plasma. Plasma may be
the preferred specimen for some methods
such as tests using gel technology. Incompletely clotted blood samples may cause
small fibrin clots that trap red cells into
aggregates that could resemble agglutinates. Plasma collected from patients with
high levels of fibrinogen or patients with
dysproteinemia may demonstrate rouleaux.
Rouleaux formation can be mistaken for
agglutination. Clotting may be incomplete
in specimens not intended to be anticoagulated, such as patients who have been
treated with heparin. Adding thrombin or
protamine sulphate to the sample usually
corrects the problem.
It is permissible to collect blood from an
infusion line. To avoid interference from residual intravenous fluid, the tubing should
be flushed with saline, and 5 mL or a volume of blood approximately twice the fluid

410

AABB Technical Manual

volume in the line should be withdrawn
and discarded before sample collection.7

Appearance of Sample
The appearance of the serum or plasma
may create difficulties in detecting antibodyinduced hemolysis. Whenever possible, a
hemolyzed sample should be replaced with
a new specimen. Test results observed with
lipemic serum can be difficult to evaluate.
On occasion, it may be necessary to use
hemoglobin-tinged or lipemic serum or
plasma. If hemolyzed samples are used, it
should be noted in the patient testing records to differentiate hemolysis as a result
of an antigen-antibody reaction. Each institution should have a procedure describing the indications for using hemolyzed
and lipemic specimens.

Age of Sample
Blood samples intended for use in crossmatching should be collected no more
than 3 days before the intended transfusion unless the patient has not been pregnant or transfused within the preceding 3
months. If the patient’s transfusion or
pregnancy history is uncertain or unavailable, compatibility tests must be performed on blood samples collected within
3 days of RBC transfusions.1(p38) This is to
ensure that the sample used for testing
reflects the recipient’s current immunologic status because recent transfusion or
pregnancy may stimulate production of
unexpected antibodies. Because it is not
possible to predict whether or when such
antibodies will appear, a 3-day limit has
been selected as an arbitrary interval expected to be both practical and safe. It is
short enough to reflect acute changes in
immunologic status but long enough to
allow the results of preadmission testing
completed on Friday (day 0) to be used
for surgical cases performed on Monday

(day 3). The 3-day requirement applies
only to patients who have been transfused
or pregnant within the last 3 months, but
many laboratories prefer to standardize
their operations by setting a 3-day limit
on all specimens used for pretransfusion
testing.
Each institution should have a policy that
defines the length of time samples may be
used. Testing of stored specimens should
be based on the specimen storage limitations in the reagent manufacturer’s information circulars. Lack of appropriate storage space may also limit the length of time
specimens are stored.

Retaining and Storing Blood Samples
The recipient’s blood specimen and a
sample of the donor’s red cells must be
stored at refrigerator temperature for at
least 7 days after each transfusion.1(p37) Donor red cells may be from the remainder
of the segment used in the crossmatch or
a segment removed before issuing the
blood. If the opened crossmatch segment
is saved, it should be placed in a tube labeled with the unit number and sealed or
stoppered. Keeping the patient’s and donor’s samples allows repeat or additional
testing if the patient experiences adverse
effects.

Serologic Testing
The patient’s ABO group and Rh type must
be determined in order to transfuse ABOand Rh-compatible components. Standards1(pp37,38) requires that the red cells of
the intended recipient be typed for ABO
and Rh and the serum/plasma be tested
for expected and unexpected antibodies
before components containing red cells
are issued for transfusion. There should
be written procedures for exceptions during emergencies. When only plasma and

Chapter 18: Pretransfusion Testing

platelets or Cryoprecipitated AHF are being infused, historical testing information
in the patient’s record may be used. Refer
to Table 18-1 for selection of the ABO types
for red cell and plasma transfusion when
ABO-identical products are not available.

ABO Grouping and Rh Typing of the
Recipient
To determine the ABO group of the recipient, red cells must be tested with anti-A
and anti-B, and the serum or plasma with
A1 and B red cells. The techniques used
and interpretation of the results are described in Chapter 13. Any discrepant results should be resolved before blood is
given. If transfusion is necessary before
resolution, the patient should receive group
O red cells.
The patient’s red cells must be tested
with anti-D, with suitable observations or
controls to avoid a false-positive interpretation. Chapter 14 contains a more extensive
discussion of Rh typing reagents, appropriate control techniques, and weak D types. If
problems in D typing arise, the patient
should be given Rh-negative blood until the
problem has been resolved. Testing a recipient’s red cells for weak D is not necessary
because giving Rh-negative cells causes no

harm to recipients with the weak D phenotype. Omitting the test for weak D prevents
misinterpretations arising from the presence
of a positive direct antiglobulin test (DAT).
However, some transfusion services test patient pretransfusion specimens for weak D
in order to identify patients who could be
given Rh-positive blood components, thus
reserving the Rh-negative components for
patients who are D–. Routine testing for
other Rh antigens is not required.

Detecting Unexpected Antibodies to Red
Cell Antigens
Before deciding upon routine procedures
for antibody detection, the blood bank director must approve which antibodies are
considered potentially clinically significant. In general, an antibody is considered potentially clinically significant if antibodies of that specificity have been
associated with hemolytic disease of the
fetus and newborn, a hemolytic transfusion
reaction, or notably decreased survival of
transfused red cells. Antibodies reactive at
37 C and/or in the antiglobulin test are
more likely to be clinically significant
than cold-reactive antibodies.8
Numerous serologic techniques have
been developed that are suitable for detec-

Table 18-1. Selection of Components When ABO-Identical Donors Are Not Available
ABO Requirements
Whole Blood
Red Blood Cells
Granulocytes Pheresis
Fresh Frozen Plasma*
Platelets Pheresis
Cryoprecipitated AHF

411

Must be identical to that of the recipient.
Must be compatible with the recipient’s plasma.
Must be compatible with the recipient’s plasma.
Must be compatible with the recipient’s red cells.
All ABO groups are acceptable; components compatible
with the recipient’s red cells are preferred.
All ABO groups are acceptable.

*Also see Table 21-5.

412

AABB Technical Manual

tion of blood group antibodies (see Chapter
12 and Chapter 19). Goals in providing
compatible blood for a recipient are to:
Detect as many clinically significant
■
antibodies as possible.
Detect as few clinically insignificant
■
antibodies as possible.
Complete the procedure in a timely
■
manner.
Standards1(p38) requires that tests for unexpected antibodies use unpooled reagent
red cells in a method that detects clinically
significant antibodies and includes an antiglobulin test preceded by incubation at 37 C.
Each negative antiglobulin test must be
followed by a control system of IgG-sensitized cells (check cells). If alternative procedures are used, there must be documentation of equivalent sensitivity, and the
manufacturer’s specified controls must be
used.
The method chosen should have sufficient sensitivity to detect very low levels of
antibody in a recipient’s serum. Transfusion
of antigen-incompatible red cells to a recipient with a weakly reactive antibody may
result in rapid anamnestic production of
antibody, with subsequent red cell destruction. The same antibody detection procedure may be used for all categories of specimens, including pretransfusion and prenatal
tests on patients and screening of donor
blood. Once a procedure has been adopted,
the method must be described in the facility’s standard operating procedures manual.

Reading and Interpreting Reactions
In serologic testing, the hemolysis or agglutination that constitutes the visible endpoint of a red cell antigen-antibody interaction must be observed accurately and
consistently. The strength of agglutination
or degree of hemolysis observed with each
cell sample should be recorded immediately after reading. All personnel in a lab-

oratory should use the same interpretations and notations and be consistent in
grading (eg, 0-4+) reactions. Some laboratories prefer to use a numeric scoring (eg,
0-12) system to indicate reaction strength.
Refer to Method 1.8 for grading and scoring. An optical aid such as a concave mirror enhances visualization in reading tube
tests. Microscopic observation is not routinely recommended in manufacturer’s inserts for enhancement media. A microscope can be useful in distinguishing
rouleaux from true agglutination. Microscopic reading may also allow for the
detection of specific patterns of agglutination that are characteristic of some ana
tibodies. For example, anti-Sd typically
produces small refractile agglutinates in a
sea of free red cells, giving the appearance
of a mixed-field appearance. If gel or solid
phase is used, the manufacturer’s directions must be followed for reading and interpreting positive and negative reactions.

Autologous Control
An autologous control or DAT is not required as a part of pretransfusion testing.
It is of limited value, even for patients
who have recently been transfused, and
should be used only when antibody identification is required.

Practical Considerations
Antibody detection tests may be performed
in advance of, or together with, a crossmatch between the patient’s serum/plasma
and donor red cells. Performing antibody
detection tests before crossmatching permits early recognition and identification
of clinically significant antibodies. This
allows for the selection of the appropriate
crossmatch procedure and acquisition of
units with special antigen blood types (eg,
e– units).

Chapter 18: Pretransfusion Testing

Comparison with Previous Records
Results of ABO and Rh tests on a current
specimen must be compared with previous transfusion service records if there has
been prior testing during the past 12
months, and the comparison must be documented.1(p39) Errors in identification and/
or testing may be detected when discrepancies are found between previous and
current ABO and Rh results.
Records are also to be reviewed for the
presence of clinically significant red cell antibodies, for difficulties in testing, for the
occurrence of significant adverse reactions,
and for special transfusion requirements.1(p39)
Clinically significant red cell alloantibodies
may become undetectable in a recipient’s
serum over time. Between 30% and 35% of
antibodies become undetectable within 1
year and nearly 50% become undetectable
after 10 or more years.9

Crossmatching Tests
Unless there is an urgent need for blood, a
crossmatch must be performed for red
cell transfusion. The crossmatch shall use
procedures to demonstrate ABO incompatibility and clinically significant antibodies to red cell antigens. Blood lacking
the relevant antigens is to be selected for
transfusion when a patient has clinically
significant antibody identified currently
or historically, even though the antibody
is presently nonreactive.1(pp39,40) The crossmatch shall include the antiglobulin test.
When no clinically significant antibodies
are detected in current antibody screening tests and there is no record of previ1(p40)
then
ous detection of such antibodies,
only a method to detect ABO incompatibility, such as an immediate-spin or computer crossmatch, is required. It is very
rare for the antiglobulin phase of the
crossmatch to detect a clinically significant

413

unexpected antibody if the patient’s antibody detection test is negative.10,11
The potential benefits of omitting a routine antiglobulin crossmatch include decreased turnaround time, decreased workload, reduced reagent costs, and more effective
use of blood inventory. Omitting the antiglobulin phase of the crossmatch for patients
who meet the criteria must be in approved
written standard operating procedures. The
methods used for serologic crossmatching
may be the same as those used for red cell
antibody detection or identification or they
may be different. For example, gel methods
may be used for both antibody detection
and identification, but the crossmatch may
be performed using a tube test.

Repeat Testing of Donor Blood
A serologic test to confirm the ABO group
of all RBC units and the Rh type of RBC
units labeled as Rh-negative must be performed before transfusion. Confirmatory
testing for weak D is not required. This
confirmatory testing is to be performed
on a sample obtained from an attached
segment. Any discrepancies are to be resolved before the unit is issued for transfusion.1(p37)

Suggested Procedures for Routine
Crossmatching
Red cells used for crossmatching must be
obtained from a segment of tubing originally attached to the blood container. For
routine tube testing, the cells may be
washed and resuspended to 2% to 5% in
saline. Washing the donor’s red cells removes small fibrin clots and some cold
agglutinins that may interfere with interpretation of results. Because the ratio of
serum to cells markedly affects the sensitivity of agglutination tests, it is important
to stay within the 2% to 5% cell suspension range or as specified by the manufac-

414

AABB Technical Manual

turer’s instructions. For example, if too many
red cells are present, weak antibodies may
be missed because too few antibody molecules bind to each cell. Many laboratorians find that a 2% to 3% concentration
yields the best results. For tests using column (gel) or solid-phase microplate systems, follow the manufacturer’s directions.
The simplest serologic crossmatch method
is the immediate-spin saline technique, in
which serum is mixed with saline-suspended red cells at room temperature and
the tube is centrifuged immediately. The
immediate-spin crossmatch method is designed to detect ABO incompatibilities between donor red cells and recipient serum.
It can be used as the sole crossmatch
method only if the patient has no present
or previous clinically significant antibodies.
Because the testing is performed at room
temperature, antibodies such as anti-M, -N,
and -P1 may be detected that were not observed if antibody detection tests omitted
room-temperature testing. A sample immediate-spin crossmatch technique is described in Method 3.1. An antiglobulin
crossmatch procedure that meets the requirements of Standards for all routine situations is described in Method 3.2.

Type and Screen (Antibody Detection Test)
Type and screen is a policy in which the
patient’s blood sample is tested for ABO,
Rh, and unexpected antibodies, then stored
in the transfusion service for future crossmatch if a unit is needed for transfusion.
Crossmatched blood is not labeled and
reserved for patients undergoing surgical
procedures that rarely require transfusion. The blood bank must have enough
donor blood available to meet unexpected
needs of patients undergoing operations
under a type and screen policy. If transfusion becomes necessary, ABO- and Rh-

compatible blood can be safely released
after an immediate-spin or computer
crossmatch, if the antibody screen is negative and there is no history of clinically
significant antibodies. However, if the antibody screen is positive, the antibody(ies)
must be identified and antigen-negative
units for the clinically significant antibodies identified must be available for use if
needed.

Routine Surgical Blood Orders
Blood ordering levels for common elective
procedures can be developed from previous records of blood use. Because surgical
requirements vary among institutions,
routine blood orders should be based on
local transfusion utilization patterns. The
surgeons, anesthesiologists, and the medical director of the blood bank should
agree on the number of units required for
each procedure. See Chapter 3 for a more
detailed discussion of blood ordering protocols. Routine blood order schedules are
successful only when there is cooperation
and confidence among the professionals
involved in setting and using the guidelines.
Once a surgical blood ordering schedule
has been established, the transfusion service routinely crossmatches the predetermined number of units for each patient undergoing the designated procedures. Routine
orders may need to be modified for patients with anemia, bleeding disorders, or
other conditions in which increased blood
use is anticipated. As with other circumstances that require rapid availability of
blood, the transfusion service staff must be
prepared to provide additional blood if the
need arises.

Computer Crossmatch
When no clinically significant antibodies
have been detected by antibody screening

Chapter 18: Pretransfusion Testing

and history review, it is permissible to omit
the antiglobulin phase of the crossmatch
and perform only a procedure to detect
ABO incompatibility. Computerized matching of blood can be used to fulfill the requirement, provided that the following
1(pp40,41)
:
conditions have been met
The computer system has been vali■
dated, on site, to ensure that only
ABO-compatible whole blood or red
cells have been selected for transfusion.
Two determinations of the recipi■
ent’s ABO group as specified in Standards are made, one on a current
specimen and a second one by one
of the following methods: by retesting the same sample, by testing a
second current sample, or by comparison with previous records.
The computer system contains the
■
unit number, component name,
ABO group, and Rh type of the component; the confirmed donor unit
ABO group; two unique recipient
identifiers; and the recipient’s ABO
group, Rh type, and antibody screen
results.
■
A method exists to verify correct entry of data before the release of
blood components.
■
The system contains logic to alert
the user to discrepancies between
the donor ABO group and Rh type
on the unit label and the interpretation of the blood group confirmatory
test, and to ABO incompatibility between recipient and donor unit.
Butch et al12,13 and Safwenberg et al14 have
described in detail a model computer crossmatch system. Potential advantages of a
computer crossmatch include decreased
workload, reduced sample volume required
for testing, reduced exposure of personnel
to blood specimens, and better use of blood
inventory.

415

Compatibility Testing for Neonates Less
than 4 Months of Age
Requirements for compatibility testing for
neonates less than 4 months of age are
discussed in Chapter 24. An initial pretransfusion specimen must be obtained
from the infant to determine ABO and Rh
type. For ABO typing, testing the cells
with anti-A and anti-B is the only test required. Serum or plasma from either the
infant or the mother may be used to detect unexpected red cell antibodies and
for crossmatching. The infant’s serum
need not be tested for ABO antibodies unless a non-group-O infant is to receive
non-group-O cells that are incompatible
with passively acquired anti-A or anti-B.
This antibody most often comes from the
mother but can be present from group O
RBCs or plasma from incompatible platelets. Test methods in this case are to include an antiglobulin phase using either
donor or reagent A1 or B red cells. If no
clinically significant unexpected antibodies are present, it is unnecessary to crossmatch donor red cells for the initial or
subsequent transfusions. Repeat testing
may be omitted for infants less than 4
months of age during any one hospital
admission as long as they are receiving
only group O cells.1(p42)

Interpretation of Antibody
Screening and Crossmatch
Results
Most samples tested have a negative antibody screen and are crossmatch-compatible with units selected. A negative antibody screen, however, does not guarantee
that the serum does not have clinically
significant red cell antibodies, only that it
contains no antibodies that react with the
screening cells by the techniques employed.

416

AABB Technical Manual

Furthermore, a compatible crossmatch
does not guarantee normal red cell survival.
Table 18-2 reviews the possible causes of
positive pretransfusion tests. Most of what
is known about red cell antigen and antibody reactions comes from work performed in tube testing. This information is
not necessarily applicable to antibody detection tests using other technologies such
as solid-phase microtiter plates or column
technologies. Spurious or unexpected reactions in these other technologies may have
the same or other causes than those seen in
the tube tests. Depending on the antigen/
antibody reaction strength and the testing
conditions, not all of the scenarios will
result in positive tests.
The cause of the serologic problems
should be identified before transfusion.
Chapter 19 reviews techniques for problem
resolution. If the patient is found to have
clinically significant antibodies, units issued for transfusion should be nonreactive
for such antigens when tested with licensed
reagents (see Selection of Units, Other
Blood Groups). When the antibody identified is considered clinically insignificant
(eg, anti-A1, -M, -N, -P1, -Lea, and/or -Leb)
15
and is not reactive at 37 C, random units
may be selected for crossmatch.

Labeling and Release of
Crossmatched Blood at the
Time of Issue
1(p44)

Standards
requires that the following
activities take place at the time of issue:
■
A tag or label indicating the recipient’s two independent identifiers,
donor unit number, and compatibility test interpretation, if performed,
must be attached securely to the
blood container.

■

A final check of records maintained
in the blood bank for each unit of
blood or component must include:
1.
Two independent identifiers,
one of which is usually the patient’s name.
2.
The recipient’s ABO group and
Rh type.
3.
The donor unit or pool identification number.
4.
Donor’s ABO and, if required,
Rh type.
5.
The interpretation of the crossmatch tests (if performed).
6.
The date and time of issue.
7.
Special transfusion requirements
(eg, cytomegalovirus-reducedrisk, irradiated, or antigen-negative).
There must be a process to confirm
■
that the identifying information, the
request, the records, and the blood
or component are in agreement and
that discrepancies have been resolved before issue.
Additional records that may be of use include those that identify the person issuing
the blood and the person to whom the
blood was issued or the destination of the
unit.
After the transfusion, a record of the
transfusion shall be made a part of the patient’s medical record. This information
may be part of a computer record or a paper form. Records must contain the identification of the person(s) performing the test
and, if blood is issued before the resolution
of compatibility problems, the final status
of the serologic findings.
There should be a system to ensure that
the proper blood component is issued for
the intended patient. Before issuing a unit
of blood or component, personnel must
ensure that the product is acceptable for
use, the unit is checked to make sure it
does not have an abnormal color or ap-

Chapter 18: Pretransfusion Testing

417

Table 18-2. Causes of Positive Pretransfusion Tests*
Negative Antibody Screen, Incompatible Immediate-Spin Crossmatch
■ Donor red cells are ABO-incompatible, caused by error in selecting donor unit, patient
specimen, or labeling the donor unit.
■ Donor red cells are ABO-incompatible, caused by failure to detect weak expressions of antigens.
■ Donor red cells are polyagglutinable.
■ Anti-A1 in the serum of an A2 or A2B individual.
■ Other alloantibodies are reactive at room temperature (eg, anti-M).
■ Rouleaux formation.
■ Cold autoantibodies (anti-I), especially if immediate spin is not tested on antibody detection
screen.
Negative Antibody Screen, Incompatible Antiglobulin Crossmatch
■ Donor red cells have a positive direct antiglobulin test.
■ Antibody reacts only with red cells having strong expression of a particular antigen either
because of dosage effect (eg, Rh, Kidd, Duffy, and MN antigens) or because of intrinsic variation
in antigen strength (eg, P1).
■ Antibody reacts with a low-incidence antigen present on the donor red cells.
■ Passively transferred antibody is present—significant levels of circulating anti-A or -B may
be present after infusion of ABO-incompatible platelets to a recipient.
Positive Antibody Screen, Compatible Crossmatches
■ Auto-anti-IH (-H).
bH
■ Anti-Le .
■ Antibodies are dependent on reagent cell diluent.
■ Antibodies demonstrating dosage effect and red cells of the unit are from heterozygotes
(ie, express a single dose of antigen).
Positive Antibody Screen, Incompatible Crossmatches, Negative Auto Control
■ Alloantibody(ies).
■ Unexpected interactions with reagent red cells.
Positive Antibody Screen, Incompatible Crossmatches, Positive Auto Control, Negative
Direct Antiglobulin Test
■ Antibody to ingredient in enhancement media.
Positive Antibody Screen, Incompatible Crossmatches, Positive Auto Control
■ Alloantibody is present and patient is experiencing either a delayed serologic or hemolytic
transfusion reaction.
■ Passively transferred alloantibody from a derivative reactive with the recipient’s cells (eg,
intravenous immune globulin).
■ Cold-reactive autoantibody.
■ Warm-reactive autoantibody.
■ Rouleaux formation.
■ Reagent-related problems.
*Causes are dependent upon serologic methods used.

418

AABB Technical Manual

pearance, the container is not leaking, and
the product is not outdated.
Final identification of the recipient and
the blood container rests with the transfusionist, who must identify the patient and
donor unit and certify that identifying information on forms, tags, and labels is in
agreement (see Chapter 22).

Selection of Units
ABO Compatibility
Whenever possible, patients should receive ABO-identical blood; however, it
may be necessary to make alternative selections. If the component to be transfused contains 2 mL or more of red cells,
the donor’s red cells must be ABO-compatible with the recipient’s plasma.1(p40) Because plasma-containing components
can affect the recipient’s red cells, the
ABO antibodies in transfused plasma
should be compatible with the recipient’s
red cells when feasible. Requirements for
components and acceptable alternative
choices are summarized in Table 18-1.

Rh Type
Rh-positive blood components should
routinely be selected for D+ recipients.
Rh-negative units will be compatible but
should be reserved for D– recipients. D–
patients should receive red-cell-containing components that are Rh-negative to
avoid immunization to the D antigen. Occasionally, ABO-compatible Rh-negative
components may not be available for D–
recipients. In this situation, the blood
bank physician and the patient’s physician should weigh alternative courses of
action. Depending on the childbearing
potential of the patient and the volume of

red cells transfused, it may be desirable to
administer Rh Immune Globulin to a D–
patient given Rh-positive blood.16

Other Blood Groups
Antigens other than ABO and D are not
routinely considered in the selection of
units of blood. However, if the recipient
has a clinically significant unexpected antibody, antigen-negative blood should be
selected for crossmatching. If the antibody is weakly reactive or no longer demonstrable, a licensed reagent should be
used to confirm that the donor units are
antigen negative. If there is an adequate
quantity of the patient’s serum, or if another patient’s serum with the same antibody specificity is available, and that antibody reacts well with antigen-positive red
cells, that serum may be used to screen
for antigen-negative units. Those units
found to be antigen negative must be
confirmed with a licensed reagent, when
available. When licensed reagents are not
available (eg, anti-Lan or anti-Yta), expired
reagents or stored serum specimens from
patients or donors can be used, provided
that controls tested on the day of use are
acceptable (see the Food and Drug Administration Compliance Program Guidance Manual, Chapter 42, Blood and Blood
Products). When crossmatch-compatible
units cannot be found, the medical director should be involved in the decision to
transfuse the patient. (See Chapter 19 and
Chapter 20 for additional information on
issuing crossmatch-incompatible units.)
Antigen-negative units are not usually
provided for the patient who has antibodies that are not clinically significant.
Sometimes, problems associated with
crossmatching units for patients with
these antibodies may be avoided by altering the serologic technique used for the
crossmatch.

Chapter 18: Pretransfusion Testing

Blood Administered in Urgent Situations
When blood is urgently needed, the patient’s physician must weigh the risk of
transfusing uncrossmatched or partially
crossmatched blood against the risk of
delaying transfusion until compatibility
testing is complete. Ideally, a transfusion
service physician should provide consultation. The risk that the transfused unit
might be incompatible may be judged to
be less than the risk of depriving the patient of oxygen-carrying capacity of that
transfusion. See Chapter 21.

Required Procedures
When blood is released before pretransfusion testing is complete, the records
must contain a signed statement of the
requesting physician indicating that the
clinical situation was sufficiently urgent
to require release of blood. 1(p46) Such a
statement does not absolve blood bank
personnel from their responsibility to issue properly labeled donor blood that is
ABO-compatible with the patient. When
urgent release is requested, blood bank
personnel should:
1.
Issue uncrossmatched blood, which
should be:
a.
Group O Red Blood Cells if the
patient’s ABO group is unknown.
It is preferable to give Rh-negative blood if the recipient’s Rh
type is unknown, especially if the
patient is female with the potential to bear children.
b.
ABO and Rh compatible, if there
has been time to test a current
specimen. Previous records must
not be used, nor should information be taken from other records such as cards, identification tags, or driver’s license.
2.
Indicate in a conspicuous fashion on
the attached tag or label that com-

419

patibility testing was not complete
at the time of issue.
Begin compatibility tests and complete them promptly (for massive
transfusion, see below). If incompatibility is detected at any stage of testing, the patient’s physician and the
transfusion service physician should
be notified immediately.

Massive Transfusion
Massive transfusion is defined as infusion,
within a 24-hour period, of a volume of
blood approximating the recipient’s total
blood volume. Exchange transfusion of an
infant is considered a massive transfusion.
Following massive transfusion, the pretransfusion sample no longer represents
the blood currently in the patient’s circulation and its use for crossmatching has limited benefit. It is only important to confirm
ABO compatibility of units administered
subsequently. The blood bank director may
implement a more limited pretransfusion
testing protocol to be used in these situations. This protocol should be in writing to
ensure consistent application by all laboratory personnel.

Blood Administered After
Non-Group-Specific Transfusion
Transfusion services sometimes release
units for transfusion during emergencies
before they receive a sample for blood
typing. When it arrives, the sample is a
pretransfusion specimen. In most cases,
the sample is tested and units of that ABO
group are issued for transfusion without
concern for anti-A and/or anti-B remaining from the initial emergency-release
units. Because most donor units are RBCs
with comparatively little supernatant
plasma, or RBCs (Additive Solution Added)
with even less residual plasma, the risks
involved in following this practice are

420

AABB Technical Manual

minimal. For example, the patient may
exhibit a transient positive DAT.
In some cases, when large volumes of
red cells are transfused, or when young
children or infants receive transfusions,
passively acquired ABO antibodies may be
detected,17 and it may be appropriate to
demonstrate compatibility of red cells of
the patient’s original ABO group with a
freshly drawn serum specimen. If the crossmatch is incompatible because of ABO antibodies, transfusion with red cells of the alternative group should be continued.
If the change in blood type involves only
the Rh system, return to type-specific blood
is simple because antibodies are unlikely to
be present in the plasma of either the recipient or the donor. If a patient has received
blood of an Rh type other than his or her
own before a specimen has been collected
for testing, it may be difficult to determine
the correct Rh type. If there is any question
about the recipient’s D type, Rh-negative
blood should be transfused if possible. The
use of Rh Immune Globulin prophylaxis
should be considered when Rh-positive
components are transfused to Rh-negative
patients. See Chapter 21.

10.
11.

12.

13.

14.

15.

16.

References
1.

17.

Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.
Code of federal regulations. Title 42 CFR
493.1241. Washington, DC: US Government
Printing Office, 2004 (revised annually).
Sazama K. Reports of 355 transfusion associated deaths: 1976 through 1985. Transfusion
1990;30:583-90.
Linden JV, Wagner K, Voytovich AE, Sheehan
J. Transfusion errors in New York State: An
analysis of 10 years’ experience. Transfusion
2000;40:1207-13.
Butch SH, Stoe M, Judd WJ. Solving the sameday admission identification problem (abstract). Transfusion 1994;34(Suppl):93S.

AuBuchon JP. Blood transfusion options: Improving outcomes and reducing costs. Arch
Pathol Lab Med 1997;121:40-7.
Procedures for the collection of diagnostic
blood specimens by venipuncture. 3rd ed.
NCCLS document H3-A2, approved standard.
Villanova, PA: National Committee for Clinical Laboratory Standards, 1991.
Issitt PD, Anstee DJ. Applied blood group serology. Durham, NC: Montgomery Scientific
Publications, 1998:873-905.
Ramsey G, Smietana SJ. Long term follow-up
testing of red cell alloantibodies. Transfusion
1994;34:122-4.
Oberman HA. The present and future crossmatch. Transfusion 1992;32:794-5.
Meyer EA, Shulman IA. The sensitivity and
specificity of the immediate-spin crossmatch.
Transfusion 1989;29:99-102.
Butch SH, Judd WJ, Steiner EA, et al. Electronic verification of donor-recipient compatibility: The computer crossmatch. Transfusion 1994;34:105-9.
Butch SH, Judd WJ. Requirements for the
computer crossmatch (letter). Transfusion 1994;
34:187.
Safwenberg J, Högman CF, Cassemar B. Computerized delivery control a useful and safe
complement to the type and screen compatibility testing. Vox Sang 1997;72:162-8.
Shulman IA, Petz LD. Red cell compatibility
testing: Clinical significance and laboratory
methods. In: Petz LD, Swisher SN, Kleinman
S, eds. Clinical practice of transfusion medicine. 3rd ed. New York: Churchill Livingstone,
1996:199-244.
Pollack W, Ascari WQ, Crispen JF, et al. Studies on Rh prophylaxis II: Rh immune prophylaxis after transfusion with Rh-positive blood.
Transfusion 1971;11:340-4.
Garratty G. Problems associated with passively transfused blood group alloantibodies.
Am J Clin Pathol 1998;109:169-77.

Suggested Reading
Beck ML, Tilzer LL. Red cell compatibility testing:
A perspective for the future. Transfus Med Rev 1996;
10:118-30.
Brecher ME. Collected questions and answers. 7th
ed. Bethesda, MD: AABB, 2001:49-56.
Butch SH for the Scientific Section Coordinating
Committee. Guidelines for implementing an electronic crossmatch. Bethesda, MD: AABB, 2003.

Chapter 18: Pretransfusion Testing

Butch SH, Oberman HA. The computer or electronic crossmatch. Transfus Med Rev 1997;11:25664.
Frohn C, Dumbgen L, Brand JM, et al. Probability
of anti-D development in D– patients receiving D+
RBCs. Transfusion 2003;43:893-8.
Garratty G. How concerned should we be about
missing antibodies to low-incidence antigens?
(editorial) Transfusion 2003;43:844-7.
Issitt PD. From kill to overkill: 100 years of (perhaps too much) progress. Immunohematology 2000;
16:18-25.

421

of erroneous blood grouping of blood bank specimens. Transfusion 1997;37:1169-72.
Padget BJ, Hannon JL. Variations in pretransfusion
practices. Immunohematology 2003;19:1-6.
Rossmann SN for the Scientific Section Coordinating Committee. Guidelines for the labeling of specimens for compatibility testing. Bethesda, MD:
AABB, 2002.
Schonewille H, van Zijl AM, Wijermans PW. Importance of antibodies against low-incidence RBC
antigens in complete and abbreviated crossmatching. Transfusion 2003;43:939-44.

Lumadue JA, Biyd JS, Ness PM. Adherence to a strict
specimen-labeling policy decreases the incidence

Chapter 19: Initial Detection and Identification of Alloantibodies to Red Cell Antigens

Chapter 19

Initial Detection and
Identification of
Alloantibodies to Red Cell
Antigens

ED CELL ALLOANTIBODIES other
than naturally occurring anti-A or
-B are called unexpected red cell
alloantibodies. Depending upon the group
of patients or donors studied and the sensitivity of the test methods used, alloantibodies can be found in 0.3% to 38% of the
population.1,2 Alloantibodies react only with
allogeneic red cells, whereas red cell autoantibodies react with the red cells of the
antibody producer. Immunization to red
cell antigens may result from pregnancy,
transfusion, transplantation or from injections with immunogenic material. In
some instances, no specific immunizing
event can be identified. These naturally
occurring antibodies are a result of exposure to environmental, bacterial, or viral
antigens that are similar to blood group
antigens. Also, antibodies detected in serologic tests can be passively acquired.
These antibodies may be acquired from
injected immunoglobulins, donor plasma,

or passenger lymphocytes in transplanted
organs or hematopoietic progenitor cells.

Significance of
Alloantibodies
Alloantibodies to red cell antigens may be
initially detected in any test that uses serum or plasma (eg, ABO test, antibody detection test, crossmatch) or in an eluate
prepared from red cells coated with alloantibody. Once an antibody is detected, its
specificity should be determined and its
clinical significance assessed.
A clinically significant red cell antibody,
although difficult to define, could be characterized as an antibody that shortens the
survival of transfused red cells or has been
associated with hemolytic disease of the fetus and newborn (HDFN). The degree of
clinical significance varies with antibodies
of the same specificity. Some antibodies
423

424

AABB Technical Manual

cause destruction of incompatible red cells
within hours or even minutes, others decrease the survival by only a few days, and
some cause no discernible red cell destruction. Antibodies of some specificities are
known to cause HDFN, whereas others may
cause a positive direct antiglobulin test
(DAT) in the fetus without clinical evidence
of HDFN.
Reported experience with other examples of antibody with the same specificity
can be used in assessing clinical significance. Table 15-1 summarizes the expected
reactivity and clinical significance of commonly encountered alloantibodies. Daniels
et al3 have published a review of these and
other specificities. For some antibodies, few
or no data exist, and decisions must be
based on the premise that clinically significant antibodies are usually those active at
37 C and/or by an indirect antiglobulin test
(IAT). It is not true, however, that all antibodies active in vitro at 37 C and/or by an
IAT are clinically significant.
Antibodies encountered in pretransfusion testing should be identified to assess
the need to select antigen-negative red cell
components for transfusion. Patients with
clinically significant antibodies should,
whenever practical, receive red cells that
have been tested and found to lack the corresponding antigen. In prenatal testing, the
specificity and immunoglobulin class of an
antibody influence the likelihood of HDFN.
The results of antibody identification tests
on donor blood may be used to characterize the units for labeling before transfusion
and to procure blood typing reagents or
teaching samples.

General Procedures
The techniques employed for antibody detection and antibody identification are

similar. Antibody identification methods
can be more focused and based on the reactivity patterns seen in the antibody detection test.
Each facility should establish which
techniques for antibody detection and
identification will be employed routinely.
Sometimes, it is valuable to develop flowcharts to clearly guide the technologist
through the process of selecting additional
techniques to identify antibody specificities. This approach is helpful in expediting
the identification process and minimizing
unnecessary testing.

Specimen Requirements
Either serum or plasma may be used for
antibody detection and identification.
Plasma is not suitable for detecting complement-activating antibodies. A 5- to
10-mL aliquot of whole blood usually
contains enough serum or plasma for
identifying simple antibody specificities;
more may be required for complex studies.
When autologous red cells are studied,
the use of a sample anticoagulated with
EDTA avoids problems associated with
the in-vitro uptake of complement components by red cells, which may occur in
clotted samples.

Medical History
It is useful to know a patient’s clinical diagnosis, history of transfusions or pregnancies, and recent drug therapy when
performing an antibody identification.
For example, in patients who have had recent red cell transfusions, the circulating
blood may contain sufficient donor cells
to make red cell phenotyping studies difficult to interpret. Special procedures to
separate the autologous red cells for typing may be required (see Method 2.15).
Other special procedures may be required
for patients with autoantibodies.

Chapter 19: Initial Detection and Identification of Alloantibodies to Red Cell Antigens

Reagents

Antibody Detection Red Cells
Group O red cells suitable for antibody
screening are commercially available and
are offered as sets of either two or three
vials of single-donor red cells. Pooled antibody detection cells (usually from two
donors) can only be used in testing serum
samples from donors.
The decision to use two or three cells in
an antibody detection test should be based
on circumstances in each individual laboratory. The reagent red cells are selected to
express the antigens associated with most
commonly encountered antibodies. Reagent cells licensed by the Food and Drug
Administration (FDA) for this purpose must
express the following antigens: D, C, E, c, e,
M, N, S, s, P1, Lea, Leb, K, k, Fya, Fyb, Jka, and
Jkb.4 Some weakly reactive antibodies react
only with red cells from donors who are homozygous for the genes controlling the expression of these antigens, a serologic phenomenon called dosage. Antibodies in the
Rh, Duffy, MNS, and Kidd systems most
commonly demonstrate dosage. Reagent
red cells should be refrigerated when not in
use and should not be used for antibody
detection beyond their expiration date.

Antibody Identification Panels
Identification of an antibody to red cell antigen(s) requires testing the serum against
a panel of selected red cell samples with
known antigen composition for the major
blood groups. Usually, they are obtained
from commercial suppliers, but institutions
may assemble their own by using red cells
from local sources. Panel cells are (except
in special circumstances) group O, allowing serum of any ABO group to be tested.
Each cell of the panel is from a different
individual. The cells are selected so that,
taking all the cells into account, a distinctive pattern of positive and negative reac-

425

tions exists for each of many antigens. To
be functional, a reagent red cell panel must
make it possible to identify with confidence
those clinically significant alloantibodies
that are most frequently encountered, such
as anti-D, -E, -K, and -Fya. The phenotypes
of the reagent red cells should be distributed such that single specificities of the
common alloantibodies can be clearly
identified and most others excluded. Ideally, the pattern of reactivity for most examples of single alloantibodies will not overlap
with any other; eg, all of the K+ samples
should not be the only ones that are also
E+. It may also be valuable to include red
cell samples with a double dose of the antigen in question for antibodies that frequently show dosage. To lessen the possibility that chance alone has caused an
apparently definitive pattern, there must be
a sufficient number of red cell samples that
lack, and sufficient red cell samples that express, most of the antigens listed in Table
19-1.
Commercially prepared panels are generally issued every 2 to 4 weeks. Each panel
contains different red cell samples with different antigen patterns, so it is essential to
use the phenotype listing sheet that comes
with the panel in use. Commercial cells
usually come as a 2% to 5% suspension in a
preservative medium that can be used directly from the vial. Washing is generally
unnecessary unless the media in which the
reagent cells are suspended are suspected
of interfering with alloantibody identification.
Panel cells should not be used beyond
the expiration date; however, this is not always
practical. Most serologists use in-date reagent
cells for initial antibody identification panels and, if necessary, use expired reagent
cells for exclusion or confirmation of specificity. Each laboratory must establish and
validate a policy for the use of expired reagent cells.5

426
AABB Technical Manual

Table 19-1. A Reagent Red Cell Panel for Alloantibody Identification
Copyright © 2005 by the AABB. All rights reserved.

Rh
Sample
Rh
#
Phenotype C
1
2
3
4
5
6
7
8
9
10

r′r
R1w
R1
R2
r″r
r
r
r
r
R0

+
+
+
0
0
0
0
0
0
0

Kell

Duffy

Kidd

Lewis

Fya Fyb

Jka Jkb

Le a Le b

0
+
0
0
0
0
0
0
0
0

+
0
0
+
+
+
+
+
+
+

0
+
+
+
0
0
0
0
0
+

0
0
0
+
+
0
0
0
0
0

+
+
+
0
+
+
+
+
+
+

0
+
0
0
0
0
+
0
0
0

+ Denotes presence of antigen; 0 denotes absence of antigen.

+
+
+
0
+
0
0
+
0
0

0
+
+
+
+
+
+
0
+
0

+
0
+
0
0
+
+
0
+
+

+
+
+
+
+
0
0
+
0
+

+
+
0
+
0
+
+
+
0
+

0
+
0
+
0
0
0
+
0
0

+
0
+
0
+
0
+
0
+
0

MNS
M

+
+
+
0
+
+
+
0
0
+

+
+
0
+
+
+
0
+
+
+

0
+
+
0
+
0
+
0
+
+

+
+
0
+
0
+
0
+
0
+

Chapter 19: Initial Detection and Identification of Alloantibodies to Red Cell Antigens

Antiglobulin Reagents
To detect clinically significant antibodies,
most antibody identification tests include
an antiglobulin phase. Either polyspecific
or IgG-specific antiglobulin reagents may
be used. Polyspecific reagents may detect,
or detect more readily, antibodies that bind
complement. This may be of value in the
detection of certain Kidd antibodies.6 Although this may be advantageous in some
instances, many serologists prefer to use
IgG-specific reagents to avoid unwanted
reactivity resulting from in-vitro complement binding by cold-reactive antibodies.

Enhancement Media
Although the test system may consist solely
of serum and red cells (reagent red cells as
provided by the manufacturer or salinesuspended red cells), most serologists use
some type of enhancement medium. Many
different media are available, including
low-ionic-strength saline (LISS), polyethylene glycol (PEG), and 22% to 30% albumin. For the initial antibody identification panel, most laboratories use the
same enhancement method used in their
routine antibody detection tests. Additional enhancement techniques may be
employed for more complex studies. Enhancement techniques are discussed later
in this chapter.

Autologous Control (Autocontrol)
It may be helpful to know how a serum
under investigation reacts with autologous
red cells. This helps determine whether
alloantibody, autoantibody, or both are
present. Serum that reacts only with the
reagent red cells usually contains only
alloantibody, whereas reactivity with both
reagent and autologous red cells suggests
the presence of autoantibody or autoantibody plus alloantibody. However, a patient with alloantibodies to antigens ex-

427

pressed on recently transfused red cells may
have circulating donor red cells coated
with alloantibodies, resulting in a positive
autocontrol. Because this result may be
misinterpreted as being due to autoantibody, a detailed history of recent transfusions should be obtained for all patients
with a positive DAT or positive autocontrol.
The autologous control, in which serum
and autologous cells undergo the same test
conditions as serum and reagent cells, is
not the same as a DAT. Incubation and the
presence of enhancement reagents may
cause reactivity in the autologous control
that is only an in-vitro phenomenon. If the
autocontrol is positive in the antiglobulin
phase, a DAT should be performed. If the
DAT is positive, elution studies should be
considered if the patient has been recently
transfused, if there is evidence of immune
hemolysis, or if the results of serum studies
prove inconclusive. A reactive DAT may
also indicate the presence of autoantibody.
If autoantibody is detected in the serum,
adsorption studies may be necessary to
detect coexisting alloantibodies.

Basic Antibody Identification
Techniques
For initial panels, it is common to use the
same methods and test phases used in the
antibody detection test or crossmatch.
Some serologists may choose to include
an immediate centrifugation reading and/
or a room temperature incubation and
reading without adding an enhancement
medium. This may enhance the detection
of certain antibodies (anti-M, -N, -P1, -I,
a
b
-Le , or -Le ) and may help to explain reactions detected at other phases. Many institutions omit these steps to avoid finding
antibodies that react only at lower temperatures and have little or no clinical sig-

428

AABB Technical Manual

nificance. Test observation after 37 C incubation may detect some antibodies (eg,
potent anti-D, -K, or -E) that can cause direct agglutination of red cells. Other antia
a
bodies (eg, anti-Le , -Jk ) may be detected
by their lysis of antigen-positive red cells
during the 37 C incubation. Some serologists believe that because clinically significant antibodies will be detected with the
IAT, the reading after 37 C can be safely
omitted. This omission will lessen the detection of unwanted positive reactions resulting from clinically insignificant coldreactive auto- and alloantibodies.7,8
The phenotype of the reactive antibody
detection cells will provide clues to the
specificity or help exclude specificities. This
information is useful for selecting cells that
would be most informative in additional
testing.
If the patient has previously identified
antibodies, this may affect panel selection.
For example, if the patient is known to have
anti-e, it will not be helpful to test the serum against a panel of 10 red cell samples,
nine of which are e+. Testing a panel of selected e– red cell samples will better reveal
any newly formed antibodies.
Sometimes, the patient’s phenotype influences the selection of reagent cells. For
example, if the patient is D– and the serum
is reactive with D+ cells in the screening
test, an abbreviated panel or select cell panel
of D– red cell samples may be tested. This
can both confirm the presence of anti-D
and demonstrate the presence or absence
of additional antibodies, while minimizing
the amount of testing required.9

be a more complex process combining
technical knowledge and intuitive skills.
Panel results generally will include both
positive and negative results at different
phases of testing, each of which should be
explained by the final conclusion. Determination of the patient’s red cell phenotype and the probability of antibody specificity can also play roles in the final
interpretation.

Positives and Negatives
Both positive and negative reactions are
important in antibody identification. Positive reactions indicate the phase and
strength of reactivity (see Method 1.8 for
grading agglutination), which can suggest
certain specificities. Positive reactions also
can be compared to the antigen patterns
expressed by the panel cells to help assign
specificity. Single alloantibodies usually
yield definite positive and negative reactions that create a clear-cut pattern with
antigen-positive and -negative reagent
red cell samples. For example, if a serum
reacts only with cells 4 and 5 of the reagent red cell panel shown in Table 19-1,
anti-E is very likely present. Both reactive
samples express E and all nonreactive
samples lack E.
Negative reactions are important in antibody identification because they allow tentative exclusion of antibodies to antigens
expressed on the nonreactive cells. Exclusion of antibodies is an important step in
the interpretation process and must be performed to ensure proper identification of
all the antibodies present.

Exclusion or “Crossing Out”

Interpreting Results
Antibody screening results are interpreted
as positive or negative based on the presence or absence of reactivity (eg, agglutination). Interpretation of panel results can

A widely used first approach to the interpretation of panel results is to exclude
specificities based on nonreactivity with
the serum tested. Such a system is sometimes referred to as a “cross-out” or “rule-

Chapter 19: Initial Detection and Identification of Alloantibodies to Red Cell Antigens

out” method. Once results have been recorded on the worksheet, the antigen profile of the first nonreactive cell is examined. If an antigen is present on the cell
and the serum did not react with the cell,
the presence of the corresponding antibody may be, at least tentatively, excluded.
Many technologists will cross out that antigen from the listing on the panel sheet
to facilitate the process. After all antigens
present on that cell have been crossed off,
interpretation proceeds with the other nonreactive cells and additional specificities
are excluded. In most cases, this process
will leave a group of antibodies that still
have not been excluded.
Next, the cells reactive with the serum
are evaluated. The pattern of reactivity for
each nonexcluded specificity is compared
to the pattern of reactivity obtained with
the test serum. If there is a pattern that
matches exactly, that is most likely the
specificity of the antibody in the serum.
However, if there are remaining specificities that have not been excluded, additional testing may be needed to eliminate
remaining possibilities and to confirm the
specificity identified. This requires testing
the serum against cells selected for specific
antigenic characteristics. For example, this
approach could be employed if the pattern
of positive reactions exactly fits anti-Jka, but
anti-K and anti-S have not been excluded.
Then serum should be tested against selected cells, ideally with the following phenotypes: Jk(a–), K–, S+; Jk(a–), K+, S–; and
Jk(a+), K–, S–. The reaction pattern with
these cells should both confirm the presa
ence of anti-Jk and include or exclude
anti-K and anti-S.
Although the exclusion (cross-out) approach often identifies simple antibody
specificities, it should be considered only a
provisional step, particularly if the cross-out
was completed based on the nonreactivity
of cells with weaker (eg, heterozygous) ex-

429

pression of an antigen (see Variations in
Antigen Expression).

Probability
To ensure that an observed pattern is not
the result of chance alone, conclusive antibody identification requires serum to be
tested against sufficient reagent red cell
samples that lack, and that express, the
antigen that corresponds to the apparent
specificity of the antibody.
A standard approach (based on Fisher’s
exact method10) has been to require, for
each specificity identified, three antigenpositive cells that react and three antigen-negative cells that fail to react. This
standard is not always possible, but it works
well in practice, especially if cells with
strong antigen expression are available. A
somewhat more liberal approach is derived
from calculations by Harris and Hochman,11
whereby minimum requirements for a
probability (p) value of 0.05 are met by having two positive and three negative cells, or
one positive and seven negative cells (or
the reciprocal of either combination). Comparative p values are shown in Table 19-2.
The use of two positive and two negative
cells is also an acceptable approach for antibody confirmation.12 Additional details on
calculating probability may be found in the
suggested readings by Race and Sanger,
Menitove, and Kanter. The possibility of
false-negative results with antigen-positive
cells must be considered as well as unexpected positives, ie, false-positive results
due either to the presence of an additional
antibody specificity or an error in the presumptive antibody identification.

Phenotype of Autologous Red Cells
Once an antibody has been tentatively
identified in a serum, it is often helpful to

430

AABB Technical Manual

Table 19-2. Probability Values
10

No. Tested

No. Positive

No. Negative

p (Fisher )

p (Harris and Hochman )

5
6
6
7
7
8
8
8
8
9
9
9
10
10
10
10
10

3
4
3
5
4
7
6
5
4
8
7
6
9
8
7
6
5

2
2
3
2
3
1
2
3
4
1
2
3
1
2
3
4
5

0.100
0.067
0.050
0.048
0.029
0.125
0.036
0.018
0.014
0.111
0.028
0.012
0.100
0.022
0.008
0.005
0.004

0.035
0.022
0.016
0.015
0.008
0.049
0.011
0.005
0.004
0.043
0.008
0.003
0.039
0.007
0.002
0.001
0.001

demonstrate the presence or absence of
the corresponding antigen on the autologous red cells. For example, if serum from
an untransfused individual appears to
contain anti-Fya but the autologous red
cells have a negative DAT and type as
Fy(a+), the data are clearly in conflict and
further testing is indicated.
Determination of the patient’s phenotype can be difficult if the patient has been
transfused recently, generally within 3
months. If a pretransfusion specimen is
available, these red cells should be used to
determine the phenotype. Alternatively, the
patient’s own red cells can be separated
from the transfused red cells and then
typed (see Methods 2.15 and 2.16). The use
of potent blood typing reagents, appropriate controls, and observation for mixedfield reactions often allow an unseparated

specimen to be phenotyped. Phenotyping
results on posttransfusion samples can be
misleading, however, and should be interpreted with caution.13 If there is little uncertainty about antibody identification, extensive efforts to separate and type the patient’s
own red cells are not necessary. Compatible
antiglobulin crossmatch,14(p40) of antigennegative donor units provides additional
confirmation of antibody specificity. Definitive testing can be performed on the patient’s red cells after a sufficient period
without red cell transfusion. In a chronically transfused patient, definitive testing
can be performed after an interval during
which only antigen-negative blood has
been given. Any antigen-positive red cells
detected after prolonged transfusion of antigen-negative blood would presumably be
the patient’s own.

Chapter 19: Initial Detection and Identification of Alloantibodies to Red Cell Antigens

Complex Antibody Problems
Not all antibody identifications are simple. The exclusion procedure does not always lead directly to an answer and additional approaches may be required. Figure
19-1 shows some approaches to identifying antibodies in a variety of situations
when the autocontrol is negative. Additional approaches may be needed if the
autocontrol is positive; they are discussed
later in this chapter.

Variations in Antigen Expression
For a variety of reasons, antibodies do not
always react with all cells positive for the
corresponding antigen. Basic interpretation by exclusion, as described previously,
may result in a given specificity being excluded because the sample is nonreactive
with an antigen-positive red cell sample,
despite the presence of the antibody.
Technical error, weak antibody reactivity,
and variant or weak antigenic expression
are all possible causes. Therefore, whenever possible, antibody specificities should
be excluded only on the basis of cells
known to bear a strong expression of the
antigen. Enhancement techniques often help
resolve problems associated with variations in antigen expression (see Methods
3.2.2, 3.2.3, 3.2.4, 3.5.5, and 3.5.6).

Zygosity
Reaction strength of some antibodies may
vary from one red cell sample to another
due to a phenomenon known as dosage,
in which antibodies react preferentially
with red cells from persons homozygous
for the gene that determines the antigen
(ie, possessing a “double dose” of the antigen). Red cells from individuals heterozygous for the gene may express less antigen
and may react weakly or be nonreactive.
Alloantibodies vary in their tendency to

431

recognize dosage. Many antibodies to antigens in the Rh, Duffy, MNS, and Kidd
systems have this trait.

Variation in Adults and Infants
a

Some antigens (eg, I, P1, Le , and Sd ) are
expressed to varying degrees on red cells
from different adult donors. This variation is unrelated to zygosity; however, the
antigenic differences can be demonstrated serologically. Certain antibodies
(eg, anti-I, -Lea) demonstrate weaker reactivity with cord red cells than with red
cells from adults (see Table 19-3).

Changes with Storage
Blood group antibodies may give weaker
reactions with stored red cells than with
fresh red cells. Some antigens (eg, Fya, Fyb,
a
a
17
M, P1, Kn /McC , Bg) deteriorate during
storage more rapidly than others and the
rate varies among red cells from different
donors. Because red cells from donors are
often fresher than commercial reagent cells,
some antibodies give stronger reactions
with suspensions of donor cells than with
reagent cells. Frozen storage of red cells
may result in antigen deterioration that
can cause misleading antibody identification results.
The pH or other characteristics of storage media can affect the rate of antigen deterioration.17,18 For example, Fya and Fyb antigens may be weakened when the cells are
stored in a suspending medium of low pH
and low ionic strength. Alternatively, certain antibodies may demonstrate stronger
or weaker reactions with red cells from different manufacturers using different suspending media. The age and nature of the
specimen must also be considered when
typing red cells. Antigens on cells from clotted samples tend to deteriorate faster than
antigens on cells collected in citrate anticoagulants such as ACD or CPD. Red cells in

432
AABB Technical Manual

Figure 19-1. Approaches for identifying antibodies (modified from Brendel ).

Chapter 19: Initial Detection and Identification of Alloantibodies to Red Cell Antigens

433

Table 19-3. Antigen Expression on Cord Red Blood Cells*
Expression

Antigens

Negative

Lea, Leb, Sda, Ch, Rg, AnWj

Weak

I, H, P1, Lua, Lub, Yta, Vel, Bg, McCa, Yka, S1a, Csa, Hy, Gy, Joa, Doa, Dob, Fy3

Strong

i, LWa, LWb

*Modified from Reid.

donor units collected into these anticoagulants generally retain their antigens throughout the standard shelf life of the blood component. EDTA samples up to 14 days old
are suitable for antigen typing; however, the
manufacturer’s instructions should be consulted when using commercial typing re19
agents.

No Discernible Specificity
Factors other than variation in antigen expression may contribute to difficulty in
interpreting results of antibody identification tests. If the reactivity obtained with
the serum is very weak and/or if the crossout process has excluded all likely specificities, alternative approaches to interpretation should be used.

Antigens Present in Common
Instead of excluding antibodies to antigens on nonreactive cells, one can observe what antigens are common to the
reactive cells. For example, if the cells reacting at room temperature are all P1+, yet
not all the P1+ cells react, the antibody
could be an anti-P1 that does not react
with cells having a weaker expression of
the antigen. (Sometimes, such cells are
marked on the panel sheet as “+w.”) With
this in mind, one could use a method to
enhance anti-P1, such as testing at colder
temperatures.

If all the reactive cells are Jk(b+), but not
all the Jk(b+) cells react, the reactive ones
might all be Jk(a–b+), with a double-dose
expression of the antigen. Enhancement
techniques, such as enzymes, LISS, or PEG,
may then help demonstrate reactivity with
all the remaining Jk(b+) cells. Typing the
patient’s cells to confirm they lack the corresponding antigen can also be very helpful.

Inherent Variability
Nebulous reaction patterns that do not
appear to fit any particular specificity are
characteristic of antibodies, such as antiBga, that react with HLA antigens on red
cells. These antigens vary markedly in
their expression on red cells from different individuals. Rarely, a pattern of clearcut reactive and nonreactive tests that
cannot be interpreted is due to the incorrect typing of reagent red cells. If the cell
is from a commercial source, the manufacturer should be notified immediately
of the discrepancy.

Unlisted Antigens
Sometimes a serum sample reacts with an
antigen not routinely listed on the antigen
profile supplied by the reagent manufacturer; Ytb is one example. Even though serum studies yield clear-cut reactive and
nonreactive tests, anti-Ytb may not be sus-

434

AABB Technical Manual

pected. In such circumstances, it is useful
to ask the manufacturer for additional
phenotype information. If the appropriate blood typing reagent is available, reactive and nonreactive red cell samples, as
well as the autologous red cells, can be
tested. These problems often have to be
referred to an immunohematology reference laboratory.

ABO Type of Red Cells Tested
A serum sample may react with many or
all of the group O reagent red cell samples, but not with red cells of the same
ABO phenotype as the autologous red
cells. This occurs most frequently with
anti-H, -IH, or -LebH. Group O and A2 red
cells have large amounts of H antigen; A1
and A1B red cells express very little H (see
Chapter 13). Sera containing anti-H or -IH
react strongly with group O reagent red
cell samples, but autologous A1 or A1B red
cells or donor cells used for crossmatching may be weakly reactive or nonreactive. Anti-LebH reacts strongly with group
O, Le(b+) red cells, but reacts weakly or
not at all with Le(b+) red cells from A1 or
A1B individuals. Such antibodies should
be suspected when the antibody screen,
which uses group O red cells, is strongly
reactive, but serologically compatible A1
or A1B donor samples can be found without difficulty.

Multiple Antibodies
When a serum contains two or more alloantibodies, it may be difficult to interpret
the results of testing performed on a single panel of reagent red cells. The presence of multiple antibodies may be suggested by a variety of test results.
1.
The observed pattern of reactive and
nonreactive tests does not fit that of a
single antibody. When the exclusion
approach fails to indicate a specific

pattern, it is helpful to see if the pattern matches any two combined
specificities. For example, if the reactive cells (see Table 19-1) are numbers 2, 4, 5, and 7, none of the specificities remaining after crossing-out
exactly fits that pattern, but if both K
and E are considered together, a pattern is discerned. Cells 2 and 7 react
because of anti-K, cells 4 and 5 because of anti-E. If the typing patterns for no two specificities fit the
reaction pattern, the possibility of
more than two antibodies must be
considered. The more antibodies a
serum contains, the more complex
the identification and exclusion of
specificities will be, but the basic
process remains the same.
Reactivity is present at different test
phases.
When reactivity occurs at several
phases, each phase should be evaluated separately. The pattern seen at
room temperature may indicate a
different specificity from the pattern
of antiglobulin results. It is also
helpful to look at variability in the
strength of reactions seen at each
phase of testing. Table 15-2 provides
information on the characteristic reactivity phase of several antibodies.
Unexpected reactions are obtained when
attempts are made to confirm the specificity of a suspected single antibody.
If a serum suspected of containing
anti-e reacts with additional samples
that are e–, another antibody may be
present or the suspected antibody
may not be anti-e. Testing a panel of
selected e– red cell samples may
help indicate an additional specificity.
No discernible pattern emerges.
When uniform or variable reaction
strengths are observed, and dosage or

Chapter 19: Initial Detection and Identification of Alloantibodies to Red Cell Antigens

other variation in antigen strength does
not provide an explanation, additional approaches and methods of
testing are indicated. Some helpful
steps include:
a.
If strong positive results were
obtained, use the exclusion
method with nonreactive cells
to eliminate some specificities
from initial consideration.
b.
If weak or questionable positive
results were obtained, test the serum against cells carrying a strong
expression of antigens corresponding to any suspected specificities and combine this with
methods to enhance reactivity.
c.
If the patient has not been recently transfused, type the patient’s red cells and eliminate
from consideration specificities
that correspond to antigens on
the autologous cells.
d.
Use methods to inactivate certain antigens on the red cells,
eg, enzyme treatment to render
cells negative for antigens such
as Fya, Fyb, and S.
e.
Use adsorption/elution methods
to separate antibodies.
f.
Enhance antibody reactivity by
using a more sensitive method
(eg, PEG). These and other methods that may be helpful are discussed below.

Antibodies to High-Incidence Antigens
If all reagent red cell samples are reactive,
but the autocontrol is nonreactive, an
alloantibody to a high-incidence antigen
should be considered, especially if the
strength and test phase of reactions are
uniform for all cells tested. Antibodies to
high-incidence antigens can be identified
by testing red cells of selected rare pheno-

435

types and by testing the patient’s autologous
red cells with sera known to contain antibodies to high-incidence antigens. Knowing
the race or ethnic origin of the antibody
producer can help in selecting additional
tests to be performed. Cells that are null
for all antigens in a system (eg, Rhnull or Ko)
or modified red cells (eg, dithiothreitoltreated cells, see Method 3.10) can help
limit possible specificities to a particular
blood group.
If cells negative for particular high-incidence antigens are not available, cells positive for lower-incidence alleles can sometimes be helpful. Weaker reactivity with
Co(a+b+) cells when compared with common Co(a+b–) cells, for instance, might
suggest anti-Coa. Antibodies to high-incidence antigens may be accompanied by
other antibodies to common antigens,
which can make identification much more
difficult. Because the availability of cells
negative for high-incidence antigens is limited, it may be necessary to refer specimens
suspected of containing antibodies to highincidence antigens to an immunohematology reference laboratory.

Serologic Clues
Knowledge of the serologic characteristics
of particular antibodies to high-incidence
antigens can help in identification.
1.
Reactivity in tests at room temperak
ture suggests anti-H, -I, -P1, -P, -PP1P
a
a
(-Tj ), -LW (some), -Ge (some), -Sd ,
or -Vel.
2.
Lysis of reagent red cells when testing with fresh serum is characteristic
of anti-Vel, -P, - PP1Pk, and -Jk3. It is
also seen with some examples of
anti-H and -I.
3.
Reduced or absent reactivity in enzyme tests occurs with anti-Ch, -Rg,
-Inb, -JMH, or -Ge2 and is seen with
a
some examples of anti-Yt .

436

AABB Technical Manual

Weak nebulous reactions in the antiglobulin phase are often associated
with anti-Kna, -McCa, -Yka, and -Csa.
Complement-binding autoantibodies,
such as anti-I or anti-IH, give similar
results when polyspecific antiglobulin reagents are used.
Antibodies such as anti-U, -McCa,
-Sla, -Jsb, -Hy, -Joa, -Tca, -Cra, and -Ata
should be considered if the serum is
from a Black individual because the
antigen-negative phenotypes occur
almost exclusively in Blacks. Individuals with anti-Kpb are almost always
White. Anti-Dib is usually found among
Asian, South American Indians, and
Native American populations.16(pp526,527)

Interpreting a Positive DAT
When a patient produces antibody directed to a high-incidence antigen after
transfusion, the posttransfusion red cells
may have a positive DAT, and both serum
and eluate may react with all cells tested.
Because this pattern of reactivity is identical to that produced by many warm-reactive autoantibodies that may also appear
after transfusion, these two scenarios can
be very difficult to differentiate. A posttransfusion alloantibody to a high-incidence antigen would be expected to produce a DAT of mixed-field appearance (ie,
some cells agglutinated among many
unagglutinated cells) because only the
transfused red cells would be coated with
antibody. In practice, however, weak sensitization and mixed-field sensitization
can be difficult to differentiate. If a pretransfusion red cell sample is not available,
it may be helpful to use cell separation
procedures to isolate autologous cells for
testing. Performing a DAT on autologous
cells and/or testing the posttransfusion
serum with DAT-negative autologous cells
may help to distinguish autoantibody

from alloantibody. Chapter 15 discusses
additional serologic characteristics of antibodies reacting with high-incidence red
cell antigens.

Antibodies to Low-Incidence Antigens
Reactions between a serum sample and a
single donor or reagent red cell sample
may be caused by an antibody to a lowa
incidence antigen, such as anti-Wr . If red
cells known to carry low-incidence antigens are available, the serum can be
tested against them, or the one reactive
red cell sample can be tested with known
examples of antibodies to low-incidence
antigens. A single serum often contains
multiple antibodies to low-incidence antigens; therefore, the expertise and resources of an immunohematology reference laboratory may be required to confirm
the suspected specificities.

Serologic Strategies
If an antibody to a low-incidence antigen
is suspected, transfusion should not be
delayed while identification studies are
undertaken. If an antibody in the serum
of a pregnant woman is thought to be directed against a low-incidence antigen,
testing the father’s red cells can predict
the possibility of incompatibility with the
fetus, and identifying the antibody is unnecessary. If a newborn has a positive
DAT, testing of the mother’s serum or an
eluate from the infant’s cells against the
father’s red cells (assuming they are ABOcompatible) can implicate an antibody to
a low-incidence antigen as the probable
cause; identifying the antibody is usually
of little importance.
Some reference laboratories do not attempt
to identify antibodies to low-incidence antigens because they are often only of academic interest. Identification may be made

Chapter 19: Initial Detection and Identification of Alloantibodies to Red Cell Antigens

when time permits and suitable reagents
are available.

Unexpected Positive Results
When a serum reacts with a panel cell
designated as positive for a low-incidence
antigen, further testing to exclude the antibody is usually unnecessary. For every
antigen of low incidence represented on a
panel, there are many more that are not
represented and are also not excluded by
routine testing. Reactivity against low-incidence antigens is not uncommon; although the antigens are rare, antibodies
against some of the low-incidence antigens are much less rare. Presumably, the
testing is being performed because the
serum contains some other antibody and
reactivity with the cell expressing the
low-incidence antigen is a coincidental
finding. This may complicate interpretation of the panel results but rarely requires confirmation of antibody specificity or typing of donor blood to ensure the
absence of the antigen. If typing is desired,
a negative crossmatch with the patient’s
serum is sufficient demonstration that the
antigen is absent. Many antibodies to
low-incidence antigens are reactive only
at temperatures below 37 C and are of
doubtful clinical significance.
When the serum reacts only with red
cells from a single donor unit or reagent
cell, the other possibilities to consider are
that the reactive donor red cells are ABOincompatible, have a positive DAT, or are
polyagglutinable.

Antibodies to Reagent Components and
Other Anomalous Serologic Reactions
Antibodies to a variety of drugs and additives can cause positive results in antibody
detection and identification tests. The
mechanisms are probably similar to those
discussed in Chapter 20.

437

Most of these anomalous reactions are
in-vitro phenomena and have no clinical
significance in transfusion therapy other
than causing laboratory problems that delay needed transfusions. They rarely cause
erroneous interpretations of ABO typing
that could endanger the patient. For a more
detailed discussion, see the suggested reading by Garratty.

Ingredients in the Preservative Solution
Antibodies that react with an ingredient
in the solution used to preserve reagent
red cells (eg, chloramphenicol, neomycin,
tetracycline, hydrocortisone, EDTA, sodium
caprylate, or various sugars) may agglutinate cells suspended in that solution. Reactivity may occur with cells from several
commercial sources or may be limited to
cells from a single manufacturer. The
autologous control is often nonreactive,
unless the suspension of autologous red
cells is prepared with the manufacturer’s
red cell diluent or a similar preservative.
Such reactions can often be circumvented
by washing the reagent cells with saline
before testing. The role of the preservative
can often be confirmed by adding the medium to the autologous control and converting a nonreactive test to a positive test.
In some cases, however, washing the reagent cells does not circumvent reactivity
and the resolution may be more complex.

Ingredients in Enhancement Media
Antibodies reactive with ingredients in
other reagents, such as commercially prepared LISS additives or albumin, can cause
agglutination in tests using reagent, donor,
and/or autologous red cells. Ingredients that
have been implicated include parabens (in
some LISS additives), sodium caprylate (in
some albumins), and thimerosal (in some
LISS/saline preparations). Antibody to ingredients in enhancement media may be

438

AABB Technical Manual

suspected if the autologous control is
positive but the DAT is negative. Omitting
the enhancement medium will usually
circumvent this reactivity.
In some cases, antibodies dependent upon
reagent ingredients will show blood group
specificity, eg, paraben-dependent anti-Jka,
caprylate-dependent anti-c. The autocontrol may be reactive if the patient’s own
red cells carry the antigen, but the DAT
should be negative.

Problems with Red Cells
The age of the red cells can cause anomalous serologic reactions. Antibodies exist
that react only with stored red cells; they
can cause agglutination of reagent red cells
by all techniques and enhanced reactivity
in tests with enzyme-treated red cells. Such
reactivity is not affected by washing the
red cells, and the autocontrol is usually
nonreactive. No reactivity will be seen in
tests on freshly collected red cells, ie, from
freshly drawn donor or autologous blood
samples.

The Patient with a Positive Autocontrol

No Recent Transfusions
Reactivity of serum with the patient’s own
cells may indicate the presence of autoantibody (see Chapter 20). If this reactivity occurs at room temperature or below,
the cause is often anti-I or another cold
autoagglutinin. Reactivity of the autocontrol in the antiglobulin phase usually
signifies a positive DAT and the possibility
of autoantibody. If, in addition, the serum
reacts with all cells tested, autoadsorption
or other special procedures may be necessary to determine whether autoantibody
in the serum is masking any significant
alloantibodies. If the serum is not reactive

or shows only weak reactivity, an eluate may
demonstrate more potent autoantibody.
A negative DAT but a positive autocontrol by an IAT is unusual and may indicate antibody to a reagent constituent
causing in-vitro reactivity with all cells, including the patient’s own. It may also indicate the presence of warm autoantibodies
or cold autoagglutinins such as anti-I, -IH,
or -Pr reacting by IAT when enhancement
media are used.
Cold Autoantibodies. Potent cold autoagglutinins that react with all cells, including the patient’s own, can create special
problems, especially when reactivity persists at temperatures above room temperature. Cold autoagglutinins may be benign
or pathologic. (See Chapter 20 for a more
detailed discussion.)
There are different approaches to testing
a serum with a potent cold agglutinin. One
approach is to determine if the thermal
amplitude is high enough (usually 30 C or
above) that the antibody has clinical significance. For identification purposes and determination of thermal amplitude, in-vitro
autoadsorption of the serum must be avoided
by keeping the freshly collected blood
warm (37 C) until the serum is separated.
For purposes of detecting potentially clinically significant antibodies, methods that
circumvent the cold autoantibody are commonly used.
Procedures for the detection of alloantibodies in the presence of cold-reactive autoantibodies are discussed in Chapter 20 and
include:
1.
Prewarmed techniques, in which red
cells and serum to be tested, and saline used for washing, are incubated
at 37 C before they are combined (see
Method 3.3).
2.
The use of anti-IgG rather than polyspecific antiglobulin serum.
3.
Cold autoadsorption, to remove autoantibodies but not alloantibodies.

Chapter 19: Initial Detection and Identification of Alloantibodies to Red Cell Antigens

Adsorption with rabbit red cells.
Dealing with Warm Autoantibodies. Patients with warm-reactive autoantibody
present in their sera create a special problem because the antibody reacts with virtually all cells tested. If such patients are to be
transfused, it is important to detect any
clinically significant alloantibodies that the
autoantibody may mask. Techniques are
discussed in Chapter 20 and Methods 4.9,
4.10, 4.11, and 4.12.
Reactivity of most warm-reactive autoantibodies is greatly enhanced by such
methods as PEG and enzymes, and to lesser
extent by LISS and albumin. It may be advantageous to perform antibody detection
tests without the enhancement media usually employed. If tests are nonreactive, the
same procedure can be used for compatibility tests, without the need for adsorptions.

Recent Transfusions
If the autocontrol is positive in the antiglobulin phase, there may be antibodycoated cells in the patient’s circulation,
causing a positive DAT, which may show
mixed-field reactivity. Elution may be helpful, especially when tests on serum are
inconclusive. For example, a recently transfused patient may have a positive autocontrol and serum that reacts weakly with
most but not all Fy(a+) red cells. It may be
possible to confirm anti-Fya specificity by
elution, which concentrates into a small
fluid volume the immunoglobulin molecules present in small numbers on the red
cells in the whole blood sample. It is rare
for transfused cells to make the autocontrol
positive at other test phases, but it can occur, especially with a newly developing or
cold-reactive alloantibody.
If the positive DAT does not have a
mixed-field appearance and, especially, if
the serum is reactive with all cells tested,
the possibility of autoantibody should be

439

considered. Detection of masked alloantibodies may require allogeneic adsorptions.
Accurate phenotyping of red cells may
be difficult if the DAT is reactive in any patient, whether or not there has been recent
transfusion. A positive DAT will cause the
cells to be reactive in any test requiring the
addition of antiglobulin serum and with
some reagent antibodies (notably those in
the Rh system) in a high protein medium.
With rare exception, most monoclonal reagents not tested by an IAT can give valid
phenotyping results despite a positive DAT.20

Immunohematology Reference
Laboratories
When antibody problems cannot be resolved or when rare blood is needed, immunohematology reference laboratories
can provide consultation and assistance
through their access to the American Rare
Donor Program (see Method 3.13).

Selecting Blood for
Transfusion
Once an antibody has been identified, it is
important to decide its clinical significance. Antibodies reactive at 37 C and/or
by IAT are potentially clinically significant
and those reactive at room temperature
and below are not; however, there are
many exceptions. For example, anti-Ch,
anti-Rg, and many of the Knops and Cost
antibodies have little or no clinical effect
despite reactivity by an IAT. Anti-Vel, -P,
and -PP1Pk (-Tja) may react only at cold
temperatures yet may cause red cell destruction in vivo. Comparison with documented cases in the literature and consultation with immunohematology reference laboratories should provide guidance
about previous examples of similar specificities.

440

AABB Technical Manual

Phenotyping Donor Units
Whenever possible, red cell units selected
for transfusion to a patient with a potentially clinically significant antibody should
be tested and found to be negative for the
appropriate antigen. Even if the antibody
is no longer detectable, the red cells of all
subsequent transfusions to that patient
should lack the antigen, to prevent a secondary immune response. The transfusion service must maintain records of all
patients in whom significant antibodies
have been previously identified.14(pp38,72) An
antiglobulin crossmatch procedure is required if the serum contains, or has previously contained, a significant antibody.
A potent example of the antibody should
be used to identify antigen-negative blood.
Often, this is a commercial antiserum, but
to save expensive or rare reagents, units can
first be tested with the patient’s serum. The
absence of antigen, in nonreactive units, can
then be confirmed with the commercial reagent. If the antibody is of unusual specificity or one for which commercial reagents
are not available, a stored sample from the
sensitized patient can be used to select
units for transfusion at a later time, especially if the patient’s later specimens lose
reactivity. If a patient’s serum is to serve as a
typing reagent, it should be well characterized and retain its reactivity after storage,
and appropriate negative and weak-positive controls should be used at the time of
testing. The FDA has established the following criteria for licensing some reagents21:
a
a
1.
Anti-K, anti-k, anti-Jk , anti-Fy , and
w
anti-C : dilution of 1:8 to give at least
1+ reaction.
2.
Anti-S, anti-s, anti-P1, anti-M, anti-I,
anti-c (saline), anti-e (saline), and
anti-A1: dilution of 1:4 to give at least
1+ reaction.
3.
Most other specificities: undiluted,
must give at least a 2+ reaction.

Reagents prepared in-house from sera
that meet these dilution criteria can be
used.

Source of Antibodies
When selecting units for patients with clinically significant antibodies, some serologists recommend typing the chosen units
with antibodies from two different sources,
but others consider it unnecessary, especially when potent reagents are available.
Different lots of antibody from the same
manufacturer and even reagents from different manufacturers may not have been
prepared from different source material
because manufacturers often share the
same resources.

Labeling Units
If a donor unit from a blood establishment
is to be labeled with the results of special
antigen typing, use of licensed (commercial) reagents is preferred. If no licensed
reagent is available, the unit may be labeled with appropriate wording (eg,
“Tested and found to be negative for XX
antigen using unlicensed typing reagents”).22 Except for results of ABO and D
typing, there is no requirement that results of antigen typing appear on the label
of donor units. The establishment may
use a tie tag attached to the unit for the
additional labeling.

When to Test
For certain antibody specificities, typing
of donor units may not be necessary and
the patient’s serum can be used to select
serologically compatible red cells. This is
especially true for antibodies that characteristically react below 37 C (eg, anti-M,
-N, -P1, -Lea, -Leb, -A1) and do not ordinarily exhibit an anamnestic response to
the transfusion of antigen-positive red
cells.

Chapter 19: Initial Detection and Identification of Alloantibodies to Red Cell Antigens

It is rarely necessary to provide antigen-negative donor units as a prophylactic
measure for patients whose cells lack an
antigen but who do not have detectable antibody. However, special consideration is
sometimes given to certain Rh antibodies.
When a patient of the R1R1 phenotype has
anti-E detected in the serum, some workers
suggest that donor blood be negative for
both the E and c antigens,23 based on the
assumption that the stimulus to produce
the anti-E may also have stimulated an
anti-c or anti-cE that remains undetected
by routine tests. For an R2R2 patient with
demonstrated anti-C, the use of C–, e– donor blood may be considered. When an
antibody has not been specifically demonstrated, but cannot conclusively be excluded, it may be appropriate to transfuse
blood that lacks the antigen.

Tests to Predict Clinical Significance
Certain laboratory procedures have been
used to predict the significance of particular antibodies. The monocyte monolayer
assay, which quantifies rosetting and/or
phagocytosis of antibody-sensitized red
cells, can be used to predict the in-vivo
clinical significance of some antibodies.
The test for antibody-dependent cellular
cytotoxicity (ADCC), which measures lysis
of antibody-coated cells, and the chemiluminescence assay, which measures the
respiratory release of oxygen radicals after
phagocytosis of antibody-coated cells,
have been helpful in predicting in-vivo
antibody reactivity, particularly for severity of HDFN. For cold-reactive antibodies,
in-vitro thermal amplitude studies can
predict the likelihood of in-vivo problems.
In-vivo tests may also be used to evaluate significance of a given antibody. The
most common technique is infusion of
radiolabeled, antigen-positive red cells,
usually tagged with 51Cr. It is possible to

441

measure survival of 1 mL or less of infused
cells. Flow cytometry can also be used to
measure the survival of infused cells, but a
larger aliquot of red cells (about 10 mL) is
generally required. Small aliquots of incompatible cells may have a faster rate of destruction than an entire unit of red cells.

When Blood of Rare Type Is Needed
Blood of a rare type includes not only units
negative for high-incidence antigens but
also blood negative for a combination of
common antigens. When a patient has
multiple antibodies, it can be helpful to
determine the frequency of compatible
donors. To calculate this, the frequency of
random donors negative for one antigen
must be multiplied by the frequency of
donors negative for each of the other antigens. For example, if a serum contains
anti-c, -Fya, and -S, and, among random
donors, 18% are c–, 34% are Fy(a–), and
45% are S–, the frequency of compatible
units would be: 0.18 × 0.34 × 0.45 = 0.028.
If the patient is group O, then, because
45% of random donors are group O, 1.3%
(0.028 × 0.45) of random donors would be
compatible with the patient’s serum. If
any of these three antibodies occurred
singly, finding compatible blood would
not be too difficult. Clearly, when all three
antibodies are present, a large number of
random donors would be necessary to
provide even one unit. The preceding calculation uses frequencies in populations
of European ethnicity. If the donor population is predominantly of a different origin, frequencies for that group, if available, should be used.
When units of rare (<1 in 5000) or uncommon (<1 in 1000) type are needed, the
American Rare Donor Program can be very
helpful. This program, which can be accessed only by personnel of an accredited

442

AABB Technical Manual

immunohematology reference laboratory,
can identify blood suppliers known either
to have units available (usually frozen red
cells) or to have eligible donors who may be
asked to donate (see Method 3.13).
Family members offer another potential
source of rare blood donors. Siblings are often the best source of serologically compatible blood for patients with multiple antibodies or antibodies to high-incidence
antigens. The absence of high-incidence
antigens usually reflects inheritance of the
same rare blood group gene from each parent, and offspring of the same parents are
far more likely to have the same two rare
genes than someone in the general population. In most cases, blood from the patient’s
parents or children (and some siblings) will
carry only a single dose of the relevant antigen; if transfusion is essential, and there is
no alternative to giving incompatible blood,
these heterozygous donors would be considered preferable to random donors. Occasionally, blood from a parent or child also
lacks the high-incidence antigen.
In HDFN or other alloantibody-associated problems in infants, the mother, if ABO
compatible, is often the logical donor. If the
mother’s red cells are transfused, it is helpful
to retain the plasma for use as a rare reagent.
If the clinical situation allows, autologous
transfusion should be considered for patients
for whom compatible blood is difficult to
find. For some patients with multiple antibodies for whom autologous transfusion is
not an option, it may be necessary to determine whether any of the antibodies is likely
to be significantly less destructive than the
others and, in a critical situation, give blood
incompatible for that particular antigen.

Frequency of Antibody Testing
Once an antibody has been identified in a
patient’s serum, how frequently should
antibody detection and identification tests

be performed? A primary antibody response will produce detectable antibody
as early as 7 to 10 days but typically over a
period of 2 weeks to several months. A
secondary immune response produces
detectable antibody in a shorter time, as
early as 2 to 7 days and usually within 20
days. Shulman24 found that, in a small
number of patients, “new” antibodies could
be detected within 1 to 2 days after transfusion. AABB Standards for Blood Banks
and Transfusion Services14(p38) requires that,
for a patient who has been pregnant or received red cells within the preceding 3
months, antibody detection and compatibility tests must be performed on a specimen obtained within 3 days of the next
scheduled transfusion. The transfusion
service may consider testing a fresher
specimen if clinical evidence suggests
failure of recently transfused red cells to
survive as expected.
If a patient has previously identified clinically significant antibodies, antigen-negative red cells must be selected for all future
transfusions, even if the antibodies are no
longer detectable. In addition, an antiglobulin crossmatch must be performed using
antigen-negative red cells.
It is rarely necessary to repeat identification of known antibodies. AABB Standards
states that in patients with previously identified antibodies, methods of testing shall
be those that identify additional clinically
significant antibodies.14(p38) Each laboratory
should define and validate methods for the
detection of additional antibodies in these
patients. Depending on the specificity of
the known antibody, repeated testing of the
patient’s serum against routine antibody
detection cells is often not informative. It is
more useful to test against cells negative for
the antigen(s) to which the patient has antibody and positive for other major antigens.
This allows detection of most additional
antibodies that might develop. Usually, ap-

Chapter 19: Initial Detection and Identification of Alloantibodies to Red Cell Antigens

propriate cells can be selected from available red cell panels. Selection of test cells
may be simplified if the patient’s cells are
known to express a given antigen. The selected cells need not be positive for that antigen because the corresponding antibody
would not be anticipated.

Selected Serologic
Procedures
Many techniques and methods may be
useful in antibody identification. Some of
the methods given here are used routinely
by many laboratories; others are alternatives that may apply only in special circumstances. It is important to remember
that no single method is optimal for detecting all antibodies in all samples. Any
laboratory performing antibody detection
or identification should have standard procedures for routine testing and have access
to at least some alternative approaches.
Additional procedures are available in a
variety of references (see Suggested Reading).

Enhancement Techniques
When a pattern of weak reactions fails to
indicate specificity, or when the presence
of an antibody is suspected but cannot be
demonstrated, use of the following procedures may be helpful. An autologous control should be included with each test
performed.

LISS and PEG
The rationale for these procedures and
some technical details are discussed in
Chapter 12 and Method 3.2. Each may be
used to enhance reactivity and reduce incubation time. LISS methods include the
use of low-ionic-strength saline for resus-

443

pension of test cells and, more commonly,
the use of commercially available lowionic-strength additive media. The use of a
LISS additive requires no preparatory
stages, but care should be taken to adhere
closely to the manufacturer’s product
insert to ensure that the appropriate proportion of serum to LISS is achieved.
Commercially prepared LISS additives
may include other enhancement components besides low-ionic-strength saline.
Commercially prepared PEG additives are
also available and may contain additional
enhancing agents. Because LISS and PEG
enhance autoantibody activity, their use
may create problems with certain sam25,26
ples.

Enzyme Techniques
Treatment of red cells with proteolytic enzymes enhances their reactivity with antibodies in the Rh, P, I, Kidd, Lewis, and
some other blood group systems and simultaneously destroys or weakens reactivity with other antibodies, most notably
those in the Duffy and MNS systems (see
Table 19-4). The clinical significance of
antibodies that react only with enzyme
techniques is questionable. The literature
indicates that “enzyme-only” antibodies
28
may have no clinical significance. Procedures for the preparation and use of proteolytic enzyme solutions are given in
Methods 3.5 through 3.5.6.

Temperature Reduction
Some alloantibodies (eg, anti-M, -N, -P1,
a
b
-Le , -Le , -A1) that react at room temperature react better at lower temperatures;
specificity may be apparent only below 22
C. An autocontrol is especially important
for tests at cold temperatures because many
sera also contain anti-I or other cold-reactive autoantibodies.

444

AABB Technical Manual

Increased Serum-to-Cell Ratio
Increasing the volume of serum incubated
with a standard volume of red cells may
enhance the reactivity of antibodies present in low concentration. One acceptable
procedure is to mix 5 to 10 volumes of serum with one volume of a 2% to 5% saline
suspension of red cells and incubate for
60 minutes at 37 C; periodic mixing during incubation promotes contact between
red cells and antibody molecules. It is
helpful to remove the serum before washing the red cells for the antiglobulin test
because the standard three or four washes
may be insufficient to remove all the unbound immunoglobulin present in the
additional volume. Additional washes are
not recommended because bound antibody molecules may dissociate. Increasing the serum-to-red cell ratio is not
appropriate for tests using a low-ionicstrength medium or requiring specific
proportions of serum and additive.

Increased Incubation Time
For most antibodies, a 15-minute incubation period is insufficient to achieve equilibrium and the observed reactions may
be weak, particularly in saline or albumin
media. Extending incubation to 30 to 60
minutes may improve reactivity and help
clarify the observed pattern of reactions.
Extended incubation may have a negative effect when LISS or PEG are used. If incubation exceeds the recommended times
for these methods, antibody reactivity may
be lost. Care must be taken to use all reagents according to the manufacturer’s directions.

Alteration of pH
Decreasing the pH of the reaction system
to 6.5 enhances the reactivity of certain
antibodies, notably some examples of
anti-M.29 If anti-M is suspected because

the only cells agglutinated are M+N–,
modifying the serum to a pH of 6.5 may
reveal a definitive pattern of anti-M reactivity. The addition of one volume of 0.1 N
HCl to nine volumes of serum brings the
pH to approximately 6.5. The acidified serum should be tested against known M–
cells as a control for nonspecific agglutination. Similarly, some examples of anti-P
may benefit from a lower pH.30
Low pH, however, significantly decreases
reactivity of some antibodies.31 If unbuffered
saline used for cell suspensions and for
washing has a pH much below 6.0, antibodies in the Rh, Duffy, Kidd, and MNS systems may lose reactivity. Use of phosphate-buffered saline (see Method 1.7) can
control pH and enhance detection of anti32
bodies poorly reactive at a lower pH.

Techniques to Isolate, Remove, or
Depress Antibody Reactivity
It is sometimes useful to decrease or eliminate the reactivity of an antibody. This
can be done by inhibiting the antibody with
specific substances, by physically removing immunoglobulin molecules, or by removing (or weakening) corresponding antigens from the red cells. Such methods can
help confirm suspected specificities and promote identification of additional antibodies.

Inhibition Tests
Soluble forms of some blood group antigens exist in such body fluids as saliva,
urine, or plasma, or can be prepared from
other sources. These substances can be used
to inhibit reactivity of the corresponding
antibody. If, for example, a suspected
anti-P1 does not give a definitive agglutination pattern, loss of reactivity after addition of soluble P 1 substance strongly
suggests that this is the specificity. A parallel dilution control with saline is essential.

Chapter 19: Initial Detection and Identification of Alloantibodies to Red Cell Antigens

Inhibition can also be used to neutralize
antibodies that mask the concomitant presence of nonneutralizable antibodies. The
following soluble blood group substances
can be used in antibody identification tests:
1.
Lewis substances. Lea and/or Leb substances are present in the saliva of
persons who possess the Le gene. Lea
substance is present in the saliva of
Le(a+b–) individuals, and Le(a–b+)
persons have both Lea and Leb substances in their saliva (see Method
2.5). Commercially prepared Lewis
substance is also available.
2.
P1 substance. Soluble P1 substance is
present in hydatid cyst fluid and can
be prepared from pigeon egg whites.
P1 substance is available commercially.
a
a
3.
Sd substance. Soluble Sd blood group
substance is present in various body
fluids; the most abundant source is
urine.33 To confirm anti-Sda specificity in a serum sample, urine from a
known Sd(a+) individual (or a pool
of urine specimens) can be used to
inhibit reactivity. Urine known to lack
Sda substance, or saline, should be used
as a negative control (see Method 3.11).

445

Chido and Rodgers substances. Ch
and Rg antigens are epitopes of the
fourth component of human complement (C4).34,35 Anti-Ch and -Rg react by an IAT with the trace amounts
of C4 present on normal red cells. If
red cells are coated in vitro with excess C4,36 these antibodies may cause
direct agglutination. A useful test to
identify anti-Ch and -Rg is by the inhibition of the antibodies with plasma
from Ch+, Rg+ individuals (see Method
3.9).
Blood group sugars. Sugars that correspond to the immunodominant
configurations of A, B, H, and some
other red cell structures can be used
to inhibit antibodies. Inhibiting anti-A
or -B may allow a serum to be tested
against non-group-O cells.

Inactivation of Blood Group Antigens
Certain blood group antigens can be destroyed or weakened by suitable treatment
of the cells (see Table 19-4). Modified cells
can be useful both in confirming the presence of suspected antibodies and in detecting additional antibodies. This can be

Table 19-4. Alteration of Antigens by Various Agents*
†

Agent

Antigens Usually Denatured or Altered

Proteolytic enzymes‡

M, N, S, Fya, Fyb, Yta, Ch, Rg, Pr, Tn, Mg, Mia/Vw, Cla, Jea, Nya,
JMH, some Ge, Inb
a
Yt , JMH, Kna, McCa, Yka, LWa, LWb, all Kell, Lutheran, Dombrock,
and Cromer blood group antigens
Alteration of all the antigens listed above

DTT
ZZAP (a combination of
DTT and proteolytic
enzymes)
27

*Modified from Wilkinson.
†
Some antigens listed may be weakened rather than completely denatured. Appropriate controls should be used with
modified cells.
‡
Different proteolytic enzymes may have different effects on certain antigens.

446

AABB Technical Manual

especially helpful if the antigen is one of
high incidence and antigen-negative cells
are rare.
Proteolytic enzymes are commonly used
to alter red cell antigens. Ficin, papain,
trypsin, and bromelin, the enzymes most
frequently used, remove antigens such as
M, N, Fya, Fyb, Xga, JMH, Ch, and Rg (see Table 19-4). Depending on the specific enzyme
and method used, other antigens may be
altered or destroyed. Antigens inactivated
by one proteolytic enzyme will not necessarily be inactivated by other enzymes.
Sulfhydryl reagents such as 2-aminoethylisothiouronium bromide (AET) or
dithiothreitol (DTT) (see Method 3.10) can
be used to weaken or destroy antigens in the
Kell system and some other anti-gens.37-39
ZZAP reagent, which contains proteolytic
40
enzyme and DTT, denatures antigens sensitive to DTT (eg, all Kell system antigens)
in addition to enzyme-sensitive antigens
(see Method 4.9). Glycine-HCl/EDTA treatment of red cells also destroys Bg and Kell
system antigens. However, with the exception of Era antigen,41 other antigens outside
the Kell system that are often destroyed by
sulfhydryl reagents remain intact (see Methods 2.14 and 4.2). Chloroquine diphosphate
can be used to weaken the expression of
Class I HLA antigens (Bg antigens) on red
cells.42 Chloroquine treatment also weakens
some other antigens, including Rh antigens
(see Method 2.13).

Adsorption
Antibody can be removed from a serum
sample by adsorption to red cells carrying
the corresponding antigen. After the antibody attaches to the membrane-bound
antigens and the serum and cells are separated, the specific antibody remains attached to the red cells. It may be possible
to harvest the bound antibody by elution.

Adsorption techniques are useful in such
situations as:
1.
Separating multiple antibodies present in a single serum.
2.
Removing autoantibody activity to
permit detection of coexisting alloantibodies.
3.
Removing unwanted antibody (often
anti-A and/or anti-B) from serum that
contains an antibody suitable for reagent use.
4.
Confirming the presence of specific
antigens on red cells through their
ability to remove antibody of corresponding specificity from previously
characterized serum.
5.
Confirming the specificity of an antibody by showing that it can be adsorbed only to red cells of a particular blood group phenotype.
Adsorption serves different purposes in
different situations; there is no single procedure that is satisfactory for all purposes.
A basic procedure for an antibody adsorption can be found in Method 3.12. The
usual serum-to-cell ratio is one volume of
serum to an equal volume of washed red
cells. To enhance antibody uptake, the proportion of antigen can be increased by using a larger volume of cells. The incubation
temperature should be that at which the
antibody is optimally reactive. Pretreating
red cells with a proteolytic enzyme may enhance antibody uptake and reduce the
number of adsorptions required for complete removal of antibody. Because some
antigens are destroyed by proteases, antibodies directed against these antigens will
not be removed by enzyme-treated red
cells.
In separating mixtures of antibodies, the
selection of red cells of the appropriate
phenotype is extremely important and depends on the object of the separation. If
none of the antibodies in the serum has
been identified, weakly reactive cells may

Chapter 19: Initial Detection and Identification of Alloantibodies to Red Cell Antigens

be used, on the assumption that they are
reactive with only a single antibody. The
phenotype of the person producing the antibody gives a clue to what specificities
might be present, and cells intended to
separate those particular antibodies can be
chosen. If one or more antibodies have
been identified, cells lacking those antigens
are usually chosen so that only one antibody is removed. Adsorption requires a
substantial volume of red cells. Vials of reagent red cells usually will not suffice, and
blood samples from staff members or donor
units are the most convenient sources.

Elution
Elution frees antibody molecules from
sensitized red cells. Bound antibody may
be released by changing the thermodynamics of antigen-antibody reactions, by
neutralizing or reversing forces of attraction that hold antigen-antibody complexes
together, or by disturbing the structure of
the antigen-antibody binding site. The
usual objective is to recover bound antibody in a usable form.
Various elution methods have been described. Selected procedures are given in
Methods 4.1 through 4.5. No single method
is best in all situations. Use of heat or
freeze-thaw elution is usually restricted to
the investigation of HDFN due to ABO incompatibility because these elution procedures rarely work well for antibodies outside the ABO system. Acid or organic solvent
methods are used for elution of warm-reactive auto- and alloantibodies.
Technical factors that influence the success of elution procedures include:
1.
Incorrect technique. Such factors as
incomplete removal of organic solvents
or failure to correct the tonicity or
pH of an eluate may cause the red
cells used in testing the eluate to
hemolyze or to appear “sticky.” The

447

presence of stromal debris may interfere with the reading of tests. Careful
technique and strict adherence to
protocols should eliminate such problems.
Incomplete washing. The sensitized
red cells must be thoroughly washed
before elution to prevent contamination of the eluate with residual serum antibody. If it is known that the
serum does not contain antibody,
saline washing may not be necessary.
Six washes with saline are usually
adequate, but more may be needed
if the serum contains a high-titer antibody. To determine the efficacy of
the washing process, supernatant
fluid from the final wash phase should
be tested for antibody activity and
should be nonreactive.
Binding of proteins to glass surfaces.
If the eluate is prepared in the same
test tube that was used during the
sensitization phase (eg, in an adsorption/elution process), antibody
nonspecifically bound to the test
tube surface may dissociate during
the elution. Similar binding can also
occur from a whole blood sample if
the patient has a positive DAT and
free antibody in the serum. To avoid
such contamination, the washed red
cells should be transferred into a clean
test tube before the elution procedure
is begun.
Dissociation of antibody before elution. IgM antibodies, such as anti-A
or -M, may spontaneously dissociate
from the cells during the wash phase.
To minimize this loss of bound antibody, cold (4 C) saline can be used
for washing. Although this is not a
concern with most IgG antibodies,
some low-affinity IgG antibodies can
also be lost during the wash phase. If
such antibodies are suspected, wash-

448

1.
2.

AABB Technical Manual

ing with cold LISS instead of normal
saline may help maintain antibody
association.
Instability of eluates. Dilute protein
solutions, such as those obtained by
elution into saline, are unstable. Eluates should be tested as soon after
preparation as possible. Alternatively,
bovine albumin may be added to a
final concentration of 6% w/v and the
preparation stored frozen. Eluates can
also be prepared directly into antibody-free plasma, 6% albumin, or a
similar protein medium instead of
into saline.
Elution techniques are useful for:
Investigation of a positive DAT (see
Chapter 20).
Concentration and purification of
antibodies, detection of weakly expressed antigens, and identification
of multiple antibody specificities.
Such studies are used in conjunction
with an appropriate adsorption technique, as described above and in
Method 2.4.
Preparation of antibody-free red cells
for use in phenotyping or autologous
adsorption studies. Procedures used
to remove cold- and warm-reactive
autoantibodies from red cells are
discussed in Method 4.6 and Method
4.9, and a discussion of autologous
adsorption of warm-reactive autoantibodies appears in Chapter 20.

Combined Adsorption-Elution
Combined adsorption-elution tests can be
used to separate mixed antibodies from a
single serum, to detect weakly expressed
antigens on red cells, or to help identify
weakly reactive antibodies. The process
consists of first incubating serum with selected cells, then eluting antibody from
the adsorbing red cells. Both the eluate

and treated serum can be used for further
testing. Unmodified red cells are generally
used for adsorption and subsequent elution; elution from enzyme- or ZZAPtreated cells may create technical problems.

Use of Sulfhydryl Reagents
Sulfhydryl reagents, such as DTT and
2-mercaptoethanol (2-ME), cleave the
disulfide bonds that join the monomeric
subunits of the IgM pentamer. Intact 19S
IgM molecules are cleaved into 7S subunits,
which have altered serologic reactivity.43
The interchain bonds of 7S Ig monomers
are relatively resistant to such cleavage
(see Chapter 11 for the structure of immunoglobulin molecules). Sulfhydryl reagents are used to diminish or destroy
IgM antibody reactivity. DTT also destroys
certain red cell antigens. The applications
of DTT and 2-ME in immunohematology
include:
1.
Determining the immunoglobulin
class of an antibody (see Method 3.8).
2.
Identifying specificities in a mixture
of IgM and IgG antibodies, particularly when an agglutinating IgM antibody masks the presence of IgG
antibodies.
3.
Determining the relative amounts of
IgG and IgM components of a given
specificity (eg, anti-A or -B).
4.
Dissociating red cell agglutinates
caused by IgM antibodies (eg, the
spontaneous agglutination of red
cells caused by potent autoantibodies)
(see Method 2.11).
5.
Dissociating IgG antibodies from red
cells using a mixture of DTT and a
proteolytic enzyme (ZZAP reagent)
(see Method 4.9).
6.
Converting nonagglutinating IgG antibodies into direct agglutinins. 44
Commercially prepared, chemically

Chapter 19: Initial Detection and Identification of Alloantibodies to Red Cell Antigens

modified, blood typing reagents for
use in rapid saline tube, slide, or
microplate tests have been manufactured in this manner (see Chapter
12).
Destroying selected red cell antigens
(eg, those of the Kell, Dombrock,
Cartwright, and LW systems) for use
in antibody investigations (see Method
3.10).

Titration
The titer of an antibody is usually determined by testing serial twofold dilutions
of the serum against selected red cell
samples. Results are expressed as the reciprocal of the highest serum dilution that
shows macroscopic agglutination. Titration values can provide information about
the relative amount of antibody present in
a serum, or the relative strength of antigen expression on red cells.
Titration studies are useful in the following situations:
1.
Prenatal studies. When the antibody
is of a specificity known to cause HDFN
or its clinical significance is unknown, the results of titration studies may contribute to the decision
about performing invasive procedures, eg, amniocentesis (see Chapter 23 and Method 5.3).
2.
Antibody identification. Some antibodies that agglutinate virtually all
reagent red cell samples may produce an indication of specificity by
demonstrating reactivity of different
strength with different samples in titration studies. For example, potent
autoanti-I may react in the undiluted state with both adult and cord
red cells, but titration may reveal reactivity at a higher dilution with
adult I+ red cells than with cord red
cells.

449

Most weakly reactive antibodies
lose reactivity when diluted even
modestly, but some antibodies that
give weak reactions when undiluted
continue to react at dilutions as high
as 1 in 2048. Such antibodies include
anti-Ch, -Rg, -Csa, -Yka, -Kna, -McCa,
-JMH, and other specificities. When
weak reactions are observed in indirect antiglobulin tests, titration may
be used to indicate specificity within
this group. Not all antibodies of the
specificities mentioned demonstrate
such “high titer, low avidity” characteristics. Thus, although demonstration of these serologic characteristics
may help point to certain specificities, failure to do so does not eliminate those possibilities. Antibodies
of other specificities may sometimes
react at high titers. Details of titration are given in Method 3.7 and
Method 3.9.
Separating multiple antibodies. Titration results may suggest that one antibody reacts at higher dilutions than
another. This information can allow
the serum to be diluted before testing against a cell panel, effectively
removing one antibody and allowing
identification of the other.

Other Methods
Methods other than traditional tube techniques may be used for antibody identification. Some are especially useful for
identifying individual antibody specificities, for dealing with small volumes of
test reagents, for batch testing, or for use
with automated systems. Such methods
include testing in capillary tubes, microplates, or by solid phase; enzyme-linked
immunosorbent assays; and column agglutination (eg, gel techniques). Other
methods useful in laboratories with spe-

450

AABB Technical Manual

cialized equipment include radioimmunoassay, immunofluorescence (including flow
cytometric procedures), immunoblotting,
and immunoelectrode biosensoring. Some
of these methods are discussed in Chapter 12.

5.
6.

10.

11.

12.

13.

14.

16.

17.

18.

References
1.

15.

Giblett ER. Blood group alloantibodies: An
assessment of some laboratory practices.
Transfusion 1977;17:299-308.
Walker RH, Lin DT, Hatrick MB. Alloimmunization following blood transfusion. Arch
Pathol Lab Med 1989;113:254-61.
Daniels G, Poole J, deSilva M, et al. The clinical significance of blood group antibodies.
Tranfus Med 2002;12:287-95.
Code of federal regulations. Title 21 CFR Part
660.33. Washington, DC: US Government
Printing Office, 2004 (revised annually).
Standards Source – 4.3.2.1. ( January 2004)
Bethesda, MD: AABB, 2004.
Howard JE, Winn LC, Gottlieb CE, et al. Clinical significance of anti-complement component of antiglobulin antisera. Transfusion
1982;22:269-72.
Judd WJ, Fullen DR, Steiner EA, et al. Revisiting the issue: Can the reading for serologic reactivity following 37 C incubation be omitted?
Transfusion 1999;39:295-9.
Issitt PD. Antibody screening: Elimination of
another piece of the test (editorial). Transfusion 1999;39:229-30.
Shulman IA, Calderon C, Nelson JM, Nakayama
R. The routine use of Rh-negative reagent red
cells for the identification of anti-D and the
detection of non-D red cell antibodies. Transfusion 1994;34:666-70.
Fisher RA. Statistical methods and scientific
inference. 2nd ed. Edinburgh, Scotland: Oliver and Boyd, 1959.
Harris RE, Hochman HG. Revised p values in
testing blood group antibodies. Transfusion
1986;26:494-9.
Kanter MH, Poole G, Garratty G. Misinterpretation and misapplication of p values in antibody identification: The lack of value of a p
value. Transfusion 1997;37:816-22.
Reid ME, Oyen R, Storry J, et al. Interpretation of RBC typing in multi-transfused patients can be unreliable (abstract). Transfusion 2000;40 (Suppl):123.
Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

Brendel WL. Resolving antibody problems.
In: Pierce SR, Wilson JK, eds. Approaches to
serological problems in the hospital transfusion service. Arlington, VA: AABB, 1985:51-72.
Reid ME, Lomas-Francis C. The blood group
antigen factsbook. 2nd ed. New York: Academic
Press, 2004.
Issitt PD, Anstee DJ. Applied blood group serology. 4th ed. Durham, NC: Montgomery
Scientific Publications, 1998.
Malyska H, Kleeman JE, Masouredis SP, et al.
Effects on blood group antigens from storage
at low ionic strength in the presence of
neomycin. Vox Sang 1983;44:375-84.
Westhoff CM, Sipherd BD, Toalson LD. Red cell
antigen stability in K3EDTA. Immunohematology 1993;9:109-11.
Rodberg K, Tsuneta R, Garratty G. Discrepant
Rh phenotyping results when testing IgGsensitized rbcs with monoclonal Rh reagents
(abstract). Transfusion 1995;35(Suppl):67.
Code of federal regulations. Title 21 CFR Part
660.25. Washington, DC: US Government
Printing Office, 2004 (revised annually).
Food and Drug Administration. Compliance
program guidance manual. Chapter 42; inspection of licensed and unlicensed blood
banks, brokers, reference laboratories, and
contractors—program 7342.001. Attachment
C—Product testing system, blood grouping
and typing (ABO and Rh), and compatibility
testing. Rockville, MD: CBER Office of Communication, Training, and Manufacturers Assistance, 2003. [Available at http://www.fda.
gov/cber/cpg.htm.]
Shirey RS, Edwards RE, Ness PM. The risk of
alloimmunization to c (Rh4) in R1R1 patients
who present with anti-E. Transfusion 1994;34:
756-8.
Shulman IA. Controversies in red blood cell
compatibility testing. In: Nance SJ, ed. Immune
destruction of red blood cells. Arlington, VA:
AABB, 1989:171-99.
Reisner R, Butler G, Bundy K, Moore SB.
Comparison of the polyethylene glycol antiglobulin test and the use of enzymes in antibody detection. Transfusion 1996;36:487-9.
Issitt PD, Combs MR, Bumgarner DJ, et al.
Studies of antibodies in the sera of patients
who have made red cell autoantibodies.
Transfusion 1996;36:481-6.
Wilkinson SL. Serological approaches to
transfusion of patients with allo- or autoantibodies. In: Nance SJ, ed. Immune destruction of red blood cells. Arlington, VA:
AABB, 1989:227-61.
Issitt PD, Combs MR, Bredehoeft SJ, et al.
Lack of clinical significance of “enzyme-only”

Chapter 19: Initial Detection and Identification of Alloantibodies to Red Cell Antigens

29.

30.
31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.
42.

43.

44.

red cell alloantibodies. Transfusion 1993;33:
284-93.
Beattie KM, Zuelzer WW. The frequency and
properties of pH-dependent anti-M. Transfusion 1965;5:322-6.
Judd WJ. A pH dependent autoagglutinin with
anti-P specificity. Transfusion 1975;15:373-6.
Bruce C, Watt AH, Hare V, et al. A serious
source of error in antiglobulin testing. Transfusion 1986;26:177-81.
Rolih S, Thomas R, Fisher F, Talbot J. Antibody
detection errors due to acidic or unbuffered
saline. Immunohematology 1993;9:15-8.
Morton J, Pickles MM, Terry AM. The Sd a
blood group antigen in tissues and body fluids. Vox Sang 1970;19:472-82.
O’Neil GJ, Yang SY, Tegoli J, et al. Chido and
Rodgers blood groups are distinct antigenic
components of human complement, C4. Nature 1978;273:668-70.
Tilley CA, Romans DG, Crookston MC. Localization of Chido and Rodgers to the C4d fragment of human C4 (abstract). Transfusion
1978;18:622.
Judd WJ, Kreamer K, Moulds JJ. The rapid
identification of Chido and Rodgers antibodies using C4d-coated red blood cells. Transfusion 1981;21:189-92.
Advani H, Zamor J, Judd WJ, et al. Inactivation of Kell blood group antigens by 2-aminoethylisothiouronium bromide. Br J Haematol
1982;51:107-15.
Branch DR, Muensch HA, Sy Siok Hian AL,
Petz LD. Disulfide bonds are a requirement
for Kell and Cartwright (Yta) blood group antigen integrity. Br J Haematol 1983;54:573-8.
Moulds J, Moulds MM. Inactivation of Kell
blood group antigens by 2-amino-ethylisothiouronium bromide. Transfusion 1983;23:274-5.
Branch DR, Petz LD. A new reagent (ZZAP)
having multiple applications in immunohematology. Am J Clin Pathol 1982;78:161-7.
Liew YW, Uchikawa M. Loss of Era antigen in
very low pH buffers. Transfusion 1987;27:442-3.
Swanson JL, Sastamoinen R. Chloroquine
stripping of HLA A,B antigens from red cells
(letter). Transfusion 1985;25:439-40.
Freedman J, Masters CA, Newlands M, et al.
Optimal conditions for use of sulphydryl
compounds in dissociating RBC antibodies.
Vox Sang 1976;30:231-9.
Romans DG, Tilley CA, Crookston MC, et al.
Conversion of incomplete antibodies to direct agglutinins by mild reduction. Evidence
for segmental flexibility within the Fc fragment of immunoglobulin G. Proc Natl Acad
Sci U S A 1977;74:2531-5.

451

Suggested Reading
Boorman KE, Dodd BE, Lincoln PJ. Blood group
serology. 6th ed. Edinburgh, Scotland: Churchill
Livingstone, 1988.
Crookston MC. Soluble antigens and leukocyte related antibodies. Part A. Blood group antigens in
plasma: An aid in the identification of antibodies.
In: Dawson RD, ed. Transfusion with “crossmatch
incompatible” blood. Washington, DC: AABB, 1975:
20-5.
Daniels G. Human blood groups. 2nd ed. Oxford,
England: Blackwell Scientific Publications, 2002.
Engelfriet CP, Overbeeke MAM, Dooren MC, et al.
Bioassays to determine the clinical significance of
red cell antibodies based on Fc receptor-induced
destruction of red cells sensitized by IgG. Transfusion 1994;14:617-26.
Garratty G. In-vitro reactions with red blood cells
that are not due to blood group antibodies: A review. Immunohematology 1998;14(1):1-11.
Issitt PD, Anstee DJ. Applied blood group serology.
4th ed. Durham, NC: Montgomery Scientific Publications, 1998.
Johnson ST, Rudmann SV, Wilson SM, eds. Serologic problem-solving strategies: A systematic approach. Bethesda, MD: AABB, 1996.
Judd WJ. Elution of antibody from red cells. In:
Bell CA, ed. A seminar on antigen-antibody reactions revisited. Washington, DC: AABB, 1982:175221.
Judd WJ. Methods in immunohematology. 2nd ed.
Durham, NC: Montgomery Scientific Publications,
1994.
Kanter MH. Statistical analysis. In: Busch MP, Brecher
ME, eds. Research design and analysis. Bethesda,
MD: AABB, 1998:63-104.
Mallory D, ed. Immunohematology methods and
procedures. Rockville, MD: American Red Cross,
1993.
Marsh WL, Reid ME, Kuriyan M, et al. A handbook
of clinical and laboratory practices in the transfusion of red blood cells. Moneta, VA: Moneta Medical Press, 1993.
Menitove JE. The Hardy-Weinberg principle: Selection of compatible blood based on mathematic
principles. In: Fridey JL, Kasprisin CA, Chambers
LA, Rudmann SV, eds. Numbers for blood bankers.
Bethesda, MD: AABB, 1995:1-11.

452

AABB Technical Manual

Mollison PL, Engelfriet CP, Contreras M. Blood
transfusion in clinical medicine. 10th ed. London:
Blackwell Scientific Publications, 1997.

Rolih S. A review: Antibodies with high-titer, lowavidity characteristics. Immunohematology 1990;
6:59-67.

Race RR, Sanger R. Blood groups in man. 6th ed.
Oxford, England: Blackwell Scientific Publications,
1975.

Telen MJ. New and evolving techniques for antibody and antigen identification. In: Nance ST, ed.
Alloimmunity: 1993 and beyond. Bethesda, MD:
AABB, 1993:117-39.

Reid ME, Lomas-Francis C. The blood group antigen factsbook. 2nd ed. New York: Academic Press,
2004.

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction

Chapter 20

The Positive Direct
Antiglobulin Test and
Immune-Mediated Red
Cell Destruction

HE DIRECT ANTIGLOBULIN test
(DAT) is generally used to determine
if red cells have been coated in vivo
with immunoglobulin, complement, or both.
A positive DAT, with or without shortened
red cell survival, may result from:
1.
2.

Autoantibodies to intrinsic red cell
antigens.
Alloantibodies in a recipient’s circulation, reacting with antigens on recently transfused donor red cells.
Alloantibodies in donor plasma,
plasma derivatives, or blood fractions that react with antigens on the
red cells of a transfusion recipient.
Alloantibodies in maternal circulation that cross the placenta and coat
fetal red cells.
Antibodies directed against certain
drugs that bind to red cell membranes (eg, penicillin).
Nonspecifically adsorbed proteins,
including immunoglobulins, associated with hypergammaglobulinemia

or recipients of high-dose intravenous
gammaglobulin,1,2 or modification of
the red cell membrane by certain
drugs, eg, some cephalosporins.
7.
Red-cell-bound complement. This may
be due to complement activation by
alloantibodies, autoantibodies, drugs,
or bacterial infection.
8.
Antibodies produced by passenger
lymphocytes in transplanted organs
3
or hematopoietic components.
A positive DAT does not necessarily
mean that a person’s red cells have shortened survival. Small amounts of both IgG
and complement appear to be present on
all red cells. A range of 5 to 90 IgG molecules/red cell4 and 5 to 40 C3d molecules/
5
red cell appears to be normal on the red
cells of healthy individuals.
The DAT can detect a level of 100 to 500
molecules of IgG/red cell and 400 to 1100
molecules of C3d/red cell, depending on
the reagent and technique used. Positive
DATs without clinical manifestations of im453

454

AABB Technical Manual

mune-mediated red cell destruction are reported in 1 in 1000 up to 1 in 14,000 blood
donors and 1% to 15% of hospital patients.4
Most blood donors with positive DATs
appear to be perfectly healthy, and most
patients with positive DATs have no obvious signs of hemolytic anemia, although
some may show slight evidence of increased red cell destruction.4,6(p222) Elevated
levels of IgG or complement have been
noted on the red cells of patients with sickle
cell disease, β-thalassemia, renal disease,
multiple myeloma, autoimmune disorders,
AIDS, and other diseases with elevated serum globulin or blood urea nitrogen (BUN)
levels with no clear correlation between a
positive DAT and anemia.1,2,7 Interpretation
of positive DATs should include the patient’s history, clinical data, and the results
of other laboratory tests.

most laboratories. If cord blood samples
are to be tested, it is appropriate to use
anti-IgG only because hemolytic disease of
the fetus and newborn (HDFN) results from
the fetal red cells becoming sensitized with
maternally derived IgG antibody and complement activation rarely occurs.3

The Pretransfusion DAT and the
Autologous Control
Neither the AABB, in the Standards for
Blood Banks and Transfusion Services,8 nor
any other accrediting agency requires a DAT
or an autologous control (autocontrol) as
part of pretransfusion testing. Studies
have shown that eliminating the DAT/
autocontrol portion of routine pretransfusion testing carries minimal risk.9

Evaluation of a Positive DAT

The Direct Antiglobulin Test
The principles of the DAT are discussed in
Chapter 12. Although any red cells may be
tested, EDTA-anticoagulated blood samples are preferred to prevent in-vitro fixation of complement. If red cells from a
clotted blood sample have a positive DAT
due to complement, the results should be
confirmed on cells from a freshly collected or EDTA-anticoagulated specimen
if those results are to be used for diagnostic purposes.
Most DATs are initially performed with
a polyspecific antihuman globulin (AHG)
reagent capable of detecting both IgG and
C3d (see Method 3.6). If positive, tests with
specific anti-IgG and anticomplement reagents may be appropriate. Occasionally,
polyspecific AHG reagents react with cellbound proteins other than IgG or C3d (eg,
IgM, IgA, or other complement components); specific reagents to distinguish
these proteins are not readily available in

Extent of Testing
Clinical considerations should dictate the
extent to which a positive DAT is evaluated. Dialogue with the attending physician is important. Interpretation of the
significance of serologic findings requires
knowledge of the patient’s diagnosis; recent drug, pregnancy, and transfusion
history; and information on the presence
of acquired or unexplained hemolytic
anemia. The results of serologic tests alone
are not diagnostic; their significance must
be assessed in conjunction with clinical
information and such laboratory data as
hematocrit, bilirubin, haptoglobin, and
reticulocyte count. When investigating a
transfusion reaction, performance of the
DAT on postreaction specimens is part of
the initial transfusion reaction investigation. The DAT may be positive if sensitized red cells have not been destroyed or
negative if hemolysis and rapid clearance
have occurred. Positive DAT results should
be further evaluated. The patient’s history

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction

is important in interpreting a posttransfusion reaction positive DAT (see Chapter 27).
Answers to the following questions may
help decide what investigations are appropriate:
1.
Is there any evidence of in-vivo red
cell destruction? Reticulocytosis,
spherocytes observed on the peripheral blood film, hemoglobinemia,
hemoglobinuria, decreased serum
haptoglobin, and elevated levels of
serum unconjugated bilirubin or
lactate dehydrogenase (LDH), especially LDH1, may be associated with
increased red cell destruction. If an
anemic patient with a positive DAT
does show evidence of hemolysis,
testing to evaluate a possible immune etiology is appropriate. IF
THERE IS NO EVIDENCE OF INCREASED RED CELL DESTRUCTION, NO FURTHER STUDIES ARE
NECESSARY, unless the patient needs
transfusion and the serum contains
incompletely identified unexpected
antibodies to red cell antigens.
2.
Has the patient been recently transfused? Many workers routinely attempt to determine the cause of a
positive DAT when the patient has
received transfusions within the previous 3 months because the first indication of a developing immune response may be the attachment of
antibody to recently transfused red
cells. Antibody may appear as early
as 7 to 10 days (but typically 2 weeks
to several months) after transfusion
in primary immunization and as
early as 2 to 7 days (but usually within 20 days) in a secondary response;
these alloantibodies could shorten the
survival of red cells already transfused or given subsequently.
Studies have shown that the positive DAT and reactive eluates can

455

persist for more than 300 days following a transfusion reaction, which
is far longer than the transfused cells
would be expected to survive, suggesting that autologous as well as
transfused red cells are sensitized
following a transfusion reaction.10,11 A
mixed-field appearance in the posttransfusion DAT may or may not be
observed.
Is the patient receiving any drugs, such
as cephalosporins, procainamide, intravenous penicillin, or -methyldopa?
Cephalosporins are associated with
positive DATs; the second- and thirdgeneration cephalosporins can be associated with immune red cell destruction.12 In one study, 21% of patients
receiving procainamide developed a
positive DAT (three of whom had evidence of hemolytic anemia).13 A high incidence (39%) of positive DATs has been
reported in patients taking Unasyn.14
Although not commonly seen in recent
years, approximately 3% of patients receiving intravenous penicillin, at very
high doses, and 15% to 20% of patients
receiving α-methyldopa will develop a
positive DAT. However, fewer than 1%
of those patients who develop a positive DAT have hemolytic anemia. Positive DATs associated with other drugs
are rare. If a positive DAT is found in a
patient receiving such drugs, the attending physician should be alerted so
that appropriate surveillance for red
cell destruction can be maintained. If
red cell survival is not shortened, no
further studies are necessary.
Has the patient received marrow, peripheral blood stem cells, or an organ
transplant? Passenger lymphocytes of
donor origin produce antibodies directed against ABO or other antigens
on the recipient’s cells, causing a positive DAT.3

456

AABB Technical Manual

Is the patient receiving IGIV or intravenous RhIG? Immune Globulin, Intravenous (IGIV) may contain ABO
antibodies, anti-D, or, sometimes, other
antibodies. Intravenous Rh Immune
Globulin (RhIG) causes Rh-positive
patients to develop a positive DAT.15
Is the patient septic? Complement
activation can occur in septic patients,
leading to intravascular hemolysis.
This is most often seen in cases of
polyagglutination resulting from organisms that produce neuraminidase.

Serologic Studies
Three investigative approaches are helpful in the evaluation of a positive DAT.
1.
Test the DAT-positive red cells with
anti-IgG and anti-C3d reagents to
characterize the types of proteins
coating the red cells.
2.
Test the serum/plasma to detect and
identify clinically significant antibodies to red cell antigens.
3.
Test an eluate (see Methods 4.1 through
4.5) prepared from the coated red
cells with a panel of reagent red cells
to define whether the coating protein
has red cell antibody activity. When
the only coating protein is complement, eluates are frequently nonreactive. However, an eluate from the patient’s red cells coated only with
complement should be tested if there
is clinical evidence of antibody-mediated hemolysis. The eluate preparation can concentrate small amounts
of IgG that may not be detectable on
direct testing using routine methods.
Results of these tests combined with
the patient’s history and clinical data
should assist in classification of the
problems involved.

See Appendix 20-1 for an example of an
algorithm for investigating a positive DAT
(excluding investigation of HDFN).

Elution
Elution frees antibody from sensitized red
cells and recovers antibody in a usable form.
Details of eluate preparation are given in
Chapter 19 and in Methods 4.1 through
4.5. Commercial elution kits are also
available. Table 20-1 lists the advantages
and disadvantages of several common
elution methods; no single elution method
is ideal in all situations. Although many
elution methods damage or destroy the
red cells, certain techniques (see Methods
2.11, 2.12, 2.13, and 2.14) remove antibody but leave the cells sufficiently intact
to allow testing for various antigens or for
use in adsorption procedures. Some antigens may be altered by elution, however,
and appropriate controls are essential.
In cases of HDFN or hemolytic transfusion reactions, specific antibody (or antibodies) is usually detected in the eluate,
which may or may not be detectable in the
serum. In the case of transfusion reactions,
newly developed antibodies initially detectable only in the eluate are usually detectable in the serum after about 14 to 21 days.
Eluate preparation from the patient’s red
cells often concentrates antibody activity
and may facilitate identification of weakly
reactive serum antibodies.
When the eluate reacts with all the cells
tested, autoantibody is the most likely explanation, especially if the patient has not
been recently transfused. WHEN NO UNEXPECTED ANTIBODIES ARE PRESENT IN
THE SERUM, AND IF THE PATIENT HAS
NOT BEEN RECENTLY TRANSFUSED, NO
FURTHER SEROLOGIC TESTING OF AN
AUTOANTIBODY IS NECESSARY.
A nonreactive eluate prepared from IgGcoated red cells may have several causes.

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction

457

Table 20-1. Antibody Elution Techniques
Method

Advantages

Disadvantages

Heat (56 C)

Good for ABO-HDFN; quick and
easy

Poor recovery of other blood
group allo- and
autoantibodies

Freeze-thaw

Good for ABO-HDFN; quick
method; requires small volume of red cells

Poor recovery of other blood
group allo- and
autoantibodies

Cold acid

Quick and easy; adequate for
most warm auto- and
alloantibodies; commercial
kits available

Possible false-positive elution
(see Leger et al16)

Digitonin acid

Nonhazardous; good recovery of
most antibodies

Time-consuming washing of
stroma

Dichloromethane/
Methylene chloride

Noncarcinogenic, nonflammable; good for IgG auto- and
alloantibodies

Vapors harmful

Compiled from Judd and South et al.

One cause may be that the eluate was not
tested against cells positive for the corresponding antigen, notably group A or
group B cells, or antigens of low incidence,
which are absent from most reagent cell
panels. If a non-group-O patient has received plasma containing anti-A or anti-B
(as in transfusion of group O platelets), and
the recipient appears to have immune
hemolysis, the eluate can be tested against
A and/or B cells. If the expected ABO antibodies are not detected, other causes of the
positive DAT should be sought. It may be
appropriate to test the eluate against red
cells from recently transfused donor units,
which could have caused immunization to
a rare antigen, or, in HDFN, against cells
from the father, from whom the infant may
have inherited a rare gene. Pursuing the
cause of a nonreactive eluate for patients
with no evidence of hemolysis is usually
not indicated. Toy et al1 showed that 79% of

hospital patients with a positive DAT have a
nonreactive eluate. It is suggested that at
least one contributing factor to these positive DAT results is nonspecific uptake of
proteins on the red cells, which occurs in
patients with elevated gamma globulin levels.1,2
Reactivity of eluates can be enhanced by
testing them against enzyme-treated cells
or by the use of enhancement techniques
such as polyethylene glycol (PEG). Antibody reactivity can be increased by the use
of a concentrated eluate, either by alteration of the fluid-to-cell ratio or by use of
commercial concentration devices. Washing the red cells with low-ionic-strength saline (LISS) or cold wash solutions may prevent the loss of antibody while the cells are
being prepared for elution.
Certain elution methods give poor results with certain antibodies. When eluates
are nonreactive yet clinical signs of red cell

458

AABB Technical Manual

destruction are present, elution by a different method may be helpful. If both serum
and eluate are nonreactive at all test phases
and if the patient has received high-dose
intravenous penicillin or other drug therapy, testing to demonstrate drug-related
antibodies should be considered.

Immune-Mediated
Hemolysis
Immune-mediated hemolysis (immune
hemolysis) is the shortening of red cell
survival by the product(s) of an immune
response. If marrow compensation is adequate, the reduced red cell survival may
not result in anemia. Immune hemolysis
is only one cause of hemolytic anemia,
and many causes of hemolysis are unrelated to immune reactions. The serologic
investigations carried out in the blood bank
do not determine whether a patient has a
“hemolytic” anemia. The diagnosis of
hemolytic anemia rests on clinical findings and such laboratory data as hemoglobin or hematocrit values; reticulocyte
count; red cell morphology; bilirubin,
haptoglobin, and LDH levels; and, sometimes, red cell survival studies. The serologic findings help determine whether the
hemolysis has an immune basis and, if so,
what type of immune hemolytic anemia is
present. This is important because the
treatment for each type is different.
The terms hemolysis and hemolytic are
frequently used to indicate both intravascular and extravascular red cell destruction;
however, this may be misleading. In-vivo
lysis of cells and release of free hemoglobin
within the intravascular compartment (ie,
resulting in hemoglobinemia and hemoglobinuria) is uncommon and, often, dramatic.
Extravascular hemolysis, which is more
common, is characterized by an increase in

serum bilirubin, but not by hemoglobinemia
and hemoglobinuria. As a description of
in-vitro antibody reactivity, hemolysis or
lysis of the red cells with release of free hemoglobin to the surrounding media is both
obvious and rare.
Immune hemolytic anemias can be classified in various ways. One classification
system is shown in Table 20-2. Autoimmune hemolytic anemias (AIHAs) are subdivided into five major types: warm antibody AIHA ( WAIHA), cold agglutinin
syndrome (CAS), mixed-type AIHA, paroxysmal cold hemoglobinuria (PCH), and
DAT-negative AIHA. Not all cases fit neatly
into these categories. Drugs (discussed in a
later section of this chapter) may also induce immune hemolysis. The prevalence of
each type can vary depending on the patient population studied. Table 20-3 shows
the serologic characteristics of the autoimmune and drug-induced hemolytic anemias.
DATs performed with IgG- and C3-specific AHG reagents as well as the serum and
eluate studies described earlier can be used
to help classify AIHAs. Three additional procedures may be useful: a cold agglutinin titer and thermal amplitude studies (Methods 4.7 and 4.8) and the Donath-Landsteiner
test for PCH (Method 4.13).
The binding of antibody to red cells does
not, in itself, damage the cells. It is the phenomena that the bound antibody-antigen
complex promotes that may eventually
damage cells. These include complement
binding, adherence to Fc receptors on
macrophages leading to phagocytosis, and
cytotoxic lysis. The IgG subclass of bound
antibody may be significant. IgG1 is the
subclass most commonly found, sometimes alone but often in combination with
other subclasses. The other IgG subclasses
occur more often in combination with
other subclasses than alone. In general,
IgG3 antibodies have the most destructive

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction

459

Table 20-2. Classification of Immune Hemolytic Anemias
Autoimmune Hemolytic Anemia (AIHA)
1. Warm autoimmune hemolytic anemia
a.

primary (idiopathic)

secondary [to such conditions as lymphoma, systemic lupus erythematosus (SLE),
carcinoma, or to drug therapy]

2. Cold agglutinin syndrome
a.

primary (idiopathic)

secondary (to such conditions as lymphoma, mycoplasma pneumonia, infectious
mononucleosis)

3. Mixed-type AIHA
a.

primary (idiopathic)

secondary (to such conditions as SLE, lymphoma)

4. Paroxysmal cold hemoglobinuria
a.

primary (idiopathic)

secondary (to such conditions as syphilis, viral infections)

5. DAT-negative AIHA
a.

primary (idiopathic)

secondary (to such conditions as lymphoma, SLE)

Drug-Induced Hemolytic Anemia
Alloimmune Hemolytic Anemia
1. Hemolytic disease of the fetus and newborn
2. Hemolytic transfusion reaction

effects, followed by IgG1. IgG2 antibodies
are associated with less destruction and
IgG4 with little to no destruction.
The number of antibody molecules per
red cell also plays a role. The number of antibody molecules on the red cells of apparently healthy blood donors with positive
DATs (<200 molecules/red cell) is far less
than that usually seen in patients with
AIHA.4,5 Some patients with apparent immune hemolysis may have negative DATs.

Warm Antibody Autoimmune Hemolytic
Anemia
The most common type of AIHA is associated with warm-reactive (37 C) antibodies. Typical serologic findings are described
below.

DAT
When IgG-specific and complement-specific AHG reagents are used, three pat-

460

WAIHA

CAS

Mixed-Type AIHA

PCH

Drug-Induced

Percent of cases

48%19 to 70%3

16%3 to 32%19

7%19 to 8%20

Rare in adults; 32% in
children21

12%3 to 18%19

DAT

IgG:
20%3 to 66%19
IgG + C3:
24%19 to 63%3
C3:
7%19 to 14%3

C3 only:
91%19 to 98%3

IgG + C3:
71%19 to 100%3
C3:
13%3

C3 only:
94%19 to 100%3

IgG:
94%19
IgG + C3:
6%19

Immunoglobulin type

IgG (sometimes IgA or
IgM, rarely alone)

IgM

IgG, IgM

IgG

Eluate

IgG antibody

Nonreactive

IgG antibody

Nonreactive

IgG antibody

Serum

57% react by saline-IAT;
13% hemolyze
enzyme-treated RBCs
at 37 C; 90%
agglutinate enzymetreated RBCs at 37 C;
30% agglutinate
untreated RBCs at
20 C; rarely
agglutinate untreated
RBCs at 37 C3

IgM agglutinating
antibody; titer usually
>1000 at 4 C; usually
react at 30 C in
albumin; monoclonal
antibody in chronic
disease3

IgG IAT-reactive antibody
plus IgM
agglutinating
antibody, usually
react at 30-37 C in
saline; high titer at
4 C (classic CAS) or
low titer (<64) at
4 C3,19,20,22

IgG biphasic hemolysin
(Donath-Landsteiner
antibody)3

IgG antibody similar to
WAIHA3

Specificity

Rh specificity; other
specificities have
been reported*

Usually anti-I but can be
anti-i; rarely anti-Pr23

Usually specificity is
unclear19,20,22
Can be anti-I, -i, or other
cold agglutinin
specificities

Anti-P (nonreactive with
p and Pk RBCs)

Specificity often Rh
related23

*See text.

AABB Technical Manual

Table 20-3. Serologic Findings in Immune Hemolytic Anemias

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction

461

terns of reactivity may be found: coating
with IgG alone, with complement alone,
or with both. In approximately 1% of cases,
the DAT will be positive with a polyspecific AHG reagent but negative with
IgG- and complement-specific AHG reagents. Some of these may be due to attachment of IgM or IgA alone if reactivity
with these immunoglobulins has not been
excluded by the manufacturer.3,19

treated cells or when PEG is used. The
eluate will usually have no serologic activity if the only protein coating the red cells
is complement components. Occasionally, antibody not detected by the DAT will
be detected in the eluate, possibly due to
the concentrating effect of eluate preparation.

Serum

The specificity of autoantibodies associated with WAIHA is complex. In routine
tests, all cells tested are usually reactive.
Some autoantibodies that have weaker or
negative reactivity with cells of rare Rh
phenotypes, such as D– – or Rhnull, appear
to have broad specificity in the Rh system.
Apparent specificity for simple Rh antigens
(D, C, E, c, e) is occasionally seen, either
as the sole autoantibody or as a predominant portion, based on stronger reactivity
with cells of certain phenotypes. Such reactivity is often termed a “relative” specificity. Such relative specificity in a serum
may be mistaken for alloantibody, but cells
negative for the apparent target antigen
can adsorb and remove the “mimicking”
23,24
specificity.
Unusual Specificities. Apart from Rh
specificity, warm autoantibodies with many
other specificities have been reported, eg,
specificities in the LW, Kell, Kidd, Duffy, and
Diego systems.24 Dilution and selective adsorption of eluates may uncover specificity
or relative specificity of autoantibodies. Patients with autoantibodies of Kell, Rh, LW,
Ge, Sc, Lu, and Lan specificities may have
depressed expression of the respective antigen and the DAT may be negative or very
24
weakly positive.
Practical Significance. Tests against red
cells of rare phenotype and by special techniques have limited clinical application. In
rare instances of WAIHA involving IgM agglutinins, determining autoantibody speci-

Autoantibody in the serum typically is IgG
and reacts by indirect antiglobulin testing
against all cells tested.3 If the autoantibody has been adsorbed by the patient’s
red cells in vivo, the serum may contain
very little free antibody. The serum will
contain antibody after all the specific antigen sites on the red cells have been occupied and no more antibody can be
bound in vivo. In such cases, the DAT is
usually strongly positive. Approximately
50% of patients with WAIHA have serum
antibodies that react with untreated saline-suspended red cells. When testing
with PEG, enzyme-treated red cells, or
solid-phase methods, over 90% of these
sera can be shown to contain autoantibody.
Approximately one-third of patients with
WAIHA have cold-reactive autoagglutinins
demonstrable in tests at 20 C, but cold agglutinin titers at 4 C are normal. The presence of this cold agglutinin does not
mean the patient has CAS in addition to
WAIHA.3

Eluate
The presence of the IgG autoantibody on
the red cells may be confirmed by elution
at least upon initial diagnosis and/or at
pretransfusion testing. (See Methods 4.1,
4.2, and 4.5.) Typically, the eluate reacts
with virtually all cells tested, with reactivity enhanced in tests against enzyme-

Specificity of Autoantibody

462

AABB Technical Manual

ficity may help differentiate such cases
from typical CAS.25 It is rarely, if ever, necessary to ascertain autoantibody specificity in
order to select antigen-negative blood for
transfusion. If apparent specificity is directed to a high-incidence antigen (eg,
anti-U), or when the autoantibody reacts
with all red cells except those of a rare Rh
phenotype (eg, D– –, Rhnull), compatible donor blood is unlikely to be available and
there is little point in determining specificity. Such blood, if available, should be
reserved for alloimmunized patients of that
uncommon phenotype.
Transfusion-Stimulated Autoantibodies.
Transfusion itself may lead to the production of autoantibodies that may persist and
cause positive DATs for some time after
transfusion yet not cause obvious red cell
destruction. Such cell-bound autoantibodies
sometimes display blood group specificity
(eg, E, K, Jka). The positive DAT may persist
long after transfused red cells should have
disappeared from the circulation, apparently adsorbed to the patient’s own antigen-negative red cells.11

Transfusion of Patients with Warm-Reactive
Autoantibodies
Inherent Risks. Patients with warm-reactive autoantibodies range from those with
no apparent decreased red cell survival to
those with life-threatening anemia. Patients with little or no evidence of significant in-vivo red cell destruction tolerate
transfusion quite well.
When autoantibody is active in serum, it
may be difficult to exclude the presence of
alloantibodies, which increases the risk of
an adverse reaction. Transfusion may stimulate alloantibody production, complicating subsequent transfusions. Transfusion
may intensify the autoantibody, inducing
or increasing hemolysis and making serologic testing more difficult. Transfusion

may depress compensatory erythropoiesis.
Destruction of transfused cells may increase
hemoglobinemia and hemoglobinuria. In patients with active hemolysis, transfused red
cells may be destroyed more rapidly than the
patient’s own red cells. In rare cases, this may
promote hypercoagulability and disseminated intravascular coagulation (DIC). Transfusion reactions, if they occur, may be difficult to investigate.
Transfusion in WAIHA. Transfusion is
especially problematic for patients with
rapid in-vivo hemolysis, who may present
with a very low hemoglobin level and hypotension. Reticulocytopenia may accompany
a rapidly falling hematocrit, and the patient
may exhibit coronary insufficiency, congestive heart failure, cardiac decompensation,
or neurologic impairment. Under these circumstances, transfusion is usually required
as a lifesaving measure. The transfused
cells may support oxygen-carrying capacity
until the acute hemolysis diminishes or
other therapies can effect a more lasting
benefit. These patients represent a significant challenge because serologic testing
may be complex while clinical needs are
acute.
Transfusion should not be withheld
solely because of serologic incompatibility.
The volume transfused should usually be
the smallest amount required to maintain
adequate oxygen delivery, not necessarily
to reach an arbitrary hemoglobin level. Volumes of about 100 mL may be appropriate.3
The patient should be carefully monitored
throughout the transfusion.
Transfusion in Chronic WAIHA. Most
patients with WAIHA have a chronic stable
anemia, often at relatively low hemoglobin
levels. Those with hemoglobin levels above
8 g/dL rarely require transfusion, and many
patients with levels of 5 g/dL (or even
lower) can be managed with bed rest and
no transfusions. Transfusion will be required if the anemia progresses or is ac-

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction

companied by such symptoms as severe
angina, cardiac decompensation, respiratory distress, and cerebral ischemia.
Most patients with chronic anemia due
to WAIHA tolerate transfusion without
overt reactions, even though the transfused
cells may not survive any better than their
own. Because transfusion may lead to circulatory overload or to increased red cell destruction, the decision to transfuse should
be carefully considered. A few patients
without acute hemolysis have had severe
hemolytic reactions after transfusion. This
may be due to the sudden availability of
large volumes of donor cells and the exponential curve of decay, by which the number of cells hemolyzed is proportional to
the number of cells present.3,6(pp230,369)
Selecting Blood. If the decision is made
to transfuse, selection of appropriate donor
blood is essential. It is important to determine the patient’s ABO and Rh type and, if
time permits, to detect potentially clinically
significant alloantibodies. Adsorption and
other special techniques described later in
this chapter can greatly reduce the risk of
undetected alloantibodies but may be
time-consuming. If clinically significant
alloantibodies are present, the transfused
cells should lack the corresponding antigen(s).
If the autoantibody has apparent and
relatively clear-cut specificity for a single
antigen (eg, anti-e) and there is active ongoing hemolysis, blood lacking that antigen
may be selected. There is evidence that, in
some patients, such red cells survive better
than the patient’s own red cells.6(p230),20 In the
absence of hemolysis, autoantibody specificity is not important, although donor units
negative for the antigen may be chosen because this is a simple way to circumvent the
autoantibody and detect potential alloantibodies. If the autoantibody shows broader
reactivity, reacting with all cells but showing some relative specificity (eg, it reacts

463

preferentially with e+ red cells), the use of
blood lacking the corresponding antigen is
debatable. It may be undesirable to expose
the patient to Rh antigens absent from
autologous cells, especially D and especially in females who may bear children
later, merely to improve serologic compatibility testing with the autoantibody (eg,
when a D– patient has autoanti-e).
In many cases of WAIHA, no autoantibody specificity is apparent. The patient’s
serum reacts with all red cell samples to the
same degree or reacts with red cells from
different donors to varying degrees for reasons seemingly unrelated to Rh phenotypes.
Even if specificity is identified, the exotic
cells used for such identification are not
available for transfusion. The most important consideration in such cases is to exclude the presence of clinically important
alloantibodies before selecting either phenotypically similar or dissimilar, crossmatchincompatible red cells for transfusion. In
extremely rare cases in which there is severe and progressive anemia, it may be essential to transfuse blood that does not react with the patient’s autoantibody.
Frequency of Testing. Although AABB
8(p38)
Standards
requires that a sample be
tested every 3 days, some serologists contend that, in these difficult cases, the continued collection and testing (to include
antibody investigation) of patient samples
are unnecessary.26 Others disagree with that
opinion. In studies of patients with WAIHA,
there was 12% to 40% alloimmunization,
with many alloantibodies developing after
recent transfusions.27,28 These two papers
offer methods to assist in the detection of
alloantibodies in the presence of autoantibodies. For patients with previously identified clinically significant antibodies, Standards8(p38) requires that methods of testing
shall be those that identify additional clinically significant antibodies. It is the exclusion of newly formed alloantibodies that is

464

AABB Technical Manual

of concern. Autoantibodies that react with
all reagent red cells, even weakly, are capable of masking alloantibody reactivity; the
serologic reactivity is not necessarily additive.29 Due to the presence of autoantibodies,
all crossmatches will be incompatible. This
is unlike the case of clinically significant
alloantibodies, where a compatible crossmatch with antigen-negative red cells can
be obtained. Monitoring for evidence of red
cell destruction due to alloantibodies is difficult in patients who already have AIHA;
the patient’s own red cells and transfused
red cells will have shortened survival. In patients who have autoantibodies without
hemolytic anemia, transfused red cells
should have normal survival.
An alternative transfusion management
protocol proposed by one group uses prophylactic antigen-matched units for patients with warm autoantibodies where feasible, in combination with streamlined
adsorption procedures.30 Such a protocol
depends on the ability to maintain an
adequate inventory of antigen-negative
units.31

IgM Warm AIHA
AIHA associated with IgM agglutinins that
react at 37 C is unusual but is characterized by severe hemolysis.25,32-34 The prognosis for these patients is poor.

DAT
The patient’s red cells are typically spontaneously agglutinated, requiring disruption of the IgM agglutinin by dithiothreitol in order to obtain accurate DAT (and
ABO/Rh) results. Complement is usually
detected on the red cells. In one series,
IgG was detected in 17% of cases and IgM
in 28%. By a more sensitive flow cytometric method, red cell-bound IgM was
detected on 82% of patients’ red cells not
reacting with anti-IgM by tube DAT.33

Serum
Warm IgM autoagglutinins are typically
weak and sometimes are enhanced in the
presence of albumin or when the serum is
acidified.33 Occasionally, optimal reactivity is between 20 C and 30 C, rather than
37 C. These antibodies have low or negligible antibody titers; a 4 C titer of <64 easily differentiates this IgM warm antibody
from those seen in CAS.

Eluate
IgM agglutinins are often detected in an
eluate when inspected at the agglutinin
phase before proceeding to the antiglobulin test.

Cold Agglutinin Syndrome
Cold agglutinin syndrome (also called cold
hemagglutinin disease, CHD) is the hemolytic anemia most commonly associated with
cold-reactive autoantibodies and accounts
for approximately 16% to 32% of all cases
of immune hemolysis.3,19 (See Table 20-3.)
It occurs as an acute or chronic condition.
The acute form is often secondary to
lymphoproliferative disorders (eg, lymphoma) or Mycoplasma pneumoniae infection. The chronic form is often seen in
elderly patients, sometimes associated
with lymphoma, chronic lymphocytic
leukemia, or Waldenstrom’s macroglobulinemia. Acrocyanosis and hemoglobinuria may occur in cold weather. CAS is
often characterized by rapid agglutination, at room temperature, of red cells in
an EDTA specimen. Clumping of red cells
may be obvious in such a sample, sometimes so strong that the cells appear to be
clotted. Problems with ABO and Rh typing
and other tests are not uncommon. Maintaining the EDTA specimen at 37 C and
washing the red cells with 37 C saline is
usually necessary to disperse the cold

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction

autoagglutinin before performing ABO and
Rh typing and the DAT.

DAT
Complement is the only protein detected
on the red cells in almost all cases. If other
proteins are detected, a negative control
for the DAT, eg, 6% to 10% albumin,
should be tested to ensure that the cold
autoagglutinin is not causing a false-positive test.
The cold-reactive autoagglutinin is usually IgM, which binds to red cells in the
comparatively low temperature of the peripheral circulation and causes complement components (C3 and C4 in particular)
to attach to the red cells. As the red cells circulate to warmer areas, the IgM dissociates,
but the complement remains. Red-cellbound C3b can react with the CR1 or CR3
receptors of macrophages in the reticuloendothelial system. More of the red cell destruction occurs in the liver. Regulatory
proteins convert the bound C3 and C4 to
C3dg and C4d, and it is the anti-C3d component of polyspecific AHG reagents that
accounts for the positive DAT. The presence
of C3dg alone does not shorten red cell survival because macrophages have no C3dg
or C3d receptors.

Serum
IgM cold-reactive autoagglutinins associated with immune hemolysis usually react ≥30 C and have a titer ≥1000 when
tested at 4 C; they rarely react with saline-suspended red cells above 32 C. If
30% bovine albumin is included in the reaction medium, 100% and 70% of clinically significant examples will react at 30
35
C or 37 C, respectively. Occasionally,
pathologic cold agglutinins will have a
lower titer (ie, <1000), but they will have a
high thermal amplitude (ie, reactive at 30
C with or without the addition of albu-

465

min). Hemolytic activity against untreated
red cells can be demonstrated sometimes
at 20 to 25 C, and, except in rare cases
with Pr specificity, enzyme-treated red
cells are hemolyzed in the presence of adequate complement.
Determination of the true thermal amplitude or titer of the cold autoagglutinin
requires that the specimen be collected and
maintained strictly at 37 C until the serum
and cells are separated, to avoid in-vitro
autoadsorption. Alternatively, plasma can
be used from an EDTA-anticoagulated
specimen that has been warmed for 10 to
15 minutes at 37 C (with repeated mixing)
and then separated from the cells, ideally at
37 C. This should release autoadsorbed antibody back into the plasma.
In chronic CAS, the IgM autoagglutinin
is usually a monoclonal protein with kappa
light chains. In the acute form induced by
Mycoplasma or viral infections, the antibody is polyclonal IgM with normal kappa
and lambda light-chain distribution. Rare
examples of IgA and IgG cold-reactive
autoagglutinins have also been described.

Eluate
Elution is seldom necessary in obvious
cases of CAS. If the red cells have been
collected properly and washed at 37 C,
there will be no immunoglobulin on the
cells and no reactivity will be found in the
eluate.

Specificity of Autoantibody
The autoantibody specificity in CAS is
usually of academic interest only. CAS is
most often associated with antibodies with
I specificity.3,23 Less commonly, i specificity is found, usually associated with infectious mononucleosis.3 On rare occasions,
cold-reactive autoagglutinins with Pr or
other specificities are seen3,23 (see Method
4.7). Dilution of the serum may be neces-

466

AABB Technical Manual

sary to demonstrate specificity of very
high-titer antibodies.
Autoantibody specificity is not diagnostic for CAS. Autoanti-I may be seen in
healthy subjects as well as patients with
CAS. The nonpathologic forms of autoanti-I, however, rarely react to titers above
64 at 4 C, and are usually nonreactive with
I– (i cord and i adult) red cells at room temperature. In contrast, the autoanti-I of CAS
may react quite strongly with I– red cells in
tests at room temperature, and equal or
even stronger reactions are observed with
I+ red cells. Autoanti-i reacts in the opposite manner, demonstrating stronger reactions with I– red cells than with red cells
that are I+. Procedures to determine the
titers and specificities of cold-reactive autoantibodies are given in Method 4.6 and
Method 4.7.
Pretransfusion Testing. Antibody detection tests should be performed in ways that
minimize cold-reactive autoantibody activity yet still permit detection of clinically significant alloantibodies. The use of albumin
and other potentiators may increase the reactivity of the autoantibodies. To avoid the
detection of bound complement, most
serologists use an IgG-specific reagent,
rather than a polyspecific AHG serum. Additionally, a prewarming technique may be
used (see Method 3.3).
Adsorption Procedures. When cold-reactive autoantibody reactivity continues to
interfere with antibody detection tests (eg,
when performed strictly at 37 C), cold
autoadsorption studies (see Method 4.6)
can be helpful. One or two cold autoadsorptions should remove enough autoantibody to make it possible to detect
alloantibodies at 37 C that were otherwise
masked by the cold-reactive autoantibody;
many cold autoadsorptions would be required to remove enough of the cold-reactive autoantibody for room temperature
testing. If the patient has been recently

transfused, rabbit red cells may be used to
remove autoanti-I and -IH from sera36; clinically significant alloantibodies, notably
anti-B, -D, -E, Vel, and others, have been removed by this method.37,38 A preparation of
rabbit red cell stroma is commercially available. Alternatively, allogeneic adsorption
studies at 4 C can be performed as for
WAIHA (see below).

Mixed-Type AIHA
Although about one-third of patients with
WAIHA have nonpathologic IgM antibodies that react to high titer at low temperature, another group of patients with
WAIHA have cold agglutinins that react at
or above 30 C. This latter group is referred
to as “mixed-type” AIHA and can be subdivided: patients with high titer, high
thermal amplitude IgM cold antibodies
(the rare WAIHA plus classic CAS) and patients with normal titer (<64 at 4 C), high
thermal amplitude cold antibodies.19,20,22,39
Patients with mixed-type AIHA often
present with hemolysis and complex serum reactivity present in all phases of
testing. Typical serologic findings are described below.

DAT
When the patient has WAIHA plus classic
CAS, both IgG and C3 are usually detectable on the patient’s red cells. When the
cold agglutinin has a normal titer, but
high thermal amplitude (greater than or
equal to 30 C), IgG and/or C3 may be detectable on the red cells.3

Serum
Both warm-reactive IgG autoantibodies
and cold-reactive, agglutinating IgM autoantibodies are present in the serum. These
usually result in reactivity at all phases of
testing, with virtually all cells tested. The
IgM agglutinating autoantibody(ies) re-

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction

acts at 30 C or above. If adsorption studies
are done to detect alloantibodies, it may
be necessary to perform adsorptions at both
warm and cold temperatures.

Eluate
A suitably prepared eluate will contain a
warm-reactive IgG autoantibody.

Specificity of Autoantibodies
The unusual cold-reactive IgM agglutinating autoantibody can have specificities
typical of CAS (ie, I or i) but often has no
apparent specificity.19,20,22 The warm-reactive IgG autoantibody often appears serologically indistinguishable from specificities encountered in typical WAIHA.

Transfusion in Mixed-Type AIHA
If blood transfusions are necessary, the
considerations in the selection of blood
for transfusion are identical to those described for patients with acute hemolysis
due to WAIHA (see above).

Paroxysmal Cold Hemoglobinuria
The rarest form of DAT-positive AIHA is
PCH. In the past, it was characteristically
associated with syphilis, but this association is now unusual. More commonly, PCH
presents as an acute transient condition
secondary to viral infections, particularly
in young children. In such cases, the
biphasic hemolysin (see below) may only
be transiently detectable. PCH can also
occur as an idiopathic chronic disease in
older people. One large study found that
none of 531 adults having well-defined
immune hemolytic anemias had DonathLandsteiner hemolysins, whereas 22 of 68
(32%) children were shown to have DonathLandsteiner hemolysins.21

467

DAT
PCH is caused by an IgG complement-fixing antibody, but, as with IgM cold-reactive autoagglutinins, it reacts with red
cells in colder areas of the body (usually
the extremities), causes C3 to bind irreversibly to red cells, and then the antibody dissociates from the red cells as the
blood circulates to warmer parts of the
body. Red cells washed in a routine manner for the DAT are usually coated only
with complement components, but IgG
may be detectable on cells that have been
washed with cold saline and tested with
3
cold anti-IgG reagent. Keeping the system
nearer its optimal binding temperature
allows the cold-reactive IgG autoantibody
to remain attached to its antigen.

Serum
The IgG autoantibody in PCH is classically
described as a biphasic hemolysin because
binding to red cells occurs at low temperatures but hemolysis does not occur until
the coated red cells are warmed to 37 C.
This is the basis of the diagnostic test for
the disease, the Donath-Landsteiner test
(see Method 4.13). The autoantibody may
agglutinate normal red cells at 4 C but
rarely to titers greater than 64. Because the
antibody rarely reacts above 4 C, the serum is usually compatible with random
donor cells by routine crossmatch procedures and pretransfusion antibody detection tests are usually nonreactive.

Eluate
Because complement components are
usually the only globulins present on circulating red cells, eluates prepared from
red cells of patients with PCH are almost
always nonreactive.

468

AABB Technical Manual

Specificity of Autoantibody
The autoantibody of PCH has most frequently been shown to have P specificity,
reacting with all red cells by the DonathLandsteiner test (including the patient’s
own red cells) except those of the very
rare p or Pk phenotypes. Exceptional examples with other specificities have been
described.6(p221),23

not causing the positive results.3,40 There
may be too few antibody molecules on
the cell for detection by routine methods
but enough to be demonstrable by methods such as flow cytometry, enzymelinked antiglobulin tests, solid phase,
PEG, direct Polybrene, column agglutination, or concentrated eluate.

Nonroutine Reagents
Transfusion in PCH
Transfusion is rarely necessary for adult
patients with PCH, unless their hemolysis
is severe. In children, especially under age
6, the thermal amplitude of the antibody
tends to be much wider than in adults
and hemolysis more brisk, so transfusion
may be required as a lifesaving measure.
Although there is some evidence that p
red cells survive better than P+ (P1+ or P1–)
red cells,6(p221) the prevalence of p blood is
approximately 1 in 200,000 and the urgent
need for transfusion usually precludes attempts to obtain this rare blood. Transfusion of random donor blood should not
be withheld from PCH patients whose
need is urgent. Red cells negative for the P
antigen should be considered only for
those patients who do not respond adequately to random donor blood.3

The causative antibody may be IgM or IgA
not detected by routine AHG reagents.
Anti-IgG, anti-C3d, and the combined
anti-C3b, -C3d reagents are the only licensed products available in the United
States for use with human red cells. AHG
reagents that react with IgA, IgM, or C4
are available commercially but have been
prepared for use with endpoints other
than agglutination. These must be used
cautiously and their hemagglutinating reactivity carefully standardized by the
user.3 Quality control must be rigorous
because agglutination with AHG reagents
is more sensitive than precipitation; a serum that appears to be monospecific by
precipitation tests may react with several
different proteins when used in agglutination tests.

Antigen Depression
DAT-Negative AIHA
Clinical evidence of hemolytic anemia is
present in some patients whose DAT is nonreactive. Frequently, autoantibody cannot
be detected in either eluate or serum.
There may be several reasons the DAT is
negative. The autoantibody may be IgA or
IgM.3,40 Antibodies with low binding affinity may dissociate from the red cells during saline washing of the cells for the DAT.
Washing with ice cold (eg, 4 C) LISS or saline may help retain antibody on the cells;
a control (eg, 6-10% albumin) is necessary
to confirm that cold autoagglutinins are

Patients with autoantibodies of Kell, Rh,
LW, Ge, Sc, Lu, and Lan specificities may
have depressed red cell expression of the
respective antigens. When this occurs, antibody may be detected in the serum and
eluate, but the DAT may be negative or very
weakly positive. This may provide in-vivo
protection of autologous cells. Donor cells
of common specific antigen type may be
destroyed, but cells lacking the corresponding antigen (usually high-incidence)
may survive well. When the autoantibody
subsides, autologous cells again express
normal amounts of antigen.6(p228),24

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction

Serologic Problems with
Autoantibodies
In pretransfusion tests on patients with autoantibodies, the following problems may
arise:
1.
Cold-reactive autoantibodies can cause
autoagglutination, resulting in erroneous determinations of ABO and
Rh type.
2.
Red cells strongly coated with globulins may undergo spontaneous agglutination with high-protein, antiRh, blood-typing reagents, and occasionally even with low-protein reagents.41
3.
The presence of free autoantibody in
the serum may make antibody detection and crossmatching tests difficult to interpret. If time permits, the
presence or absence of unexpected,
clinically important alloantibody(ies)
should be determined (see Methods
4.9 through 4.12) before blood is transfused.
Although resolving these serologic problems is important, delaying transfusion in
the hope of finding serologically compatible blood may cause greater danger to the
patient in some cases. Only clinical judgment can resolve this dilemma; therefore,
dialogue with the patient’s physician is important.

Resolution of ABO Problems
There are several approaches to the resolution of ABO typing problems associated
with cold-reactive autoagglutinins. Often,
it is only necessary to maintain the blood
sample at 37 C immediately after collection and to wash the red cells with warm
(37 C) saline before testing. It is helpful to
perform a parallel control test, using 6%
to 10% bovine albumin in saline, to determine if autoagglutination persists. If the

469

control test is nonreactive, the results obtained with anti-A and anti-B are usually
valid. If autoagglutination still occurs, it
may be necessary to treat the red cells
with sulfhydryl reagents.
Because cold-reactive autoagglutinins
are almost always IgM and sulfhydryl reagents denature IgM molecules, reagents
such as 2-mercaptoethanol (2-ME) or
dithiothreitol (DTT) can be used to abolish
autoagglutination (see Method 2.11). Treating the red cells with ZZAP reagent as in the
preparation for adsorptions can also be used
(see Method 4.10). Appropriate controls are
essential for all tests.
When the serum agglutinates group O
reagent red cells, the results of serum tests
may be unreliable. Repeating the tests using prewarmed serum and group A, B, and
O red cells at 37 C will often resolve any discrepancy, but weak anti-A and/or -B in
some patients’ sera may not react at 37 C.
Alternatively, adsorbed serum (either autoadsorbed or adsorbed with allogeneic
group O red cells) can be used. Because
rabbit red cells express a B-like antigen,
sera adsorbed with rabbit red cells or
stroma may not contain anti-B, and sera
adsorbed in this manner should not be
used for ABO serum tests.

Resolution of Rh Problems
Autoagglutination of red cells by cold- or
spontaneous agglutination of red cells by
warm-reactive autoantibodies may also
cause discrepant Rh typing. The same
procedures described for the resolution of
ABO problems, with the exception of using ZZAP-treated red cells, may be useful.
Also, IgG antibody can be dissociated from
the cells by treatment with chloroquine
diphosphate (Method 2.13), or by glycineHCl/EDTA (Method 2.14), methods that
leave red cells intact for subsequent typing. IgM-coated cells can be treated with

470

AABB Technical Manual

sulfhydryl reagents (such as 2-ME or DTT,
Method 2.11) to circumvent autoagglutination and spontaneous agglutination.

Detection of Alloantibodies in the
Presence of Warm-Reactive
Autoantibodies
If the patient who has warm-reactive
autoantibodies in the serum needs transfusion, it is important to evaluate the
possible simultaneous presence of alloantibodies to red cell antigens. Some
alloantibodies may make their presence
known by reacting more strongly or at different phases than the autoantibody, but
quite often studies may not suggest the
29
existence of masked alloantibodies. It is
helpful to know which of the common red
cell antigens are lacking on the patient’s
red cells, to predict which clinically significant alloantibodies the patient may have
produced or may produce. Antigens absent from autologous cells could well be
the target of present or future alloantibodies. When the red cells are coated with
IgG, antiglobulin-reactive reagents cannot be used to test IgG-coated cells unless
the IgG is first removed (see Methods 2.13
and 2.14). Low-protein antisera (eg, monoclonal reagents) that do not require an
antiglobulin test may be helpful in typing
the DAT-positive red cells. Cell separation
procedures (see Methods 2.15 and 2.16)
may be necessary if the patient has been
transfused recently.
Methods to detect alloantibodies in the
presence of warm-reactive autoantibodies
attempt to remove, reduce, or circumvent
the autoantibody. Antibody detection methods that use PEG, enzymes, column agglutination, or solid-phase red cell adherence
generally enhance autoantibodies. Testing
LISS- or saline-suspended red cells may
avoid autoantibodies but allow detection of
most significant alloantibodies. Other pro-

cedures involve adsorption, the principles
of which are discussed in Chapter 19. Two
widely used approaches are discussed below.

Autologous Adsorption
In a patient who has not been recently
transfused, autologous adsorption (see
Method 4.9) is the best way to detect alloantibodies in the presence of warm-reactive autoantibodies. The adsorbed serum
can be used in the routine antibody detection procedure.
Autoadsorption generally requires some
initial preparation of the patient’s red cells.
At 37 C, in-vivo adsorption will have occurred and all antigen sites on the patient’s
own red cells may be blocked. It may be
necessary, therefore, to remove autoantibody from the red cells to make sites available for adsorption. A gentle heat elution at
56 C for 5 minutes can dissociate some of
the bound IgG. This can be followed by
treatment of the autologous red cells with
proteolytic enzymes to increase their capacity to adsorb autoantibody. Treatment
of the red cells with ZZAP, a mixture of
papain or ficin and DTT (see Method 4.9)
accomplishes both of these actions in one
step; the sulfhydryl component makes the
IgG molecules more susceptible to the protease and dissociates the antibody molecules from the cell. Multiple sequential
autoadsorptions with new aliquots of red
cells may be necessary if the serum contains high levels of autoantibody. Once the
autoantibody has been removed, the adsorbed serum is examined for alloantibody
activity.
If the patient is to be transfused, it can
be advantageous to collect and save additional aliquots of pretransfusion cells, to be
used for later adsorptions.
Autologous adsorption is not recommended for patients who have been re-

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction

cently transfused, because they may have
an admixture of transfused red cells that
might adsorb alloantibody. Red cells normally live for about 110 to 120 days. In patients with AIHA, autologous and transfused red cells can be expected to have
shortened survival. However, determining
how long transfused red cells remain in circulation in patients who need repeated
transfusions is not feasible. It has been
demonstrated that very small amounts
(<10%) of antigen-positive red cells are capable of removing alloantibody reactivity in
in-vitro studies42; therefore, it is recommended to wait for 3 months after transfusion before autologous adsorptions are performed.

Allogeneic Adsorption
The use of allogeneic red cells for adsorption may be helpful when the patient has
been recently transfused or when insufficient autologous red cells are available.
The goal is to remove autoantibody and
leave the alloantibody in the adsorbed serum. The adsorbing cells must not have
the antigens against which the alloantibodies react. Because alloantibody specificity is unknown, red cells of different
phenotypes will usually be used to adsorb
several aliquots of the patient’s serum.
Given the number of potential alloantibodies, the task of selecting cells may appear formidable. However, the selected
cells need only demonstrate those few alloantibodies of clinical significance likely to
be present. These include the common Rh
antigens (D, C, E, c, and e), K, Fya and Fyb,
Jka and Jkb, and S and s. Cell selection is
made easier by the fact that some antigens
can be destroyed by appropriate treatment
(eg, with enzymes) before use in adsorption
procedures. Antibodies to high-incidence
antigens cannot be excluded by allogeneic

471

adsorptions because the adsorbing cells
will almost invariably express the antigen
and adsorb the alloantibody along with
autoantibody.
Patient’s Phenotype Unknown. When
the patient’s phenotype is not known,
group O red cell samples of three different
Rh phenotypes (R1R1, R2R2, and rr) should
be selected (see Method 4.10). One should
lack Jka and another Jkb. If treated with
ZZAP, these cells would also lack all antigens of the Kell system and enzyme-sensitive antigens (see Table 19-3). If ZZAP is not
available, cells treated only with proteolytic
enzyme can be used, but at least one of the
adsorbing cells must be K– because Kell
system antigens will not be destroyed. Untreated cells may be used, but antibody
may be more difficult to remove and the
adsorbing cells must, at a minimum, include at least one negative for the S, s, Fya,
b
Fy , and K antigens in addition to the Rh
and Kidd requirements above.
Each aliquot may need to be adsorbed
two or three times. The fully adsorbed
aliquots are tested against reagent red cells
known either to lack or to carry common
antigens of the Rh, MNS, Kidd, Kell, and
Duffy blood group systems. If an adsorbed
aliquot is reactive, that aliquot (or an additional specimen similarly adsorbed) should
be tested to identify the antibody. Adsorbing several aliquots with different red cell
samples provides a battery of potentially
informative specimens. For example, if the
aliquot adsorbed with Jk(a–) red cells subsequently reacts only with Jk(a+) red cells,
the presence of alloanti-Jka can confidently
be inferred.
Patient’s Phenotype Known. If the patient’s Rh and Kidd phenotypes are known
or can be determined, adsorption can be
performed with a single sample of allogeneic ZZAP-treated red cells of the same
Rh and Kidd phenotypes as the patient (see
Method 4.11).

472

AABB Technical Manual

Problems Encountered. Occasionally
autoantibody will not be removed by three
sequential adsorptions. Further adsorptions
can be done, but multiple adsorptions have
the potential to dilute the serum. If the adsorbing cells do not appear to remove the
antibody, the autoantibody may have an
unusual specificity that does not react with
the cells used for adsorption. For example,
a
autoantibodies with Kell, LW, or En FS
specificity would not be removed by ZZAPtreated cells (see Table 19-3 for a list of antigens altered by various agents).

Detection of Alloantibodies in the
Presence of Cold-Reactive Autoantibodies
Cold-reactive autoagglutinins rarely mask
clinically significant alloantibodies if serum tests are conducted at 37 C and if
IgG-specific reagents are used for the
antiglobulin phase. In rare instances, it
may be necessary to perform autoadsorption at 4 C (see Method 4.6). Achieving the
complete removal of potent cold-reactive
autoagglutinins is very time-consuming
and is usually unnecessary. Removal of
sufficient cold autoagglutinins may be facilitated by treating the patient’s cells with
enzymes or ZZAP before adsorption.

Autoantibodies Mimicking Alloantibodies
Sometimes, autoantibodies have patterns
of reactivity that are easily mistaken for
alloantibody. For example, the serum of a
D– patient may have apparent anti-C and
-e reactivity. The anti-C reactivity may reflect warm-reactive autoantibody even if
the patient’s cells lack C. The autoantibody
nature of the reactivity can be demonstrated by autologous and allogeneic adsorption studies. In this case, the apparent alloanti-C would be adsorbed by C–
red cells, both autologous and allogeneic.
This is quite unlike the behavior of a true
alloanti-C, which would be adsorbed only
by C+ red cells. In one study,43 the serum
prepared from an initial autoadsorption
would often retain autoantibodies that
mimicked alloantibodies in addition to
the true alloantibody(ies) present. Serum
prepared from an initial alloadsorption
most often contains only alloantibodies.
The differences in the auto- or alloantibody nature of specificities detected in
the autoadsorbed serum as compared to
the alloadsorbed serum reflect an inefficiency of autologous adsorption. This is
primarily due to limited volumes of autologous cells available for removing all
autoantibody reactivity from the serum.43

Drug-Induced Immune
Hemolytic Anemia
Drugs sometimes induce the formation of
antibodies, either against the drug itself
or against intrinsic red cell antigens, that
may result in a positive DAT, immune red
cell destruction, or both. Some of the antibodies produced appear to be dependent on the presence of the drug (ie, drug
dependent) for their detection or destructive capability, whereas others do not (ie,
drug independent). In some instances, a
reactive DAT may result from nonimmunologic effects of the drugs. Drugs that
have been reported to cause hemolytic
anemia and/or a positive DAT are listed in
Appendix 20-2.

Theories of the Immune Response and
Drug-Dependent Antibodies
Numerous theories have been suggested
to explain how drugs induce immune responses and what relation such responses
may have to the positive DAT and immunemediated cell destruction observed in some
patients. For many years, drug-associated

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction

positive DATs were classified into four
mechanisms: drug adsorption (penicillin-type), immune complex formation,
autoantibody production, and nonspecific adsorption. Such classification has
been useful, but many aspects lacked definitive proof. In addition, some drugs
created immune problems involving aspects of more than one mechanism. More
recent theories, still unproven, tend to44-48
ward a more comprehensive approach.
Most drugs are probably capable of
binding loosely, or firmly, to circulating
cells, which can lead to an immune response. Figure 20-1 illustrates this concept.
Antibodies can be formed to the drug itself
or to the drug plus membrane components.
When an antibody is formed to the drug
plus membrane components, the antibody
may recognize primarily the drug or primarily the membrane. One or all three of these
antibody populations may be present.

473

Serologic and Clinical Classification
Drug-induced antibodies can be classified into three groups according to their
clinical and serologic characteristics.3 In
one group, the drug binds firmly to the
cell membrane and antibody is apparently largely directed against the drug itself. This was called the drug adsorption
mechanism. Antibodies to penicillin are
the best described of this group.
The second group of drug-dependent
antibodies reacts with drugs that do not
bind well to the cell membrane (eg, quinidine, ceftriaxone). The reactive mechanism
of these antibodies was previously thought
to be due to drug/antidrug immune complex formation, but the theory has never
been proven.48 Antibodies in this group may
cause acute intravascular hemolysis and
may be difficult to demonstrate serologically. Testing for this type of drug antibody

Figure 20-1. Proposed unifying theory of drug-induced antibody reactions (based on a cartoon by
Habibi as cited by Garratty 23 ). The thicker darker lines represent antigen-binding sites on the F(ab) region of the drug-induced antibody. Drugs (haptens) bind loosely, or firmly, to cell membranes and antibodies may be made to: a) the drug [producing in-vitro reactions typical of a drug adsorption (penicillin-type) reaction]; b) membrane components, or mainly membrane components (producing in-vitro
reactions typical of autoantibody); or c) part-drug, part-membrane components (producing an in-vitro
reaction typical of the so-called immune complex mechanism). 23(p55)

474

AABB Technical Manual

is still referred to as the “immune complex”
method.
Antibodies of the third group (eg, methyldopa, procainamide, and fludarabine) have
serologic reactivity independent of the drug,
despite the fact that it was the drug that
originally induced the immune response.
Serologically, they behave as autoantibodies.

Drug-Dependent Antibodies Reactive with
Drug-Treated Red Cells: Penicillin-Type
Antibodies
The clinical and laboratory features of druginduced immune hemolytic anemia operating through this mechanism are:
1.
The DAT is strongly positive due to
IgG coating. Complement coating may
also be present.
2.
Antibody eluted from the patient’s
red cells reacts with drug-treated red
cells but not with untreated red cells.
3.
The serum contains a high-titer IgG
antibody (especially when the target
is penicillin or cefotetan) reactive with
the drug-treated red cells but not with
the untreated red cells, unless the
patient also has alloantibodies to red
cell antigens.
4.
For penicillin, the hemolysis-inducing dose is millions of units daily for
a week or more; for other drugs, eg,
cefotetan, a single 1 to 2 g dose has been
implicated in immune hemolysis.
5.
Hemolysis develops gradually but
may be life-threatening if the etiology is unrecognized and drug administration is continued.
6.
Discontinuation of the drug is usually followed by increased cell survival, although hemolysis of decreasing severity may persist for several
weeks.
Approximately 3% of patients receiving
large doses of penicillin intravenously (ie,
millions of units per day) will develop a

positive DAT; only occasionally will these
patients develop hemolytic anemia.6(p231) A
possible mechanism for the positive DAT is
given in Fig 20-2. The penicillin becomes
covalently linked to the red cells in vivo. If
the patient has antibodies to penicillin, they
bind to the penicillin bound to the red cells.
The result is that the penicillin-coated red
cells become coated with IgG. If cell destruction occurs, it takes place extravascularly,
probably in the same way that red cells
coated with IgG alloantibodies are destroyed. Intravascular hemolysis is rare.
Many cephalosporins, which are related
to penicillins, behave in a similar manner.
The cephalosporins are generally classified
by “generations,” based on their effectiveness against gram-negative organisms (see
Table 20-4). Approximately 4% of patients
receiving first- or second-generation cephalosporins develop a positive DAT.48 Dramatically reduced red cell survival has been
associated with second- and third-generation cephalosporins.12,47,49-53 The prevalence
and severity of cephalosporin-induced immune red cell destruction appear to be increasing.3

Drug-Dependent Antibodies Reacting by the
“Immune Complex” Mechanism
Many drugs have been reported as causing hemolytic anemia by this mechanism.
Some of the second- and third-generation
cephalosporins react by this mechanism;
anti-ceftriaxone has been detected only
by the immune complex method.49 The
following observations are characteristic:
1.
Complement may be the only globulin easily detected on the red cells,
but IgG may be present.
2.
The serum antibody can be either
IgM or IgG, or IgM with IgG.
3.
A drug (or metabolite) must be present in vitro for demonstration of the

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction

475

Figure 20-2. The drug-adsorption mechanism. The drug binds tightly to the red cell membrane proteins. If a patient develops a potent drug antibody, it will react with the cell-bound drug. Such red cells
will yield a positive result in the DAT using anti-IgG reagents. Complement is usually not activated and
lysis is primarily extravascular in nature. Penicillin-G is the prototype drug.

4.
5.

antibody in the patient’s serum. Antibodies may cause hemolysis, agglutination, and/or sensitization of
red cells in the presence of the drug.
The patient need only take a small
amount of the drug.
Acute intravascular hemolysis with
hemoglobinemia and hemoglobinuria
is the usual presentation. Renal failure is quite common.
Once antibody has been formed, severe hemolytic episodes may recur
after exposure to very small quantities of the drug.

Drug-Independent Antibodies: Autoantibody
Production
Some drugs induce autoantibodies that
appear serologically indistinguishable
from those of WAIHA. Red cells are coated
with IgG, and the eluate as well as the serum react with virtually all cells tested in
the absence of the drug. Blood group specificity has been demonstrated at times,
similar to that seen in AIHA. The antibody
has no in-vitro activity with the drug, directly or indirectly.
The best studied of such cases are those
induced by α-methyldopa. A closely related

476

AABB Technical Manual

Table 20-4. Some Cephalosporins
Generic Name

Trade Name*

First Generation
cefadroxil

Duricef

cefazolin

Ancef, Kefzol,
Zolicef

cephalexin

Keflex

cephalothin

Keflin

cephapirin

Cefadyl

cephradine

Anspor

Second Generation
cefaclor

Ceclor

cefamandole

Mandol

cefmetazole

Zefazone

cefonicid

Monocid

cefotetan

Cefotan

cefoxitin

Mefoxin

cefuroxime

Zinacef, Kefurox,
Ceftin

cefuroxime axetil

Ceftin

Third Generation
cefixime

Suprax

cefoperazone

Cefobid

cefotaxime

Claforan

ceftazidime

Fortaz, Ceptaz,
Pentacef, Tazicef,
Tazidime

ceftizoxime

Ceftizox

ceftriaxone

Rocephin

Fourth Generation
cefepime

Maxipime

*Several forms are marketed under other trade names.
This list is intended to be informative, not inclusive.

drug, L-dopa, has been implicated, as have
several drugs unrelated to α-methyldopa,
including procainamide, nonsteroidal
anti-inflammatory drugs (eg, mefenamic
acid), second- and third-generation cephalosporins, and fludarabine. In some
cases, drug-dependent antibodies are also
present.
Proof that a drug causes autoantibody
production is difficult to obtain. Sufficient
evidence would include: demonstration
that autoantibody production began after
drug administration; resolution of the immune process after withdrawal of the drug;
and recurrence of hemolytic anemia or
autoantibodies if the drug is readministered. The last requirement is crucial and
the most difficult to demonstrate.

Nonimmunologic Protein Adsorption
The positive DAT associated with some
drugs is due to a mechanism independent
of antibody production. Hemolytic anemia associated with this mechanism occurs rarely.
Cephalosporins (primarily cephalothin)
are the drugs with which this was originally
associated. Red cells coated with cephalothin (Keflin) and incubated with normal
plasma will adsorb albumin, IgA, IgG, IgM,
and C3 in a nonimmunologic manner. If
this occurs, a positive indirect antiglobulin
test will be seen with AHG reagents.
Other drugs that may cause nonimmunologic adsorption of proteins and a positive
DAT include diglycoaldehyde, suramin,
cisplatin, clavulanate (in Timentin and
Augmentin), sulbactam in Unasyn,54 and
55,56
tazobactam (in Tazocin and Zosyn).

Laboratory Investigation of Drug-Induced
Antibodies
The drug-related problems most commonly encountered in the blood bank are
those associated with a positive DAT. Typ-

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction

ical DAT results are shown in Table 20-3.
Recent red cell transfusions and/or dramatic hemolysis may result in a weak DAT
by the time hemolysis is suspected.
The patient’s serum should be tested for
unexpected antibodies by routine procedures. If the serum does not react with untreated red cells, the tests should be repeated against ABO-compatible red cells in
the presence of the drug(s) suspected of
causing the problem. Techniques are given
in Method 4.14 and Method 4.15.
If the drug has already been reported as
causing hemolytic anemia, testing methods
may be available in the case reports. If such
information is not available, an initial
screening test can be performed with a solution of the drug at a concentration of approximately 1 mg/mL in phosphate-buffered saline at a pH optimal for solubility of
the drug.
If these tests are not informative, attempts can be made to coat normal red cells
with the drug, and the patient’s serum and
an eluate from the patient’s red cells can be
tested against the drug-coated red cells.
This is the method of choice when penicillin or cephalosporins are thought to be implicated. Results definitive for a penicillin-induced positive DAT are reactivity of
the eluate against penicillin-coated red
cells and absence of reactivity between the
eluate and uncoated red cells.
The immune response may be due to a
metabolite of a drug rather than the drug itself. If the clinical picture is consistent with
immune-mediated hemolysis and the above
tests are noninformative, it may be helpful
to test drug metabolites (see Method 4.16).
Normal sera commonly agglutinate and/
or sensitize cephalosporin-treated red cells
due to the nonspecific uptake of protein
discussed above. This problem can be overcome by testing a 1 in 20 dilution of the patient’s serum and a normal serum control
against the cephalosporin-treated red cells.

477

During the testing of cefotetan-treated red
cells, a 1 in 100 dilution of the patient’s serum should be tested because it has been
shown that some normal sera appear to
contain “naturally occurring” antibodies to
cefotetan, some of which still react weakly at
a 1 in 20 dilution.57,58 Cefotetan antibodies
associated with drug-induced immune
hemolytic anemia have very high antiglobulin titers (4000 to 256,000).49
Two other observations have been made
regarding the testing of cefotetan antibodies: 1) the last wash from the eluate preparation may react with cefotetan-treated red
cells (possibly due to the high-titer antibodies and/or the antibody affinity), and 2)
drug-independent antibodies may be detected
in the serum and eluate and hemolysis may
be inadvertently attributed to idiopathic
49
WAIHA.

References
1.

4.
5.

Toy PT, Chin CA, Reid ME, Burns MA. Factors
associated with positive direct antiglobulin
tests in pretransfusion patients: A case control study. Vox Sang 1985;49:215-20.
Heddle NM, Kelton JG, Turchyn KL, Ali MAM.
Hypergammaglobulinemia can be associated
with a positive direct antiglobulin test, a nonreactive eluate, and no evidence of hemolysis.
Transfusion 1988;28:29-33.
Petz LD, Garratty G. Immune hemolytic
anemias. 2nd ed. Philadelphia: Churchill
Livingstone, 2004.
Garratty G. The significance of IgG on the red
cell surface. Transfus Med Rev 1987;1:47-57.
Freedman J. The significance of complement
on the red cell surface. Transfus Med Rev
1987;1:58-70.
Mollison PL, Engelfriet CP, Contreras M.
Blood transfusion in clinical medicine. 10th
ed. Oxford, England: Blackwell Scientific Publications, 1997.
Clark JA, Tanley PC, Wallas CH. Evaluation of
patients with positive direct antiglobulin
tests and nonreactive eluates discovered during pretransfusion testing. Immunohematology
1992;8:9-12.
Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.

478

10.

11.

12.
13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

AABB Technical Manual

Judd WJ, Barnes BA, Steiner EA, et al. The
evaluation of a positive direct antiglobulin
test (autocontrol) in pretransfusion testing
revisited. Transfusion 1986;26:220-4.
Salama A, Mueller-Eckhardt C. Delayed
hemolytic transfusion reactions. Evidence for
complement activation involving allogeneic
and autologous red cells. Transfusion 1984;
24:188-93.
Ness PM, Shirey RS, Thoman SK, Buck SA.
The differentiation of delayed serologic and
delayed hemolytic transfusion reactions: Incidence, long-term serologic findings, and
clinical significance. Transfusion 1990;30:
688-93.
Garratty G. Immune cytopenia associated with
antibiotics. Transfus Med Rev 1993;7:255-67.
Kleinman S, Nelson R, Smith L, Goldfinger D.
Positive direct antiglobulin tests and immune
hemolytic anemia in patients receiving procainamide. N Engl J Med 1984;311:809-12.
Lutz P, Dzik W. Very high incidence of a positive direct antiglobulin test (+DAT) in patients receiving Unasyn® (abstract). Transfusion 1992;32(Suppl):23S.
Garratty G. Problems associated with passively transfused blood group alloantibodies.
Am J Clin Pathol 1998;109:769-77.
Leger RM, Arndt PA, Ciesielski DJ, Garratty G.
False-positive eluate reactivity due to the
low-ionic wash solution used with commercial acid-elution kits. Transfusion 1998;38:
565-72.
Judd WJ. Elution—dissociation of antibody
from red blood cells: Theoretical and practical considerations. Transfus Med Rev 1999;
13:297-310.
South SF, Rea AE, Tregellas WM. An evaluation of 11 red cell elution procedures. Transfusion 1986;26:167-70.
Sokol RJ, Hewitt S, Stamps BK. Autoimmune
haemolysis: An 18 year study of 865 cases referred to a regional transfusion centre. Br Med
J 1981;282:2023-7.
Shulman IA, Branch DR, Nelson JM, et al. Autoimmune hemolytic anemia with both cold
and warm autoantibodies. JAMA 1985;253:
1746-8.
Göttsche B, Salama A, Mueller-Eckhardt C.
Donath-Landsteiner autoimmune hemolytic
anemia in children. A study of 22 cases. Vox
Sang 1990;58:281-6.
Sokol RJ, Hewitt S, Stamps BK. Autoimmune
haemolysis. Mixed warm and cold antibody
type. Acta Haematol 1983;69:266-74.
Garratty G. Target antigens for red-cellbound autoantibodies. In: Nance SJ, ed. Clinical and basic science aspects of immunohematology. Arlington, VA: AABB, 1991:33-72.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

Garratty G. Specificity of autoantibodies reacting optimally at 37 C. Immunohematology
1999;15:24-40.
Freedman J, Wright J, Lim FC, Garvey MB.
Hemolytic warm IgM autoagglutinins in autoimmune hemolytic anemia. Transfusion
1987;27:464-7.
Judd WJ. Investigation and management of
immune hemolysis: Autoantibodies and drugs.
In: Wallace ME, Levitt JS, eds. Current applications and interpretations of the direct antiglobulin test. Arlington, VA: AABB, 1988:47103.
Leger RM, Garratty G. Evaluation of methods
for detecting alloantibodies underlying warm
autoantibodies. Transfusion 1999;39:11-16.
Branch DR, Petz LD. Detecting alloantibodies
in patients with autoantibodies (editorial).
Transfusion 1999;39:6-10.
Church AT, Nance SJ, Kavitsky DM. Predicting
the presence of a new alloantibody underlying a warm autoantibody (abstract). Transfusion 2000;40(Suppl):121S.
Shirey RS, Boyd JS, Parwani AV, et al. Prophylactic antigen-matched donor blood for patients with warm autoantibodies: An algorithm
for transfusion management. Transfusion
2002;42:1436-41.
Garratty G, Petz LD. Approaches to selecting
blood for transfusion to patients with autoimmune hemolytic anemia (editorial). Transfusion 2002;42:1390-2.
Garratty G, Arndt P, Domen R, et al. Severe
autoimmune hemolytic anemia associated
with IgM warm autoantibodies directed
against determinants on or associated with
glycophorin A. Vox Sang 1997;72:124-30.
Garratty G, Arndt P, Leger R. Serological findings in autoimmune hemolytic anemia associated with IgM warm autoantibodies (abstract). Blood 2001;98(Suppl 1):61a.
Nowak-Wegrzyn A, King KE, Shirey RS, et al.
Fatal warm autoimmune hemolytic anemia
resulting from IgM autoagglutinins in an infant with severe combined immunodeficiency. J Pediatr Hematol Oncol 2001;23:2502.
Garratty G, Petz LD, Hoops JK. The correlation of cold agglutinin titrations in saline and
albumin with haemolytic anemia. Br J Haematol
1975;35:587-95.
Marks MR, Reid ME, Ellisor SS. Adsorption of
unwanted cold autoagglutinins by formaldehyde-treated rabbit erythrocytes (abstract).
Transfusion 1980;20:629.
Dzik W, Yang R, Blank J. Rabbit erythrocyte
stroma treatment of serum interferes with
recognition of delayed hemolytic transfusion
reactions (letter). Transfusion 1986;26:303-4.

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

Mechanic SA, Maurer JL, Igoe MJ, et al. AntiVel reactivity diminished by adsorption with
rabbit RBC stroma. Transfusion 2002;42:
1180-3.
Garratty G, Arndt PA, Leger RM. Serological
findings in autoimmune hemolytic anemia
(AIHA) associated with both warm and cold
autoantibodies (abstract). Blood 2003;102
(Suppl 1):563a.
Garratty G. Autoimmune hemolytic anemia.
In: Garratty G, ed. Immunobiology of transfusion medicine. New York: Marcel Dekker,
1994:493-521.
Garratty G, Postoway N, Nance SJ, Brunt DJ.
Spontaneous agglutination of red cells with a
positive direct antiglobulin test in various
media. Transfusion 1984;24:214-7.
Laine EP, Leger RM, Arndt PA, et al. In vitro
studies of the impact of transfusion on the
detection of alloantibodies after autoadsorption. Transfusion 2000;40:1384-7.
Issitt PD, Combs MR, Bumgarner DJ, et al.
Studies of antibodies in the sera of patients
who have made red cell autoantibodies.
Transfusion 1996;36:481-6.
Salama A, Mueller-Eckhardt C. Immune-mediated blood cell dyscrasias related to drugs.
Semin Hematol 1992;29:54-63.
Petz LD, Mueller-Eckhardt C. Drug-induced
immune hemolytic anemia. Transfusion
1992;32:202-4.
Shulman NR, Reid DM. Mechanisms of druginduced immunologically mediated cytopenias. Transfus Med Rev 1993;7:215-29.
Christie DJ. Specificity of drug-induced immune cytopenias. Transfus Med Rev 1993;7:
230-41.
Garratty G. Drug-induced immune hemolytic
anemia. In: Garratty G, ed. Immunobiology of
transfusion medicine. New York: Marcel
Dekker, 1994:523-51.
Arndt PA, Leger RM, Garratty G. Serology of
antibodies to second- and third-generation
cephalosporins associated with immune
hemolytic anemia and/or positive direct
antiglobulin tests. Transfusion 1999;39:123946.
Gallagher NI, Schergen AK, Sokol-Anderson
ML, et al. Severe immune-mediated hemolytic
anemia secondary to treatment with cefotetan. Transfusion 1992;32:266-8.
Garratty G, Nance S, Lloyd M, Domen R. Fatal
immune hemolytic anemia due to cefotetan.
Transfusion 1992;32:269-71.
Stroncek D, Procter JL, Johnson J. Drug-induced hemolysis: Cefotetan-dependent
hemolytic anemia mimicking an acute
intravascular immune transfusion reaction.
Am J Hematol 2000;64:67-70.

53.

54.

55.

56.

57.

58.

479

Viraraghavan R, Chakravarty AG, Soreth J.
Cefotetan-induced haemolytic anaemia. A review of 85 cases. Adv Drug React Toxicol Rev
2002;21:101-7.
Garratty G, Arndt PA. Positive direct antiglobulin tests and haemolytic anemia following
therapy with beta-lactamase inhibitor containing drugs may be associated with nonimmunologic adsorption of protein onto red
blood cells. Br J Haematol 1998;100:777-83.
Broadberry RE, Farren TW, Kohler JA, et al.
Haemolytic anaemia associated with Tazobactam (abstract). Vox Sang 2002;83(Suppl 2):
227.
Arndt PA, Leger RM, Garratty G. Positive direct antiglobulin tests and haemolytic anaemia following therapy with the beta-lactamase inhibitor, tazobactam, may also be
associated with non-immunologic adsorption of protein onto red blood cells (letter).
Vox Sang 2003;85: 53.
Arndt P, Garratty G. Is severe immune hemolytic anemia, following a single dose of cefotetan, associated with the presence of “naturally-occurring” anti-cefotetan? (abstract)
Transfusion 2001;41(Suppl):24S.
Arndt PA. Practical aspects of investigating
drug-induced immune hemolytic anemia
due to cefotetan or ceftriaxone—a case study
approach. Immunohematology 2002;18:2732.

Suggested Reading
Dacie J. Historical review. The immune haemolytic
anaemias: A century of exciting progress in understanding. Br J Haematol 2001;114:770-85.
Engelfriet CP, Overbeeke MAM, von dem Borne
AEGKr. Autoimmune hemolytic anemia. Semin
Hematol 1992;29:3-12.
Garratty G. Novel mechanisms for immune destruction of circulating autologous cells. In: Silberstein
LE, ed. Autoimmune disorders of blood. Bethesda,
MD: AABB, 1996:79-114.
Garratty G. Autoantibodies induced by blood
transfusion (editorial). Transfusion 2004:445-9.
Mack P, Freedman J. Autoimmune hemolytic anemia: A history. Transfus Med Rev 2000;14:223-33.
Petz LD, Garratty G. Immune hemolytic anemias.
2nd ed. Philadelphia: Churchill Livingstone, 2004.
Petz LD. A physician’s guide to transfusion in autoimmune haemolytic anaemia. Br J Haematol 2004;
124:712-16.

480

Appendix 20-1. An Example of an Algorithm for Investigating a Positive DAT (Excluding Investigation of HDFN)

AABB Technical Manual

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction

481

Appendix 20-2. Some Drugs Associated with Immune Hemolysis and/or Positive
DATs Due to Drug-Induced Antibodies
Drug

Therapeutic Category

Possible Mechanism

Acetaminophen
Aminopyrine
Amphotericin B
Ampicillin
Antazoline
Apazone (azapropazone)
Buthiazide (butizide)
Carbenicillin
Carbimazole
Carboplatin
Carbromal
Catergen
Cephalosporins
First generation
Second generation
Third generation
Chaparral
Chlorpropamide
Chlorpromazine
Cisplatin
Cladribine
(chlorodeoxyadenosine)
Clavulanate potassium
Cyanidanol
Cyclofenil
Cyclosporine
Diclofenac
Diethylstilbestrol
Diglycoaldehyde
Dipyrone
Elliptinium acetate
Erythromycin
Etodolac
Fenfluramine
Fenoprofen
Fludarabine
Fluorescein
Fluorouracil
Glafenine
Hydralazine
Hydrochlorothiazide
Ibuprofen
Insulin
Interferon
Isoniazid
Levodopa
Mefenamic acid
Mefloquine

Analgesic, antipyretic
Analgesic, antipyretic
Antifungal, antibiotic
Antibacterial
Antihistamine
Anti-inflammatory, analgesic
Diuretic, antihypertensive
Antibacterial
Thyroid inhibitor
Antineoplastic
Sedative; hypnotic
Diarrheal astringent, treatment of hepatic disease
Antibacterials

DD-IC
DD-IC
DD-IC
DD-IC
DD-IC
DI, DD-DA
DD-IC
DD-DA
DD-IC
DD-DA, DD-IC
DD-DA
DI

Antidiabetic
Antipsychotic
Antineoplastic
Antineoplastic
β-lactamase inhibitor/antibacterial
Gonad-stimulating principle
Immunosuppressive
Anti-inflammatory
Estrogen
Antineoplastic
Analgesic, antipyretic
Antineoplastic
Antibacterial
Anti-inflammatory, analgesic
Anorexic
Anti-inflammatory, analgesic
Antineoplastic
Injectable dye
Antineoplastic
Analgesic
Antihypertensive
Diuretic
Anti-inflammatory
Antidiabetic
Antineoplastic, antiviral
Antibacterial, tuberculostatic
Antiparkinsonian, anticholinergic
Anti-inflammatory
Antimalarial

NIA, DD-DA
DD-IC, DD-DA, DI
DD-IC, DD-DA, DI
DI
DD-IC
DI, DD-IC
NIA
DI
NIA
DI, DD-DA, DD-IC
DI
DI
DI, DD-IC
DD-IC
NIA
DD-IC, DD-DA
DD-IC
DD-DA
IC
?
DI, DD-IC
DI
DD-DA, DD-IC
DD-IC
DI, DD-IC
DD-IC
DD-IC
DI
DD-DA?, DD-IC
DI
DD-DA?, DD-IC
DI
DI
DD-IC
(cont’d)

482

AABB Technical Manual

Appendix 20-2. Some Drugs Associated with Immune Hemolysis and/or Positive
DATs Due to Drug-Induced Antibodies (cont’d)
Drug

Therapeutic Category

Possible Mechanism

Melphalan
6-Mercaptopurine
Methadone
Methicillin
Methotrexate
Methyldopa
Moxalactam (latamoxef)
Nafcillin
Nomifensine
p-Aminosalicylic acid
Penicillin G
Phenacetin
Piperacillin
Podophyllotoxin
Probenecid
Procainamide
Propyphenazone
Pyramidon
Quinidine
Quinine
Ranitidine
Rifampin (rifampicin)
Sodium pentothal
Stibophen
Streptomycin
Sulbactam sodium
Sulfonamides
Sulfonylurea derivatives
Sulindac
Suprofen
Suramin
Temafloxacin
Teniposide
Tetracycline
Thiopental
Tolbutamide
Tolmetin
Triamterene
Trimellitic anhydride
Zomepirac

Antineoplastic
Antineoplastic
Narcotic analgesic
Antibacterial
Antineoplastic, antimetabolite
Antihypertensive
Antibacterial
Antibacterial
Antidepressant
Antitubercular
Antibacterial
Analgesic, antipyretic
Antibacterial
Antineoplastic, cathartic
Uricosuric
Cardiac depressant, antiarrhythmic
Analgesic, antipyretic, anti-inflammatory
Analgesic, antipyretic
Cardiac depressant, antiarrhythmic
Antimalarial
Antagonist (to histamine H2 receptors)
Antibacterial, antitubercular
Anesthetic
Antischistosomal
Antibacterial, tuberculostatic
β-lactamase inhibitor/antibacterial
Antibiotics
Antidiabetic
Anti-inflammatory
Anti-inflammatory, analgesic
Antitrypanosomal, antifilarial
Antibacterial
Antineoplastic
Antibacterial, antirickettsial, antiamebic
Anesthetic
Antidiabetic
Anti-inflammatory
Diuretic
Used in preparation of dyes, resins, etc
Analgesic, anti-inflammatory

DD-IC
DD-DA
?
DD-DA
DD-IC
DI
DD-IC, DI
DD-DA
DI, DD-IC
DD-IC
DD-DA
DI, DD-IC
DD-DA, DD-IC
?
DD-IC
DI
DD-IC
DD-IC
DD-DA, DD-IC
DD-IC
?
DD-IC
DD-IC
DD-IC
DI, DD-DA, DD-IC
NIA
DD-IC
DD-IC
DD-DA, DI
DD-IC, DI
NIA
DD-IC
DI, DD-IC
DD-DA?, DD-IC
DD-IC
DD-DA
DI, DD-IC
DD-IC
?
DD-DA, DD-IC, DI

3,44-48

Mechanisms listed are based on descriptions in the literature.
DAT = Direct antiglobulin test.
DD-DA = Drug-dependent. Drug adsorbed onto red cells; antibody reacts with drug on cells.
DD-IC = Drug-dependent. “Immune complex mechanism.” Requires drug, serum, and red cells for serologic
demonstration. For most of these drugs, there are only single or very few case reports.
DI = Drug-independent. Associated with autoantibodies similar to those in AIHA. Drug not required for in-vitro
demonstration. Mechanisms of autoantibody production may vary.
NIA = Nonimmunologic adsorption of proteins.
? = Mechanism unclear or unknown.

Chapter 21: Blood Transfusion Practice

Chapter 21

Blood Transfusion Practice
21

HE DECISION TO transfuse, like
any other therapeutic decision,
should be based on the risks, benefits, and alternatives of treatment. Unfortunately, data regarding the indications
for transfusion are frequently not available
and recipients run the risk of both overtransfusion and undertransfusion. Transfusions based solely on laboratory test
“triggers,” in particular, are problematic.
Consensus statements on the use of blood
components such as those produced by
the National Institutes of Health (NIH)
help guide therapy but cannot substitute
for clinical judgment.

Red Blood Cell Transfusion
Physiologic Principles
The primary indication for transfusion of
Red Blood Cells (RBCs) is to restore or maintain oxygen-carrying capacity to meet tis-

sue demands; transfusion to replace red
cells destined for destruction in hemolytic disease of the newborn is discussed in
Chapter 24. Because demand for oxygen
varies greatly among different individuals
in different clinical circumstances, measurement of only the hematocrit or the
hemoglobin concentration (“the hemoglobin”) cannot accurately assess the need
1-3
for transfusion.

Normal Oxygen Supply and Demand
Tissues at rest have a baseline demand for
oxygen, particularly the heart, kidneys,
brain, liver, and gastrointestinal tract;
consumption by muscle is very low at
rest. The oxygen content of blood (mL
O2/mL blood) is determined by the hemoglobin, the binding coefficient of hemoglobin for oxygen, the oxygen saturation
of hemoglobin, and a small quantity of
483

484

AABB Technical Manual

oxygen dissolved in the plasma. This is
described as:
O2 content = (Hb × 1.39 × %sat)
+ (pO2 × 0.003)
Tissue oxygen consumption is calculated
as the difference between oxygen delivery
in the arterial blood and oxygen return by
the venous blood:
O2 consumption = Cardiac output ×
Hb × 1.39 × (%satarterial – %satvenous)/100
which is expressed as

O2 supply (Calculation assumes a hemoglobin of 140 g/L and a pO2 of 100)
= Cardiac output × O2 contentarterial
= 5 L/minute × [(140 × 1.39 × 100%)
+ (100 × 0.003)]
= 5 L/minute × 200 mL O2/L
= 1000 mL O2/minute
O2 consumption = Cardiac output × (O2
contentarterial – O2 contentvenous)
= 5 L/minute × (200 mL O2/L –
150 mL O2/L)
= 5 L/minute × 50 mL O2/L =
250 mL O2/minute

Compensation for Anemia

(mL O2/minute) =
L/minute × g/L × mL O2/g
The oxygen saturation of arterial and venous hemoglobin varies with the partial
pressure of oxygen. Under normal circumstances the pO2 falls from 100 mm Hg in the
arteries to 40 mm Hg in the veins as the tissues extract oxygen, and hemoglobin saturation falls from near 100% in the arteries to
approximately 75% in the veins; thus, the
oxygen extraction ratio is 0.25. That is, the
hemoglobin “gives up” only 25% of its oxygen. When tissue demand for oxygen increases or the supply of oxygen decreases,
the tissues extract a greater fraction of oxygen from the plasma and from hemoglobin;
this results in a lower venous pO2 and decreased oxygen saturation of the venous
blood. Studies in primates suggest that a
critical point of limited oxygen delivery is
reached when the oxygen extraction ratio
approaches twice normal or 0.50.2
Under normal resting conditions, the
body has a large reserve of oxygen supply
relative to demand. In the average adult,
approximately 1000 mL/minute is available
to the tissues and only 250 mL/minute is
consumed as follows:

The above equations demonstrate that any
decrease in oxygen content due to anemia
can be compensated for by an increase in
cardiac output.1,2 This occurs because of
increased cardiac work and also because
anemia decreases blood viscosity, and
thus peripheral vascular resistance. The
increase in oxygen supply provided by increased cardiac output is augmented by
increased oxygen extraction. Oxygen extraction is augmented acutely by a decrease in tissue oxygen tension, acting at
the steep portion of the hemoglobin-oxygen dissociation curve1 (see Fig 8-1), and
by acidosis, which promotes oxygen dissociation from hemoglobin. Over time, an
increase in red cell 2,3-diphosphoglycerate (2,3-DPG) concentration also has a
significant positive effect on oxygen unloading.

Measuring the Adequacy of Oxygen Supply
As shown above, multiple factors determine oxygen delivery to the tissues, except at the lower extremes. Therefore,
measurements in addition to the hemoglobin must be used to guide most transfusion decisions. The adequacy of the
oxygen supply depends on the partial

Chapter 21: Blood Transfusion Practice

485

pressure of inspired oxygen, gas exchange
in the lungs, the patient’s cardiac performance, hemoglobin, oxygen-hemoglobin
affinity, and current oxygen demand; all
but the oxygen-hemoglobin affinity are
subject to substantial variation. For patients in an intensive care unit or in the
operating room, direct measurement of
the cardiac output and mixed venous oxygen tension, in association with hemoglobin level, may serve as more physiologic
guides for transfusion decisions than hemoglobin alone. Nonetheless, most such
decisions will continue to be made based
on hemoglobin and standard clinical assessment.
Determinants of cardiac performance
include the patient’s intravascular volume,
the anemia-related reduction in peripheral
vascular resistance, the presence of coronary artery disease or other forms of heart
disease, and the patient’s age. Tissue oxygen debt results when oxygen demand exceeds supply; tissues convert to anaerobic
metabolism and produce increased quantities of lactic acid. Metabolic acidosis, in
turn, impairs cardiac performance, further
decreasing perfusion and tissue oxygen delivery, leading to greater tissue hypoxia in a
vicious cycle.

Assuming that oxygen extraction remains
constant, oxygen delivery could be normalized by an increase in the hemoglobin concentration to 9 g/dL, or by an increase in
the cardiac index to 7 L/minute/m 2. A
smaller increase in hemoglobin would
blunt the required increase in cardiac index.

Treating Inadequate Oxygen Supply

Red Blood Cells

RBC transfusion is the most direct means
of raising the hemoglobin concentration.
Other ways to improve oxygen supply relative to demand include increasing tissue
perfusion (maximizing cardiac performance), increasing oxygen saturation
with supplemental oxygen or mechanical
ventilation, and decreasing tissue oxygen
demands with bed rest, antipyretics, and
avoidance of hypertension.
For example, a patient with a hemoglobin of 6 g/dL and a cardiac index of 5 L/
2
minute/m has decreased oxygen delivery.

Red cell components are indicated for the
treatment of patients who require an increase in oxygen-carrying capacity and
red cell mass.5 The transfusion of red cells
increases oxygen-carrying capacity with
less expansion of blood volume per unit
than Whole Blood. This is important for
patients who are at risk for circulatory
overload (eg, neonates or patients with
congestive heart failure). In a typical adult
in the absence of bleeding, hemolysis, or
major fluid shifts, one RBC unit is expected to raise the hemoglobin concen-

Red-Cell-Containing Components

Whole Blood
Whole Blood provides oxygen-carrying
capacity, stable coagulation factors (the
concentrations of Factors V and VIII decrease during storage), and blood volume
expansion. Thus, it is useful for patients
with concomitant red cell and volume
deficits, such as actively bleeding patients, and will help support coagulation
in appropriate clinical settings such as
liver transplantation.4 In fact, Whole Blood
is rarely available for allogeneic transfusion; RBCs and asanguinous solutions
have become the standard for most cases
of active bleeding in trauma and surgery,
with supplementation of hemostatic elements as needed. The major use of Whole
Blood in the United States today is for
autologous transfusion (see Chapter 5).

486

AABB Technical Manual

tration by approximately 1 g/dL, or the
hematocrit by 3%.

lutions take precedence over restoration
of oxygen-carrying capacity and should
be started immediately. In situations of
acute bleeding, guidelines suggest transfusing patients who have lost 30% to 40%
of their blood volume in conjunction with
other measures to correct and maintain
total blood volume.3,6 Patients with cardiac or other disease may require replacement sooner. Healthy resting adults have
been demonstrated to tolerate acute isovolemic hemodilution to hemoglobin
concentrations as low as 5 g/dL without
demonstrating evidence of inadequate
10
oxygenation. In patients who refuse
transfusion on religious grounds and undergo surgery, mortality increases progressively below postoperative hemoglobin levels of 5 or 6 g/dL, particularly for
11
those with cardiovascular disease.
Perioperative transfusion accounts for
12
55% to 65% of red cell component use.
Randomized trials have demonstrated the
safety of a “transfusion trigger” of 8 g/dL of
hemoglobin in patients undergoing cardiovascular surgery,13,14 orthopedic surgery,15
16
and acute gastrointestinal bleeding. Even
before most of this evidence was available,
an NIH consensus conference on periopera7
tive red cell transfusion emphasized that a
hemoglobin of 10 g/dL was inappropriate
as a guideline or transfusion trigger in the
perioperative setting and suggested a he-

Selection of Whole Blood and Red Cell
Components
Whole Blood must be ABO identical. RBC
units need not be ABO identical but must
be compatible with ABO antibodies in the
recipient’s plasma. Rh-negative recipients
should receive Rh-negative Whole Blood
or red cell components. In cases of
trauma or massive transfusion, it may be
necessary to use Rh-positive components,
as discussed below. See Table 21-1 for
blood group selection in red cell transfusion. Selection of RBC units for patients
with blood group alloantibodies is discussed in Chapter 19.

Indications for Transfusion
Several organizations have published guide3,6,7
lines for RBC transfusion.
Reviews of
indications for transfusion are also available.1,8,9

Blood Loss and Perioperative Transfusion
For actively bleeding patients, the goal of
initial treatment should be to prevent the
development of hypovolemic shock by
stopping the bleeding and restoring intravascular volume. Efforts to restore volume
by the infusion of crystalloid or colloid so-

Table 21-1. Suggested ABO Group Selection Order for Transfusion of RBCs
Component ABO Group
Recipient
ABO Group

1st Choice

2nd Choice

3rd Choice

4th Choice

AB
A
B
O

A
O
O

Chapter 21: Blood Transfusion Practice

moglobin of 7 g/dL as a level at which
transfusion was frequently required in otherwise healthy individuals with acute anemia. Factors to be considered in making an
individual transfusion decision included
“the duration of the anemia, the intravascular volume, the extent of the operation,
the probability of massive blood loss, and
the presence of coexisting conditions such
as impaired pulmonary function, inadequate cardiac output, myocardial ischemia,
or cerebrovascular or peripheral circulatory
disease.”7 The guidelines also emphasized
that transfusion does not improve wound
healing, which depends on pO2 rather than
total oxygen content of the blood.
A guideline by an American Society of
3
Anesthesiologists task force cited a hemoglobin below 6 g/dL as “almost always” indicating transfusion, a hemoglobin above
10 g/dL as rarely indicating transfusion,
and the range in between as the realm of
clinical judgment. A task force of the College of American Pathologists reached a
similar conclusion and proposed several
objective measures that might indicate red
cell transfusion for hemoglobin levels in the
range of 6 to 10 g/dL, including tachycardia
or hypotension in the face of normovolemia, a mixed venous pO2 of <25 torr, an oxygen extraction ratio >50%, or a total oxygen consumption of <50% of baseline.6
In the past, there was some concern that
transfusions of a single RBC unit were likely
to represent unnecessary intervention. However, if transfusion of a single unit will
achieve the desired clinical outcome, then
only one unit should be transfused. Transfusing additional units in this setting will
increase the risk of transfusion without any
additional benefit.

Anemia
Among medical patients, those with cardiovascular and malignant diseases ac-

487

count for a large proportion of those re13
ceiving RBC units. In a prospective randomized trial of red cell transfusion, anem i c b u t e u vo l e m i c p a t i e n t s i n t h e
intensive care unit (ICU) were assigned to
either “restrictive” or “liberal” transfusion
regimens that maintained the hemoglobin between 7 and 9 g/dL or between 10
17
and 12 g/dL, respectively. The mortality
rate during hospitalization (but not at 30
days) was significantly lower in the restrictive-strategy group (22.3% vs 28.1%, p
= 0.05). No difference in mortality rate
was seen among all patients with clinically significant cardiac disease. One third
of the restrictive group avoided transfusion, and total red cell use was half that of
the liberal group. These authors concluded that a restrictive strategy of red
cell transfusion is at least as effective as,
and possibly superior to, a liberal transfusion strategy in critically ill patients, with
the possible exception of the subset of patients with acute myocardial infarction
(MI) and unstable angina. Reanalysis of
the patients in this study with cardiovascular disease showed a trend, albeit not
statistically significant, toward increased
mortality in the restrictive group among
18
patients with MI and unstable angina.
A retrospective study of a large number
of elderly (>65 years old) patients hospitalized with acute MI divided into groups according to admission hematocrit compared
30-day mortality rates in patients who received transfusion and those who did not.19
Transfusion appeared beneficial in patients
with a hematocrit <30%. This result persisted when data were adjusted for multiple
clinical and institutional factors.
Patients with chronic anemia tolerate a
low hemoglobin better than those with
acute anemia because of cardiovascular
compensation and increased oxygen extraction. Moreover, patients at bed rest
who are not febrile, who do not have con-

488

AABB Technical Manual

gestive heart failure, and who are not
hypermetabolic have low oxygen requirements and may tolerate anemia remarkably well. However, the high oxygen needs
of cardiac muscle may precipitate angina
in patients with cardiac disease and anemia. A hemoglobin concentration of 8 g/
dL adequately meets the oxygen needs of
most patients with stable cardiovascular
disease.
Although it is desirable to prevent unnecessary transfusions, anemic patients
who are symptomatic should receive appropriate treatment. Anemia may cause
symptoms of generalized weakness, headache, dizziness, disorientation, breathlessness, palpitations, or chest pain, and signs
include pallor (not cyanosis) and tachycardia. Elwood and coworkers20 could not correlate symptoms with the hemoglobin level
in patients with chronic iron deficiency
anemia and a hemoglobin as low as 8 g/dL.
A study of the use of erythropoietin demonstrated improvement in symptoms as hemoglobin is raised to 10 g/dL, but no
change above that level.21 Patients with
chronic hypoproliferative anemia who are
known to be transfusion dependent should
be maintained at a level that prevents
symptoms by establishing a transfusion
schedule and then adjusting it as needed.

Platelet Transfusion
Physiologic Principles
Hemostasis occurs in four phases: the
vascular phase, the formation of a platelet
plug, the development of fibrin clot on
the platelet plug, and the ultimate lysis of
the clot. Platelets are essential to the formation of the primary hemostatic plug
and provide the surface upon which fibrin
forms. Deficiencies in platelet number
and/or function can have unpredictable
effects that range from clinically insignifi-

cant prolongation of the bleeding time to
major life-threatening hemorrhage. Platelet plug formation results from the combined processes of adhesion, activation and
release, aggregation, and procoagulant
activity.22 Platelet adhesion to damaged
endothelium is mediated largely by the
von Willebrand factor (vWF), which binds
to the surface glycoprotein (GP) receptor
GPIb-IX-V complex. The process of activation and release causes a dramatic
change in platelet shape, with extension
of pseudopod-like structures, a change in
the binding properties of membrane activation proteins, secretion of internal granule contents, and activation of several
metabolic pathways. These changes have
many effects, including the recruitment of
additional platelets, which aggregate with
the help of fibrinogen or vWF binding to
platelet surface glycoproteins. Finally, the
platelet membrane procoagulant activity
localizes and directs formation of fibrin.

Assessing Platelet Function
Decreased platelet numbers may result
from decreased production, increased destruction, or splenic sequestration. Platelet function may be adversely affected by
such factors as drugs, liver or kidney disease, sepsis, fibrin(ogen) degradation
products, cardiopulmonary bypass, and
primary marrow disorders. Platelet hemostasis is assessed by the medical history,
physical examination, and laboratory
tests including platelet count and bleeding time or in-vitro platelet function assays (eg, PFA100). Patients with inadequate
platelet number or function may demonstrate petechiae, easy bruising, mucous
membrane bleeding, nose and gum bleeding, and hematuria. Preprocedure platelet
counts have predicted bleeding in some
23,24
25-27
studies but not in others.

Chapter 21: Blood Transfusion Practice

Bleeding time measures both the vascular phase and the platelet phase of hemostasis. Although the bleeding time may be a
useful diagnostic test in the evaluation of
patients with known or suspected abnormalities of platelet function, it is a poor pre28
dictor of surgical bleeding and is not a reliable indicator of the need for platelet
transfusion therapy.29 The in-vitro measurement of platelet function is useful but, like
the bleeding time, is a poor predictor of
bleeding.30

489

Chapter 16. Two consecutive poor responses suggest platelet refractoriness.

Platelet Components

Platelets
A single unit of Platelets prepared from an
individual unit of Whole Blood may be
adequate for transfusion to neonates or
infants, but, for adults, 4 to 6 units are ordinarily pooled for transfusion to achieve
a dose greater than 3.0 × 1011 platelets.
This should increase the platelet count by
30,000 to 60,000/µL.

Platelet Life Span and Kinetics

Platelets Pheresis

Platelets normally circulate with a life span
31
of 10.5 days, and platelets that have been
properly collected and stored have a near
normal residual mean life span of 4 to 5
days when reinfused into the original donor. Conditions that shorten platelet life
span include splenomegaly, sepsis, drugs,
disseminated intravascular coagulation
(DIC), auto- and alloantibodies, endothelial cell activation, and platelet activation
(eg, cardiopulmonary bypass or intra-aortic balloon pumps). Because a relatively
constant number of platelets (7,00010,000/µL/day) are consumed by routine
plugging of minor endothelial defects, the
fraction of the circulating platelet pool required for maintenance functions increases
as the total number of platelets declines.
Therefore, the life span of native and
transfused platelets decreases with progressive thrombocytopenia. 3 1 The response to platelet transfusion is best assessed by observing whether bleeding
stops and by measuring the posttransfusion platelet increment. The posttransfusion increment is generally measured
between 10 minutes and 1 hour after
completion of the transfusion and is expressed as a corrected count increment
(CCI) or percent recovery, as outlined in

Units of platelets prepared by apheresis
technology (“single-donor platelets”) have
a platelet content similar to that of pooled
platelets from four to six donors and, depending on the equipment used, may
have a reduced leukocyte content. The
fact that a single such unit can provide an
entire transfusion facilitates provision of
compatible platelets to recipients who are
refractory because of alloimmunization.
Transfusion of recipients who are refractory to platelet transfusions is discussed
in Chapter 16.

Selection of Platelets

ABO Matching
Because ABO antigens are present on the
platelet surface, recovery of group A platelets transfused into group O patients is
somewhat decreased,32 but this effect is
not usually clinically significant (see
Chapter 16). Transfusion of ABO-incompatible plasma present in platelet components may also result in a blunted post33
transfusion platelet count increment.
Hemolysis occurs rarely in this setting but
it frequently causes a positive direct
antiglobulin test (DAT), which may increase costs and charges to the patient for

490

AABB Technical Manual

serologic investigation. Moreover, a retrospective analysis suggested that survival
after marrow transplantation was significantly reduced in patients who received
substantial amounts of ABO-incompatible plasma from platelet transfusion, 34
and it has been suggested that infusion of
soluble A and B antigen in platelet or
plasma components may have a similar
adverse effect mediated by immune complex function.35 Therefore, it may be prudent to use ABO-matched platelets, particularly for patients requiring repeated
transfusions. However, urgently needed
transfusions should not be delayed in order to obtain them.
For infants, it is desirable to avoid administration of plasma that is incompatible
with the infant’s red cells; if platelets containing compatible plasma are not available, the plasma can be reduced (see
Method 6.15). This is rarely necessary in
adults or older children, although significant hemolysis has been reported after
transfusion of group O Platelets Pheresis
with high-titer anti-A or B.36 If transfused
ABO antibodies are detectable in the recipient, it may become necessary to use group
O RBC units.

Matching for Rh
The D antigen is not detectable on platelets, and posttransfusion survival of platelets from Rh-positive donors is normal in
recipients with anti-D. However, platelet
components contain small numbers of
red cells so Rh-negative individuals may
become alloimmunized by platelet components from Rh-positive donors. For immunocompetent normal Rh-negative females of childbearing potential, it is
especially desirable to avoid administration of platelets from Rh-positive donors;
however, if this is unavoidable, Rh Immune Globulin (RhIG) should be admin-

istered. If hematoma formation is an issue, an intravenous form of RhIG is available. A full dose of RhIG, which is considered immunoprophylactic for up to 15 mL
of Rh-positive red cells, should protect
against the red cells in a minimum of 30
units of Rh-positive Platelets or 7 units of
Rh-positive Platelets Pheresis.

Therapeutic Platelet Transfusion
Significant bleeding due to thrombocytopenia or abnormal platelet function is
an indication for “therapeutic” platelet
transfusion.37 The decision to transfuse
platelets depends on the cause of bleeding, the patient’s clinical condition, and
the number and function of the circulating platelets.3,38-40 Platelet transfusions are
most likely to be of benefit when thrombocytopenia is the primary hemostatic defect. The goal is to maintain counts
>50,000/µL. Other blood components
may also be required in patients with
multiple defects. Bleeding due to the defects in platelet function that follow
cardiopulmonary bypass surgery or to the
ingestion of aspirin-containing compounds, glycoprotein IIb/IIIa antagonists
(eg, abciximab), and P2 inhibitors (eg,
clopidigrel and ticlopidine) often responds to platelet transfusion. Other defects such as those found in uremia or von
Willebrand disease respond less well because the transfused platelets tend to acquire the same defect.

Prophylactic Platelet Transfusion
Indications for prophylactic platelet
transfusion are more controversial than
for therapeutic rationales. A threshold of
20,000/µL or less for patients with chemotherapy-induced thrombocytopenia has
been used by many physicians, but prospective randomized trials have shown
that a threshold of 10,000/µL in stable pa-

Chapter 21: Blood Transfusion Practice

tients is equally safe and results in significant decreases in platelet usage. 41-43 A
higher transfusion trigger is often used for
patients with fever, evidence of rapid consumption, high white cell counts, coagu38
lation defects, and intracranial lesions.
In contrast, many stable thrombocytopenic patients can tolerate platelet counts
as low as 5000/µL.41
Despite the widespread use of prophylactic platelet transfusions, few studies have
documented their clinical benefit. One
study comparing patients given prophylactic transfusions with patients transfused
only for clinically significant bleeding demonstrated a significant decrease in bleeding, but the number of patients in the study
was too small to show a difference between
the groups in overall survival or in deaths
44
due to bleeding. Of interest, the prophylactic group received twice as many platelet
transfusions, and there was a suggestion
that refractoriness developed more often in
this group. This observation raises the caveat that prophylactic platelet transfusion
may be most relevant to patients in whom
thrombocytopenia is expected to be a temporary condition.38 If thrombocytopenia or
platelet dysfunction will be prolonged, the
development of refractoriness may limit the
response to platelets, particularly if immune function is normal as it is in patients
with aplastic anemia or congenital thrombocytopathies.
Patients with severe preoperative thrombocytopenia are generally assumed to benefit
from prophylactic platelet transfusion, but
this has not been demonstrated in experimental studies. The threshold for such prophylaxis is typically set at platelet counts
3
between 50,000 and 100,000/µL. Prophylactic transfusion of platelets has been investigated in circumstances in which
thrombocytopenia is expected to develop
intraoperatively, either because of dilution45
46
or cardiopulmonary bypass ; in both cases,

491

prophylaxis was ineffective. Although it endorsed the logic of prophylactic platelet
transfusion for thrombocytopenic patients
undergoing surgery, the NIH consensus
38
panel suggested that such transfusions
were most appropriate for patients in
whom hemorrhage could not be observed
or in whom it occurred at a site where it
could be critical in small amounts (eg, in
the central nervous system). Published
guidelines3,39 suggest a platelet transfusion
trigger of 50,000/µL for most major surgery,
with counts “near” 100,000/µL possibly required for patients undergoing neurosurgery or ophthalmic procedures.39 Prophylactic platelet transfusion may also be
useful for patients who are having surgery
39
and who have a platelet function defect,
including that due to treatment with
47
abciximab.

Refractoriness to Platelet Transfusion
Platelet refractoriness, defined as a poor
increment following a dose of platelets, can
result from either immune or nonimmune
mechanisms and is discussed in detail in
Chapter 16 and other reviews.48,49 The antibodies that cause immune refractoriness
may have either allo- or autoreactivity, with
alloantibodies most commonly directed
against Class I HLA antigens. Autoantibodies
occur in immune thrombocytopenic purpura (ITP) (see Chapter 16). Nonimmune
causes of the refractory state include infection, splenomegaly, drugs (particularly
amphotericin B), and accelerated platelet
consumption (see Table 16-3).

Contraindications to Platelet Transfusion
There are several conditions for which
platelet transfusions may be requested
but are contraindicated. Relative contraindications include conditions in which
the likelihood of benefit is remote; transfusion in this setting merely wastes a valu-

492

AABB Technical Manual

able component. Examples include prophylactic platelet transfusions in stable
patients with ITP50 or platelet refractoriness.
For ITP patients undergoing splenectomy,
transfusion of platelets should be delayed
until the vascular pedicle is clamped.
Platelet transfusion should be avoided for
patients with thrombotic thrombocytopenic purpura (TTP) or active heparin-induced thrombocytopenia except in life- or
organ-threatening hemorrhage. These
conditions are associated with platelet
thrombi, and major thrombotic complications may follow platelet transfusions.51

Granulocyte Transfusion
The use of granulocyte transfusions for
adult recipients is rare. New antibiotics,
adverse effects attributable to granulocyte
transfusions, the advent of recombinant
growth factors, and difficulty demonstrating efficacy have contributed to this decline. Nevertheless, in selected patients,
transfused granulocytes may produce
clinical benefits,52 particularly with the
larger granulocyte doses available from
donors treated with granulocyte colonystimulating factor. 53 Attention to HLA
compatibility is also required for alloimmunized recipients.54 The preparation,
storage, and pretransfusion testing of
granulocytes are discussed in Chapter 6,
and their use in neonates is discussed in
Chapter 24.

Indications and Contraindications
The goals of granulocyte transfusion should
be clearly defined before a course of therapy is initiated. In general, the patient
should meet the following minimum conditions:
1.
Neutropenia (granulocyte count less
than 500/µL).

Fever for 24 to 48 hours, positive
bacterial or fungal blood cultures, or
progressive parenchymal infection
unresponsive to appropriate antibiotic therapy.
3.
Myeloid hypoplasia.
4.
A reasonable chance for recovery of
marrow function.
Patients with documented granulocyte
dysfunction, such as those with chronic
granulomatous disease, may also be candidates to receive granulocyte transfusions
during life-threatening episodes of infection or while awaiting hematopoietic progenitor cell transplantation.

Other Considerations
Granulocyte components contain significant amounts of red cells, which must be
crossmatch compatible and Rh specific,
particularly for females with childbearing
potential. Granulocytes should be irradiated to avoid the risk of graft-vs-host disease (GVHD). If cytomegalovirus (CMV)
transmission is an issue, its risk can be reduced by the use of a CMV-seronegative
donor (leukocyte reduction filters are
contraindicated). For alloimmunized recipients, donors should be matched by HLA
typing or leukocyte crossmatching.52,54

Special Cellular Blood
Components
Leukocyte Reduction
The approximate leukocyte content of common blood components is summarized in
Table 21-2.55-59 Leukocyte reduction has
been used for some time for select groups
of patients. Current federal guidelines57,58
and AABB Standards for Blood Banks and
59(pp25,28,29,31,32)
Transfusion Services
define a
leukocyte-reduced component as one
with <5 × 106 residual donor leukocytes

Chapter 21: Blood Transfusion Practice

Table 21-2. Approximate Leukocyte
Content of Blood Components (per
55-59
Unit)
Whole Blood

109

RBCs

108

RBCs Washed

107

RBCs Deglycerolized

106-107

RBCs Leukocytes Reduced
(by filtration)*

<5 × 106

Platelets Pheresis

106-108

Platelets

107

Platelets Pheresis
Leukocytes Reduced*

<5 × 106

Platelets Leukocytes Reduced

<8.3 × 105

Platelets Pooled
Leukocytes Reduced

<5 × 106

FFP Thawed

<0.6 × 106 1.5 × 107

*Leukocyte reduction with third-generation leukocyte adsorption filter.

per final product (this includes RBCs;
Platelets Pheresis; and pooled Platelets).
59(p31)
5
requires <8.3 × 10
AABB Standards
leukocytes in Platelets Leukocytes Reduced, which are prepared from a single
unit of Whole Blood, to achieve the requirement for a pool of 6 platelet units.
Draft guidance from the Food and Drug
Administration (FDA) recommends quality control to indicate with 95% confidence that more than 95% of blood units
meet these criteria. By comparison, European guidelines define leukocyte-reduced
6
components as those with <1 × 10 residual leukocytes per unit and require that
there should be no more than a 10% failure rate in the process.
Published data demonstrate that leukocyte reduction reduces the risk of:
1.
Febrile nonhemolytic reactions (see
Chapter 27).

493

CMV transmission.60
HLA alloimmunization that may lead
to patients becoming refractory to
platelet transfusions.61
Controversial and unproven indications
for leukocyte reduction include:
1.
Reduction of immunomodulation that
may lead to an increased risk of cancer recurrence or bacterial infections.62
2.
Reduction in the risk of prion disease.
3.
Reduction in the risk of Yersinia enterocolitica contamination of RBC units.63
Many studies have investigated the possibility that leukocyte reduction can reduce
the incidence of clinical outcomes due to
transfusion-related immunomodulation,
but the results are contradictory.62 One such
proposal was that savings related to reduced immunomodulation could offset the
costs of leukocyte reduction, but this was
not demonstrable in a large prospective
randomized trial.64 Nonetheless, several
countries have converted to a leukocyte-reduced blood supply, and this subject remains controversial.65
2.
3.

Irradiation
Irradiation of a cellular blood component
is the only accepted method to prevent
GVHD. GVHD has been reported after
transfusion of leukocyte-reduced components.66 For more details on GVHD and indications for irradiation, see Chapter 27.

Replacement of Coagulation
Factors
Physiologic Principles
Coagulation results from a complex but
ordered enzyme cascade occurring on the
surface of platelets and cells that express
tissue factor (see Fig 21-1). The coagulation cascade is typically divided into the
intrinsic and the extrinsic pathways, the

494

AABB Technical Manual

Roberts HR, Monroe DM III, Hoffman M. Molecular biology and biochemistry of the coagulation factors and pathways of
hemostasis. In: Beutler E, Lichtman MA, Coller BS, et al. Williams’ hematology. 6th ed. New York: McGraw-Hill, 2001:1409-34.

Figure 21-1. (A) The classic cascade model of coagulation reactions was based on in-vitro experimental data in cell-free systems. The term extrinsic reflects the fact that tissue factor does not circulate in
plasma. (B) More recent evidence emphasizes that the coagulation reactions occur on the surfaces of
tissue factor-bearing cells at the site of injury and on the surface of platelets that are subsequently recruited. HK = high-molecular-weight kininogen, PK = prekallikrein, TF = tissue factor, TFPI = tissue
factor pathway inhibitor. (Adapted with permission from Roberts et al.67 )

in-vitro activity of which can be measured
by the activated partial thromboplastin
time (aPTT) and prothrombin time (PT),
respectively, but, in vivo, the cascades are
interdependent.67 The central procoagulant enzyme is thrombin, which is activated by both pathways.
Minimal levels of coagulation factors (see
Table 21-3) are required for normal formation of fibrin and hemostasis, so normal
plasma contains coagulation factors in excess, a reserve that usually allows patients
to tolerate replacement of one or more
blood volumes of red cells and crystalloid
without needing Fresh Frozen Plasma (FFP).
Patients with liver disease have less physiologic reserve and are more susceptible to
dilutional coagulopathy.

Monitoring Hemostasis
The PT, aPT T, and measurement of
fibrinogen level are commonly used to
monitor coagulation. Results should be
interpreted with four considerations in
mind: 1) mild prolongations of the PT or
aPTT occur before the residual factor concentration falls below the level normally
needed for hemostasis; 2) conversely, the
PT and aPTT are relatively insensitive to
low fibrinogen levels; 3) significant deficiencies of coagulation factors (or the
presence of coagulation factor inhibitors)
cause clearly prolonged values for the PT
or aPTT; and 4) an infusion of FFP that increases the concentration of factors by 20%
will have a far greater impact on a greatly

Table 21-3. Coagulation Factors

Factor Name

X
XI
XIII
AT

Fibrinogen
Prothrombin
Labile factor, Proaccelerin
Stable factor, Proconvertin
Antihemophilic factor
Plasma thromboplastin
component, Christmas factor
Stuart-Prower factor
Plasma thromboplastin
antecedent (PTA)
Fibrin stabilizing factor
Antithrombin

In-vitro, 4 C
Half-life

% of Normal
Needed for
Hemostasis

% In-vivo
Recovery

3-6 days
2-5 days
4.5-36 hours
2-5 hours
8-12 hours
18-24 hours

Years
>21 days
10-14 days
>21 days
7 days
>21 days

12-50
10-25
10-30
>10
30-40
15-40

50-70
50
~80
100
60-70
20

1 bag cryoprecipitate/7 kg body weight
10-20 units/kg body weight
10-20 mL plasma/kg body weight
10-20 units/kg body weight
See Table 21-6
See Table 21-6

20-42 hours
40-80 hours

>21 days
>28 days

10-40
20-30

50-95
90

10-20 units/kg body weight
10-20 mL/kg body weight

12 days
60-90 hours

>21 days
>42 days

<5
80-120

50-100
50-100

500 mL plasma every 3 weeks
40-50 IU/kg body weight

Initial Therapeutic Dose

Notes:
1. All dosings are provided as a general guideline for initial therapy; the exact loading dose and maintenance intervals should be individualized for each patient.
2. One unit of coagulation factor is present in each mL of Fresh Frozen Plasma.
3. DDAVP is the treatment of choice for patients with hemophilia A who are responders.
4. Composite data from the following references:
a. Beutler E, Lichtman MA, Coller BS, Kipps TL, eds. Williams’ hematology. 5th ed. New York: McGraw-Hill, 1995:1413-58, 1657.
b. Mollison PL, Engelfriet CP, Contreras M. Blood transfusion in clinical medicine. 10th ed. Oxford: Blackwell Scientific Publications, 1997:459-88.
c. Huestis DW, Bove JR, Case J, eds. Practical blood transfusion. 4th ed. Boston, MA: Little Brown and Co, 1988:319.
d. Counts RB, Haisch C, Simon TL, et al. Hemostasis in massively transfused trauma patients. Ann Surg 1979;190:91-9.
e. Package inserts.

Chapter 21: Blood Transfusion Practice

I
II
V
VII
VIII
IX

In-vivo
Half-life

495

496

AABB Technical Manual

prolonged PT or aPTT than on a mildly
prolonged PT or aPTT. For example, the
infusion of two units of FFP in a patient
with a PT of 14.5 seconds (normal: 11 to
13 seconds) is unlikely to provide any clinical benefit and is also unlikely to correct
the PT to the normal range.
Guidelines typically cite a PT 1.5 times
3
the upper limit of normal or the midpoint
of the normal range39 and an aPTT 1.5 times
the upper limit of normal3,39 as thresholds at
which therapeutic or prophylactic replacement may be indicated in an appropriate
clinical setting. Of note, however, studies
have consistently shown that the PT and
aPTT, even when elevated to this degree,
have little predictive value for bleeding
complications of invasive procedures including paracentesis or thoracentesis,25 liver
biopsy,26,27 angiography,23 or central venous
catheter placement.24

Components and Products Available for
Coagulation Factor Replacement
The plasma components that are available differ according to the timing of
freezing and/or thawing and variations in
preparation (see Chapter 8). FFP contains
all the clotting factors, including labile
Factors V and VIII. Other forms of plasma
have lower levels of labile factors but
could substitute for FFP for most of the
indications for which the latter is transfused. Plasma Cryoprecipitate Reduced
has a decreased content of vWF and is
used specifically for treatment of TTP (see
Chapter 6). Pooled Plasma, Solvent/Detergent-Treated is no longer available in
the United States.
Cryoprecipitated AHF (CRYO) is a concentrate of high-molecular-weight plasma
proteins that precipitate in the cold, including vWF, Factor VIII, fibrinogen, Factor XIII,
and fibronectin (see Chapter 8).

Plasma derivatives are concentrates of
specific plasma proteins from large pools of
plasma or cryoprecipitate. Cohn fractionation, which relies on the precipitation of
various plasma proteins in cold ethanolwater mixtures, was developed during
World War II and is still used with some
modifications.68 After fractionation, derivatives undergo further processing to purify
and concentrate the proteins and inactivate
contaminating viruses. Virus-inactivation
procedures include heat treatment, microfiltration, the use of chemical solvents and
detergents, and affinity column purification. Factors VIII, IX, VIIa, and antithrombin are also produced by recombinant DNA technology. These products
appear to be efficacious and are not known
to carry any infectious risk.
The specific activity (factor units/mg
protein) of presently available concentrates
has been dramatically increased in concentrates prepared with affinity columns or by
recombinant technology. Moreover, HIV,
HBV, and HCV transmission appear to be
absent in patients with hemophilia treated
exclusively with new preparations.69 Unfortunately, their cost has also increased due
to the increased complexity of manufacturing and the protein losses resulting from
extensive manipulation. Coagulation factor
concentrates are supplied in lyophilized
form and the factor activity is stated on the
label.69 Currently available products for replacement of Factors VIII and IX are listed
in Table 21-4.

Selection of ABO-Compatible Plasma
Because of its long shelf life, group-specific or compatible plasma (see Table
21-5) is typically available. (Note that
platelet transfusions usually contain a
volume of plasma equivalent to one unit
of FFP, and limitations in availability may

Chapter 21: Blood Transfusion Practice

497

Table 21-4. Available Coagulation Factor Concentrates
Product Type

Product

Approved Indications

Comment

Factor VIII, fractionated

Humate-P
Koate-HP
Koate-DVI
Alphanate

Factor VIII and vWF repl.
Factor VIII replacement
Factor VIII replacement
Factor VIII replacement

Contains vWF
Contains vWF
Contains vWF
Contains vWF

Factor VIII, affinity purified
Factor VIII, immunaffinity Hemofil-M
purified
Monarc M
Monoclate-P
Factor VIII, recombinant Kogenate FS
Helixate FS
Recombinate
(Bioclate)
ReFacto

Factor VIII replacement
Factor VIII replacement
Factor VIII replacement
Factor VIII replacement
Factor VIII replacement
Factor VIII replacement

Factor VIII, porcine
Factor IX, affinity
purified
Factor IX, immunaffinity
purified
Factor IX, recombinant

Hyate C
AlphaNine-SD

Factor VIII inhibitor tx.
Factor IX replacement

Mononine

Factor IX replacement

BeneFIX

Factor IX replacement

Factor IX complex

Bebulin VH

Factor IX replacement

Konyne 80

Factor IX replacement
Factor VIII inhibitor tx.
Warfarin reversal
Factor IX replacement

Profilnine SD

Proplex T

Factor IX complex,
activated

Factor VIIa,
recombinant

Contains trace amounts
of human albumin

Autoplex-T

Factor IX and Factor VII
replacement
Factor VIII inhibitor tx.
Factor VIII inhibitor tx.

FEIBA VH

Factor VIII inhibitor tx.

NovoSeven

Factor VIII inhibitor tx.

Does not contain human
albumin

Does not contain human
albumin
Contains Factor II,
Factor X, trace
Factor VII
Contains Factor II,
Factor X, some
Factor VII
Contains Factor II,
Factor X, some
Factor VII
Contains Factor II,
Factor VII, and
Factor X
Contains Factor lIa,
Factor VIIa, and
Factor Xa
Contains Factor IIa,
Factor VIIa, and
Factor Xa
Does not contain human
albumin

498

AABB Technical Manual

Table 21-5. Suggested ABO Group Selection Order for Transfusion of Plasma
Component ABO Group

Recipient
ABO Group

1st Choice

2nd Choice

3rd Choice

4th Choice

AB
A
B
O

(A)
AB
AB
A

(B)
(B)
(A)
B

(O)
(O)
(O)
AB

(Blood groups in parentheses represent choices with incompatible plasma, listed in “least incompatible” order.)

require infusion of incompatible plasma
in this context.)

Indications for FFP
Guidelines exist for the appropriate use of
FFP.3,39,70 FFP is the only approved component for clinically significant deficiency of
Factors II, V, X, and XI. Plasma is most often used in patients with multiple factor
deficiencies, including those with liver
disease, dilutional and consumption
coagulopathy, or a need for rapid reversal
of warfarin treatment. It is of limited clinical benefit in patients with inhibitors to
any coagulation factor. Plasma Cryoprecipitate Reduced may be more effective
than FFP for some patients receiving
plasma exchange treatment for TTP or
hemolytic-uremic syndrome71 (see Chapter 6).

Vitamin K Deficiency and Warfarin Reversal
The most common cause of multiple coagulation factor abnormalities among
hospitalized patients is deficiency of the
vitamin-K-dependent factors due to treatment with the vitamin K antagonist
warfarin (Coumadin) or nutritional defi67
ciency. Vitamin K is a fat-soluble vitamin
required for hepatocellular synthesis of
coagulation Factors II, VII, IX, and X, as
well as the anticoagulant proteins C and
S. Body stores of vitamin K last only 2

weeks, so deficiency may occur in hospitalized patients unable to tolerate normal
food intake. Absorption of vitamin K requires precursor metabolism by bacteria
in the intestine and the action of bile salts;
therefore, deficiencies can occur with antibiotic use, obstructive jaundice, and fat
malabsorption syndromes.
Vitamin K depletion or warfarin usually
cause a prolongation of the PT that is out of
proportion to the aPTT because Factor VII,
which has the shortest half-life of the vitamin-K-dependent factors, has little effect
on the aPTT. Deficiency of vitamin K is best
managed by treatment of the underlying
condition and by administration of vitamin
K.67 If liver function is adequate, coagulation
factors will return to effective levels in
about 12 hours.
Although most patients with vitamin K
deficiency do not require plasma, plasma
transfusion is occasionally needed to treat
active bleeding or to prepare for emergency
invasive procedures.67 Transfusion of 10 to
15 mL of plasma per kg of body weight will
generally achieve hemostatic coagulation
factor levels in patients with warfarin-induced coagulopathy. One form of Factor IX
complex concentrate is licensed for warfarin reversal (see Table 21-4) but carries a significant risk of thrombotic complications and
is rarely used for this indication. Concurrent
vitamin K supplementation should also be
given unless only transient correction is de-

Chapter 21: Blood Transfusion Practice

sired. Although the International Normalized
Ratio can provide useful information about
response to therapy, the need for additional
treatment should be guided by the clinical response and not by the results of laboratory
tests. As discussed above, it is rarely necessary to correct the PT or aPTT to normal to
achieve adequate hemostasis.

Liver Disease
Patients with liver disease have multiple
derangements that contribute to an increased bleeding tendency. These abnormalities include portal hypertension and
engorgement of systemic collateral venous shunts; splenomegaly with secondary thrombocytopenia; decreased synthesis of all coagulation factors except Factor
VIII; dysfibrinogenemia; decreased clearance of fibrin, fibrinogen degradation
products, and fibrinolytic activators; and
decreased synthesis of inhibitors of the
fibrinolytic system. As in vitamin K deficiency, the short half-life of Factor VII
causes the PT to be prolonged more than
the aPTT, and, for the same reason, FFP
infusion corrects the PT for only about 4
hours. 72 Because the defect in hepatocellular disease is in primary protein synthesis, supplemental vitamin K will not
usually correct the abnormality. However,
because it is one of the few treatable causes
of coagulopathy in liver disease, a trial of
replacement vitamin K may be indicated.
FFP corrects coagulation factor deficiencies found in severe liver disease, but is often used inappropriately. The most common error is to attribute all bleeding to
coagulopathy and to give systemic treatment when the cause is localized bleeding.
For example, bleeding at the operative site
after cardiac surgery usually responds
better to local hemostatic measures than to
intravenous infusion of FFP. A second common error in treating liver-associated

499

coagulopathy is overdependence on the PT.
Again, a normal PT is rarely, if ever, required for the cessation of serious bleeding,
and the goal of FFP therapy in severe liver
disease should be to correct or prevent
bleeding complications. If FFP is to be used
prophylactically before an invasive procedure, it should be given immediately before
the procedure.
Patients with liver disease may also have
abnormalities of platelet plug formation
and fibrinolysis. In addition, severe splenomegaly may impair the response to platelet
transfusions. Platelet function in some patients with liver disease can be enhanced by
administration of 1-deamino-8-D-arginine
vasopressin (desmopressin, DDAVP).73 Cryoprecipitated AHF should be given if there is
severe hypofibrinogenemia or bleeding related to dysfibrinogenemia. The increase in
systemic fibrinolysis associated with severe
liver disease may not respond to FFP alone,
and antifibrinolytic agents in combination
with plasma therapy can be useful in these
patients (see below).

Dilutional Coagulopathy
Massive blood loss and replacement with
crystalloid and/or colloid solutions may
produce a dilutional coagulopathy,74 but
most patients can tolerate loss and replacement of at least one blood volume
without developing impaired hemostasis.75 Shock accompanying traumatic
hemorrhage also contributes to the coagulopathy (see Chapter 27). In the setting
of trauma, thrombocytopenia generally
develops before plasma clotting factors are
diluted to the point of causing impaired
hemostasis, and adequate platelet replacement generally has priority.75 However,
in elective surgical patients, coagulation
factor deficits may predominate.74 FFP may
be beneficial if the PT and/or aPTT are
greater than 1.5 times normal.3,39 If surgi-

500

AABB Technical Manual

cal hemostasis has not been achieved and
significant continued bleeding is expected,
FFP may be indicated.39
Patients undergoing plasmapheresis
without plasma replacement develop a variety of coagulation factor deficits, particularly hypofibrinogenemia, depending on
the volume and frequency of the ex76
changes. Although these changes can be
striking, most authors have concluded that
routine supplements with FFP in patients
with normal liver function are unnecessary,
particularly for alternate-day regimens.

Other Conditions
Plasma exchange is lifesaving in TTP (see
Chapter 6). FFP may be an adjunct to treatment of DIC. Hereditary angioneurotic
edema results from a congenital deficiency of C1-esterase inhibitor, an inhibitory protein that regulates complement
activation. Patients with this condition
develop localized edema and may experience lifethreatening obstruction of the
upper respiratory tract following complement activation. FFP or Liquid Plasma
contain normal levels of C1-esterase inhibitor and FFP transfusion appeared to
prevent attacks at the time of oral surgery
in one study.77 There are rare anecdotal reports of exacerbation of angioneurotic
edema with FFP administration. The need
for treatment of isolated deficiency of
Factors II, V, VII, X, or XI is uncommon;
guidelines for initial therapy are given in
Table 21-3 (however, for a more complete
treatment, refer to one of the standard hematology or coagulation texts).
Plasma can also be used to replace proteins C and S, and antithrombin; these are
discussed separately below.

Misuse of Fresh Frozen Plasma
Plasma should not be used as a volume
expander, as a nutritional source, or to

38,70,75

Transfusing
enhance wound healing.
plasma for volume expansion carries a
risk of infectious disease transmission and
other transfusion reactions (eg, allergic)
that can be avoided by using crystalloid or
colloid solutions. Plasma is also not a
suitable source of immunoglobulins for
patients with severe hypogammaglobulinemia because more effective preparations exist (immunoglobulin for intravenous or intramuscular use).
FFP is often given prophylactically to patients with mild to moderate prolongation
of the PT or aPTT before invasive procedures, but there is little or no evidence that
this prevents bleeding complications. Because these tests do not accurately predict
the risk of bleeding when mildly prolonged,
there is little logic for a transfusion intended to “improve” the results.

Cryoprecipitated AHF
Transfusion
CRYO is the only concentrated fibrinogen
product currently available for systemic
use, and intravenous supplementation of
fibrinogen is its primary clinical use, particularly in DIC. A second major use has
been in patients with severe von Willebrand disease, but there are Factor VIII
concentrates that contain vWF and are
more appropriate if available (see Table
21-4). CRYO can be used in isolated Factor XIII deficiency and to ameliorate the
platelet dysfunction associated with uremia. It is also used topically as a fibrin
sealant, although a commercial preparation is available. CRYO is seldom used for
patients with hemophilia because Factor
VIII concentrates are available commercially and have been processed to reduce
or eliminate the risk of blood-borne viral
infection; CRYO is used as a last resort for
this indication. Because CRYO contains

Chapter 21: Blood Transfusion Practice

ABO antibodies, consideration should be
given to ABO compatibility when the infused volume will be large relative to the
recipient’s red cell mass.

Calculating the CRYO Dose for Fibrinogen
Replacement
On average, one unit of CRYO contains
approximately 250 mg of fibrinogen; the
minimum required by AABB Standards is
150 mg.59(p30) The amount of transfused
CRYO required to correct the fibrinogen
level depends upon the nature of the
bleeding episode and the severity of the
initial deficiency and can be calculated as
follows:
1.
Weight (kg) × 70 mL/kg = blood volume (mL).
2.
Blood volume (mL) × (1.0 – hematocrit) = plasma volume (mL).
3.
Mg of fibrinogen required = (Desired
fibrinogen level in mg/dL – initial
fibrinogen level in mg/dL) × plasma
volume (mL) ÷ 100 mL/dL.
4.
Bags of CRYO required = mg of fibrinogen required ÷ 250 mg fibrinogen/
bag of CRYO
This calculation assumes that 100% of
administered fibrinogen is recovered as
measurable intravascular fibrinogen, but,
because the content of CRYO is variable,
further refinements are unproductive.

von Willebrand Syndromes
von Willebrand syndromes are the most
common major inherited coagulation abnormalities.78 These conditions are usually autosomal dominant and represent a
collection of quantitative and qualitative
abnormalities of vWF, the most important
protein mediating platelet adhesion to
damaged endothelial surfaces. The protein also transports Factor VIII. As a result, patients with von Willebrand syndromes have varying degrees of abnormal

501

platelet plug formation and partial deficiency of Factor VIII. The former may
manifest as a prolonged bleeding time
and the latter as a prolonged aPTT, but
these abnormalities vary between syndromes. vWF exists in the plasma as a
family of multimeric molecules with a
wide range of molecular weights. The
high-molecular-weight species of vWF are
the most hemostatically effective. Laboratory evaluation demonstrates a specific deficiency in the level of vWF, often measured as ristocetin cofactor activity because
vWF is required for the platelet-agglutinating effect of ristocetin in vitro.
Mild cases of von Willebrand syndrome
can often be treated with DDAVP, which
causes a release of endogenous stores of
Factor VIII and vWF. Many Factor VIII concentrates do not contain therapeutic levels
of vWF, but several with satisfactory levels
are commercially available and one is licensed for this indication (see Table 21-4).
In the absence of a suitably therapeutic virus-inactivated concentrate, severe von
Willebrand syndrome can be treated with
CRYO. The quantity of CRYO required to
treat bleeding episodes or to prepare for
major surgery varies greatly among patients
with von Willebrand syndromes. In addition to the clinical response of the patient,
the template bleeding time, the level of Factor VIII, or the ristocetin cofactor activity
may help to guide therapy.

Fibrinogen Abnormalities
Hypofibrinogenemia may occur as a rare
isolated congenital deficiency, may be acquired as part of the DIC syndrome, or may
be due to obstetric complications such as
abruptio placentae. Dysfibrinogenemias
may be congenital or acquired and represent conditions in which fibrinogen is
present as measured by immunoassays
but functionally defective as measured by

502

AABB Technical Manual

the thrombin time. Patients with severe
liver disease frequently exhibit a dysfibrinogenemia.

in the setting of hemorrhage that results
from DIC once the fibrinogen is above 100
mg/dL.

Disseminated Intravascular Coagulation

Topical Use

DIC occurs when circulating thrombin induces widespread fibrin formation in the
microcirculation and consumption of platelets and coagulation factors, particularly
fibrinogen, prothrombin, Factor V, and
Factor VIII. Fibrin strands in the microcirculation may cause mechanical damage
to red cells, a condition called microangiopathic hemolysis, manifest as schistocytes
(fragmented red cells) in the circulation.
Widespread microvascular thrombi promote tissue ischemia and release of tissue
factor, which further activates thrombin.
Lysis of microvascular fibrin causes increased quantities of fibrin degradation
products to enter the bloodstream.
Several clinical conditions can initiate
DIC, including shock, tissue ischemia, sepsis, hemolytic transfusion reactions, disseminated cancer (particularly adenocarcinoma), acute promyelocytic leukemia,
tumor lysis syndrome, and obstetric complications such as amniotic fluid embolism.
The common precipitating event is a procoagulant signal for thrombin production that
exceeds the normal physiologic defenses
against disseminated thrombin activity.
Treatment of DIC depends on correcting
the underlying problem and preventing
further hypotension and tissue ischemia.
Replacement therapy focuses on the building blocks of the thrombus (platelets and
fibrinogen), and secondarily on other coagulation factors, including Factors VIII, XIII,
V, and II. Thus, in bleeding patients, platelet
transfusion is indicated when the platelet
count falls below 50,000/µL, and cryoprecipitate is supplemented if the fibrinogen
level is below 100 mg/dL. FFP is indicated

The fibrinogen in CRYO has been used
during surgery as a topical hemostatic
preparation, so-called fibrin sealant or fibrin glue, made from one or two units of
CRYO, which may be of autologous origin.
The fibrinogen is converted to fibrin by
the action of bovine thrombin at the site
that is bleeding or to be “glued.” Commercial preparations of fibrin sealant are
available that have a higher fibrinogen
concentration than that of CRYO and include human thrombin and bovine aprotinin (see below) to decrease the lysis of
the resulting fibrin. These pooled, virusinactivated products have been licensed
for the reduction of bleeding in cardiovascular surgery, 7 9 for repairing splenic
trauma, and for colostomy closure. Fibrin
sealants have also been used for a variety
of indications in which it is desired to
bind two tissue surfaces together, including repair of the dura mater or eardrum.
Use of bovine thrombin can stimulate
the formation of antibodies against thrombin
and other contaminant proteins including
Factor V.80 These antibodies can cross-react
with human thrombin and Factor V, causing abnormal clotting times and, in some
cases, bleeding. For this reason, it has been
suggested that use of “homemade” fibrin
sealants be replaced by use of the commercial product.80

Hemophilia A
Each unit of CRYO prepared from a single
blood donation should contain a minimum
of 80 international units (IU) of Factor
VIII.59(p30) Although no longer the component of choice, CRYO can serve as replace-

Chapter 21: Blood Transfusion Practice

ment therapy for patients with hemophilia A if virus-inactivated Factor VIII
39
concentrates are unavailable. If CRYO is
used, the amount required to provide a
therapeutic dose of Factor VIII is based on
calculations similar to those used for AHF.
Hemophilia A is a sex-linked recessive
trait (ie, affected males, carrier females)
causing deficiency of Factor VIII (antihemophilic factor, AHF).81 The responsible
genes usually produce a protein with reduced activity, so immunologic measurement of Factor VIII antigen gives normal results despite deficient Factor VIII coagulant
activity. In contrast, antigen is typically depressed in von Willebrand disease. Characteristic laboratory findings include a prolonged aPTT, normal PT and template
bleeding time, and decreased Factor VIII
activity.
The severity of hemophilia A depends on
the patient’s level of Factor VIII activity, and
this varies; patients with severe hemophilia
have Factor VIII levels below 1%, whereas
those with moderate disease typically have
1% to 5% activity, and severity is mild with
levels of 6% to 30%.81 One unit of Factor VIII
activity is defined as the Factor VIII content
of 1 mL of fresh, citrated, pooled normal
plasma. The measured level of Factor VIII
can be expressed as a concentration, as
percent activity, or as a decimal fraction.
For example, a patient with mild hemophilia with one-tenth the normal activity of
Factor VIII can be said to have a Factor VIII
level of 10 units/dL or 0.1 unit/mL or 10%
activity.
Patients with mild-to-moderate hemophilia can often be managed without replacement therapy.81 Careful attention to local hemostasis and the use of topical
antifibrinolytics may prevent the need for
further replacement. Systemic levels of Factor VIII can be raised in mild hemophilia
with the use of DDAVP, which stimulates
the release of endogenous Factor VIII from

503

storage sites. However, DDAVP is ineffective
in patients with severe hemophilia A; in
such cases, Factor VIII replacement is required. The amount of Factor VIII infused
depends upon whether therapy is intended
to prevent bleeding or, if bleeding has occurred, the nature of the bleeding episode
and the severity of the initial deficiency (see
Table 21-6). For example, treatment for
hemarthrosis ordinarily requires more Factor VIII than epistaxis.

Calculating the Dose of Factor VIII
When the desired result is determined
(see Table 21-6), the amount of Factor VIII
required for transfusion can be calculated
by one of the following formulas:
Factor VIII dose (IU/kg) =
Desired factor increase (%) × 0.5 (1
IU/kg typically raises the Factor VIII
level by 2%)
Total dose =
(Patient mass × 70mL/kg) × (1 – Hct) ×
(Desired activity – current activity)
Example: A 70-kg patient with severe hemophilia has an initial Factor VIII level of
2% (0.02 unit/mL) and a hematocrit of 40%.
How many units of Factor VIII concentrate
should be given to raise his Factor VIII level
to 50% (0.5 unit/mL)?
(70 × 70) × (1 – 0.4) × (0.5 – 0.02) =
1411 units
The therapy of choice for severe hemophilia A is a Factor VIII concentrate (see Table 21-4). CRYO could be used to supply
1411 units of Factor VIII, but, at 80 IU per
bag, this would require at least 18 bags (and
18 allogeneic donor exposures). The halflife of Factor VIII is about 12 hours, so infusions are repeated at 8- to 12-hour intervals

504

AABB Technical Manual

Table 21-6. General Factor Replacement Guidelines for the Treatment of Bleeding
in Hemophilia

Indication
Severe epistaxis, oral
mucosal bleeding†
Hemarthrosis, hematoma,
persistent hematuria,‡
gastrointestinal tract
bleeding, retroperitoneal
bleeding
Trauma without signs of
bleeding, tongue/
retropharyngeal
bleeding†
Trauma with bleeding,
surgery,§ intracranial
bleeding§

Initial
Minimum
Desired
Factor Level
(%)

Factor VIII
Dose*
(IU/kg)

Factor IX
Dose*
(IU/kg)

Duration
(days)

20-30

10-15

20-30

1-2

30-50

15-25

30-50

1-3

40-50

20-25

40-50

2-4

100

10-14

Data from USP.
*Dosing intervals are based on a half-life for Factor VIII of 8 to 12 hours (2 to 3 doses/day) and a half-life for Factor IX
of 18 to 24 hours (1 to 2 doses/day). Maintenance doses of one half the initial dose may be given at these intervals. The
frequency depends on the severity of bleeding, with more frequent dosing for serious bleeding.
†
In addition to antifibrinolytics.
‡
Painless spontaneous hematuria usually requires no treatment. Increased oral or intravenous fluids are necessary to
maintain renal output.
§
Continuous factor infusion may be administered. Following the initial loading dose, a continuous infusion at a dose of 3
IU/kg per hour is given. Subsequent doses are adjusted according to the plasma factor levels.

to maintain hemostatic levels. The duration
of treatment with Factor VIII infusions depends upon the type and location of the
hemorrhage or the reason for prophylaxis,
and the clinical response of the patient (see
Table 21-6). After major surgery, the Factor
VIII level should be maintained above 40%
to 50% for at least 10 days. When elective
surgery is planned, Factor VIII assays
should be made available to serve as a
guide to therapy.

Treatment of Inhibitors of Factor VIII
About 10% to 35% of patients with hemophilia A, typically, those with severe dis-

ease or genetic defects involving large
portions of the molecule, develop a de68,81
tectable inhibitor to human Factor VIII.
These antibodies are directed against the
active site of Factor VIII, rendering the patient relatively unresponsive to infusion.
Patients having an elective invasive procedure should be screened for such inhibitors. Management is difficult; approaches have included attempts to
overwhelm the inhibitor with very large
doses of human Factor VIII; use of porcine Factor VIII, which has low cross-reactivity with human Factor VIII antibody81;
use of Factor VIII bypassing agents in-

Chapter 21: Blood Transfusion Practice

cluding Factor IX complex, activated Factor IX complex, and activated Factor VII
(see Table 21-4); and desensitization therapy. The latter includes large daily doses
of AHF in conjunction with corticosteroids or Immunoglobulin Intravenous
(IGIV) and cyclophosphamide. Success rates
of 50% to 80% are reported. If hemorrhage
is life-threatening, intensive plasmapheresis to remove the inhibiting antibody, coupled with immunosuppression,
as well as infusions of Factor VIII and possibly antifibrinolytic therapy (see below),
can be employed.

Hemophilia B
Factor IX deficiency (hemophilia B, Christmas disease) is clinically indistinguishable from Factor VIII deficiency in that
both are sex-linked disorders that cause a
prolonged aPTT in the presence of a normal PT and bleeding time.81 The disorder
is confirmed by specific measurement of
Factor IX activity. Factor IX complex concentrate has been used for treatment of
hemophilia B for the past 2 decades, but
the new, more pure forms of Factor IX
concentrate (see Table 21-4) are preferred
because they carry much less risk of in82
ducing thrombosis.
The formula for calculating of Factor IX
dosage is similar to that for Factor VIII, but
the units to be given should be doubled because only half of the infused Factor IX
dose is recovered in the vascular space. The
biologic half-life is 18 to 24 hours, so doses
are given 1 or 2 times/day. As with hemophilia A, recommended dose and treatment
schedules vary with the severity and type of
bleeding (see Table 21-6).

Immunoglobulin, Intravenous
IGIV is prepared from modified Cohn fraction II and subjected to virus inactivation.
Preparations intended for intramuscular

505

administration contain aggregates that
may activate the complement and kinin
systems and produce hypotensive and/or
anaphylactoid reactions if administered
intravenously, but the intravenous product contains almost exclusively mono83
meric IgG molecules.
The indications for the use of IGIV are
83,84
evolving. Some conditions in which IGIV
is used are listed in Table 21-7. Infusion of
IGIV can induce such reactions as headache, vomiting, volume overload, allergic
reactions, renal failure, and pulmonary reactions, but they can usually be prevented
by infusing slowly and pretreating with
diphenhydramine and/or hydrocortisone.
Passively transferred blood group allo- and
autoantibodies and/or therapy-induced
hypergammaglobulinemia may cause a
positive DAT result in recipients, but significant hemolysis is rarely noted.

Antiprotease Concentrates
Antithrombin, also known as heparin cofactor, is a serine protease inhibitor syn85
thesized in the liver. It circulates in normal plasma at a concentration of 15 mg/
dL but is typically measured as % activity,
with a normal range of 84% to 116%. Antithrombin inactivates serine proteases including thrombin, and Factors IXa, Xa,
XIa, and XIIa by covalently bonding at the
serine site, followed by a conformation
86
change. This activity is greatly accelerated by heparin, which induces a conformation change in antithrombin and helps
approximate thrombin and antithrombin
as well.
Patients who are deficient in antithrombin are prone to thromboembolic
complications.87 Such deficiency can be
congenital or acquired. Acquired deficiency
occurs in a wide variety of disease states,
including decreased synthesis due to liver

506

AABB Technical Manual

Table 21-7. Potential Indications and Clinical Uses for Intravenous Immunoglobulin
Preparations
Congenital immune deficiencies
Hypogammaglobulinemia and agammaglobulinemia
Selective antibody deficiency
IgG subclass deficiency and recurrent infection
Premature newborns
Acquired antibody deficiency
Malignancies with antibody deficiency and recurrent infection: multiple myeloma,
chronic lymphocytic leukemia
Protein-losing enteropathy
Drug- or radiation-induced humoral immunodeficiency
Prophylaxis or treatment of bacterial and viral diseases
Pediatric HIV infection for prevention of bacterial and secondary viral infections
Cytomegalovirus infection in transplant recipients
Neonatal sepsis
Other
HIV-related immune thrombocytopenic purpura
Immune cytopenias (ITP, NAIT, PTP, WAIHA)
Kawasaki syndrome
Guillain-Barré syndrome and chronic inflammatory demyelinating neuropathy
Acquired Factor VIII inhibitors
Myasthenia gravis
Multiple sclerosis

disease or malnutrition; losses due to
nephrotic syndrome and gastrointestinal
states; accelerated consumption as in DIC,
surgery or trauma, and preeclampsia; and
associated with pharmacotherapy including heparin, L-asparaginase, and oral
contraceptives.
Antithrombin is stable in plasma so deficiency can be treated with FFP or with Liquid or Thawed Plasma. A heat-treated concentrate of antithrombin is also available;
recombinant and transgenic sources are
under investigation.
Clinical uses of antithrombin have re87
cently been reviewed. Antithrombin is approved for use in hereditary antithrombin
deficiency as part of the treatment for

thromboembolic episodes and for prophylactic use in perioperative, postoperative,
and peripartum settings. Several off-label
uses of antithrombin exist, eg, patients with
low antithrombin levels and DIC, neonates
born to mothers with antithrombin deficiency, and liver transplant recipients.
The half-life of purified antithrombin is
long, approximately 60 to 90 hours,88 but is
abbreviated when replacement is for consumptive states. The dose is calculated on
the basis of an expected increment of 1.4%
per U per kg, with an initial target of 120%.
Other available concentrates of antiproteases include alpha-1-proteinase inhibitor
(alpha-1-antitrypsin). C1-esterase inhibitor
is not available in the United States.

Chapter 21: Blood Transfusion Practice

Protein C and Protein S
Protein C and protein S are vitamin-K-dependent proteins with anticoagulant effects.86 Protein S is a cofactor for activated
protein C, which, in turn, inactivates Factor Va and Factor VIIIa. Patients with deficiencies of protein C or protein S have a
predisposition to thrombotic complications
85
and are often treated with anticoagulants.
Warfarin treatment, however, can cause
these vitamin-K-dependent proteins to
decrease to dangerously low levels, leading to skin necrosis and exacerbating
thrombosis. Transfusion of FFP can serve
as an immediate source of supplemental
protein C or protein S for patients with severe deficiencies, and a human protein C
concentrate is under development.
Patients with heterozygous protein C deficiency have plasma levels 40% to 60% of
normal and characteristically have minimal
symptoms, rarely requiring treatment with
protein C supplementation.85 If treatment is
needed for a thrombotic episode, anticoagulants suffice. Homozygous protein C deficiency causes neonatal purpura fulminans,
which requires immediate administration of
protein C, along with complex regulation of
the rest of the coagulation cascade. The
half-life of infused protein C is 6 to 16 hours.

Colloid Solutions
Human albumin (5% and 25%) and plasma
protein fraction (PPF) provide volume expansion and colloid replacement without
risk of transfusion-transmitted viruses.89
PPF has a greater concentration of nonalbumin plasma proteins than 5% albumin.
Pharmacologic agents such as hydroxyethyl
starch or dextran are also commonly used
for volume expansion.

Physiology of Albumin
The total body albumin mass is about 300
g, of which 40% (120 g) is in the plasma.

507

Daily albumin synthesis in a normal adult
approximates 16 g. Albumin has complex
roles in normal physiology and disease in
addition to its obvious one of maintenance of intravascular volume.90 Hypoalbuminemia resulting from decreased
synthesis, increased catabolism or losses,
and shifts between different fluid compartments is common in acute and chronic
illness.

Replacement
Albumin solutions are effective volume
expanders, with the promise of better
intravascular fluid retention than simpler
and less expensive crystalloids (eg, normal saline or lactated Ringer’s solution).
Because hypoalbuminemia is involved in
the pathogenesis of many disease states
and may correlate with their prognosis, it
has been tempting to try to alter their
course by exogenous supplementation,
particularly in view of the perceived low
risk status of albumin solutions. However,
this low-risk is in question.
Indications for albumin approved by a
consensus panel91 include:
1.
Volume replacement in nonhemorrhagic shock unresponsive to crystalloid or in the presence of capillary
leak syndromes.
2.
Volume replacement after the first
day in patients with extensive burns
(>50%) unresponsive to crystalloid.
3.
Replacement after removal of large
volumes (>4 L) of ascitic fluid in patients unresponsive to crystalloid.
4.
Replacement of ascitic fluid or postoperative treatment of ascites and
peripheral edema in hypoalbuminemic liver transplant recipients.
5.
Replacement during large-volume
plasma exchange.
6.
Volume replacement in patients with
severe necrotizing pancreatitis.

508

AABB Technical Manual

Diarrhea (>2 L/day) in hypoalbuminemic (<2.0 g/dL) patients on enteral
feedings, unresponsive to short chain
peptide supplementation.
Nonalbumin colloid solutions were considered less expensive first alternatives in
several of these situations.
In spite of the conceptual attractiveness,
the long history of albumin replacement,
and the consensus on its use, a number of
prospective randomized trials have suggested that albumin use was ineffective or
increased mortality. A meta-analysis of 30
randomized trials including 1419 patients
grouped according to indication (namely,
hypovolemia, burns, and hypoproteinemia)
demonstrated higher mortality with albumin treatment for each of the groups.92 In
response to this outcome, the authors of the
meta-analysis called for an immediate review of albumin use in critically ill patients.

Special Transfusion
Situations
Thalassemia
Thalassemia and sickle cell disease are inherited syndromes characterized by deficient or abnormal hemoglobin structures
and anemia. Thalassemia is caused by a
deficiency in alpha or beta chain production that ranges from mild to severe. Total
absence of synthesis of one of the alpha
chains is lethal in utero; absence of beta
chain synthesis (thalassemia major) results in a progressive anemia in the newborn period. In an attempt to compensate
for significant degrees of anemia, hematopoietic tissue expands, causing characteristic bone abnormalities and enlargement
of the liver and spleen. Tissue iron accumulates as a result of increased adsorption and transfusion (see Chapter 27). The
only current cure for thalassemia is hemato-

poietic transplantation, but because the
anemia can be controlled with red cell
transfusions and concurrent iron chelation therapy, the use of this expensive and
potentially hazardous therapy is controversial. Transfusion of thalassemia patients is discussed in Chapter 24.

Sickle Cell Disease
Sickle cell disease (SCD) results from a
single base substitution in the gene for
the beta chain of hemoglobin. The hemoglobin of individuals homozygous for this
abnormality can irreversibly polymerize
and cause red cells to deform or “sickle.”
Such cells may initiate blockage of the
microvasculature directly or in association with endothelial damage and thrombosis. They also have a decreased life
span, so SCD patients have a variably
compensated hemolytic anemia. Sickling,
which can be triggered by fever, infection,
or hypoxia, can lead to a variety of complications or “crises,” including pain crisis
due to musculoskeletal or other tissue
ischemia, splenic or pulmonary sequestration crisis (chest syndrome), aplastic
crisis due to transient marrow suppression
by viruses (particularly parvoviruses), leg
ulcers, priapism, tissue infarction, and
stroke.
Most patients with sickle cell disease are
asymptomatic most of the time and do not
require routine transfusion. Although
sickling can be prevented or reversed by
maintaining the level of normal hemoglobin above 50% to 70%, the risks from alloimmunization, iron overload, and disease
transmission outweigh the benefits of prophylactic transfusion in most patients.
Moreover, uncomplicated pain crises do not
respond well to simple transfusion. Simple
transfusion is indicated for symptomatic
anemia, aplastic crises, and blood loss.
Sometimes, patients with a history of stroke

Chapter 21: Blood Transfusion Practice

or pulmonary or cardiac disease are sometimes treated with a hypertransfusion protocol or chronic red cell exchange program to
maintain their hematocrit at 25% to 30%
and the proportion of hemoglobin S below
about 30%. Care should be taken to avoid
raising the hematocrit above 35% because
of the risks of hyperviscosity. Red cell exchange is used to manage and/or prevent
life- or organ-threatening complications,
particularly stroke and pulmonary crisis.
Red cell exchange has also been used to
prepare patients for surgery, but a randomized controlled trial did not support this
measure over simple transfusion to a hemoglobin of 10 g/dL.93
The clinical management of sickle cell
disease is complex and the reader is referred to recent reviews for more details.94,95
Patients with thalassemia and sickle cell
disease can receive standard red cell components. However, leukocyte reduction is
generally offered to avoid febrile, nonhemolytic transfusion reactions. Phenotyping the patient’s red cells and providing
antigen-matched units for transfusion
helps reduce alloimmunization to red cell
antigens and delayed hemolytic transfusion
reactions, although the cost and logistics of
such a program may be impractical for
many institutions. A frequent compromise
is to match for Rh system and K antigens.
Patients with SCD should be given hemoglobin-S-negative RBC units. For more
details, see Chapter 24.

Transfusing Known Incompatible Blood
Clinicians must occasionally transfuse a
patient for whom no serologically compatible RBC units are available. This most
often occurs in patients with autoantibodies, which typically react with all red
cells; however, once alloantibodies are
ruled out, the transfused cells are expected to survive as long as autologous

509

cells. Other situations in which all units
appear incompatible include the presence
of alloantibodies to high-incidence antigens and multiple antibody specificities.
If serologic testing fails to resolve the
problem, or if the problem is identified but
time is not sufficient for acquisition of
compatible units, the physician must weigh
the risks and benefits of transfusion and
consider what alternative therapies are
suitable. If the need is sufficiently urgent,
incompatible red cells of the patient’s ABO
and Rh type may have to be given. Depending on the alloantibody, incompatible
transfusion does not always result in immediate hemolysis, and the incompatible cells
may remain in the circulation long enough
to provide therapeutic benefit.96
If time permits and if equipment is available, the survival of a radiolabeled aliquot
of the incompatible cells can be determined. Alternatively, an “in-vivo crossmatch” can be performed by cautiously
transfusing 25 to 50 mL of the incompatible
cells, watching the patient’s clinical response, and checking a 30-minute posttransfusion specimen for hemoglobintinged serum. Such assessment does not
guarantee normal survival, but it can indicate whether an acute reaction will occur. If
no adverse symptoms or hemolysis are observed, the remainder of the unit can be
transfused slowly with careful clinical monitoring. If the transfusion need is lifethreatening, RBC units may sometimes be
given without special testing, but clinical
staff should be prepared to treat any
reaction that may result.

Transfusing Patients with Autoimmune
Hemolytic Anemia
Because of the serologic difficulties that
accompany autoimmune hemolytic anemia and the expected short red cell survival, a conservative approach to trans-

510

AABB Technical Manual

fusion is recommended. The presence of
underlying alloantibodies should be investigated before beginning transfusions,
time permitting. It is very helpful to establish the patient’s phenotype before
transfusion in order to simplify subsequent investigation for the presence of
possible alloantibodies. Chapter 20 contains a more complete discussion.

Massive Transfusion
Massive transfusion is defined as replacement approximating or exceeding the patient’s blood volume within a 24-hour interval. The most important factor in
supporting tissue oxygenation is maintenance of adequate blood flow and blood
pressure by infusing a sufficient volume
of crystalloid or blood components to
correct or prevent hypovolemic shock.

Emergency Issue
The transfusion service should establish a
standard operating procedure for emergency provision of blood. Immunohematologic testing is relatively time-consuming, so it may have to be abbreviated in
trauma cases or other hemorrhagic emergencies. Because ABO-compatible components are entirely compatible in the
vast majority of cases, particularly if the
patient has never been transfused before,
group O RBCs are often used for emergency transfusion before completion of
any compatibility tests. In this situation,
Rh-negative RBCs should be used for females of childbearing potential because
of the concern for immunizing such individuals and possibly causing hemolytic
disease of the newborn in the future. For
women beyond their childbearing years or
men, only the current presence of anti-D
is a concern. Because this antibody is no
more common than certain other blood

group alloantibodies, Rh-positive RBCs can
be used with similar safety.
The use of so-called “universal donor”
RBCs, as discussed above, has several drawbacks. Group O RBCs are typically in short
supply because of demographic differences
between the donor and recipient populations in the United States, and such universal donor practices accentuate this problem. ABO typing can be performed very
quickly, and, in most emergencies, there is
time for ABO typing of the recipient and
provision of ABO-specific components.
Second, transfusion of large quantities of
group O RBCs before a recipient blood
sample is obtained may obscure subsequent immunohematologic testing. Therefore, even if universal donor RBCs are to be
used, a blood sample should be obtained
before transfusion; this is possible in all but
the most dire cases of exsanguinating hemorrhage. Finally, unexpected blood group
antibodies can cause fatal transfusion reactions, particularly in multitransfused patients such as those with SCD or liver disease who present to an emergency room
with severe anemia or bleeding. In such
situations, a call to another hospital transfusion service at which the patient was previously transfused can be life-saving. Individuals caring for such patients must
understand the above principle concerning
the priority of volume deficits over anemia.
Emergency situations should not be
construed as justification for exceptions to
strict identification of recipient blood samples. On the contrary, increased attention to
patient identification is warranted, and one
indication for use of universal donor RBCs is
in situations when multiple trauma victims
arrive concurrently, when there is a high
risk of recipient misidentification.
AABB Standards requires physicians who
order transfusion before completion of
standard compatibility tests to document the
need by signing some type of “emergency

Chapter 21: Blood Transfusion Practice

59(p46)

release form.”
This documentation should
be required only after the emergency is
over, typically within 24 hours, and signatures on such forms should not be construed as a release of the transfusion service’s responsibility. The transfusion service
must insist on strict identification of samples, documentation of unit disposition,
and documentation of the emergency status of the transfusion. However, it also has
the responsibility to perform testing on a
STAT basis, provide consultation, and avoid
unnecessarily restrictive practices.

Changing Blood Types
The transfusion service should establish
guidelines for switching blood types during massive transfusion. An alternative to
ABO-identical RBCs is the use of ABOcompatible units (see Table 21-1). The age
and sex of the patient are important considerations. For example, when transfusing a young group B, Rh-negative woman,
it is preferable to switch to group O, Rhnegative RBCs before switching to group
B, Rh-positive cells. The clinical situation
should be evaluated by the transfusion
service’s physician. If the continuing
transfusion requirement is expected to
exceed the available supply of Rh-negative blood, evaluation of the change to
Rh-positive blood should be made early,
to conserve blood for other recipients. Once
the patient receives one or more Rh-positive units, there may be little advantage in
returning to Rh-negative blood.

Coagulation Support During Massive
Transfusion
Massive transfusion is often associated with
coagulation abnormalities that may manifest as microvascular bleeding (MVB) in
the form of oozing from multiple IV sites,
failure of blood shed into body cavities to
clot, and bleeding from tissue surfaces on

511

which hemostasis was previously obtained.
These situations have been attributed to
the dilution of platelets or coagulation
factors, but consumptive coagulopathy
also plays a role (see Chapter 27). Inadequate volume resuscitation and poor tissue perfusion not only promote the release of tissue procoagulant material
leading to DIC but also result in lactic acidosis, acidemia, and poor myocardial
performance. If MVB occurs, the results of
platelet counts, fibrinogen level, PT, and
aPTT ideally should guide the need for
hemostatic components. Empiric therapy
with platelets and/or plasma may be initiated immediately after specimens are obtained. Additional tests may be indicated
to evaluate the possibility of DIC. In this
situation, a platelet count less than
50,000/µL and a fibrinogen level less than
100 mg/dL are better predictors of hemorrhage than the PT and aPTT.97 PT results
below 1.5 times normal are usually associated with adequate hemostasis during
surgery.3 In most adult patients, these levels are encountered only after transfusion
of 15 to 20 RBC units (1.5 to 2 red cell volumes). FFP or platelets should not be administered in a fixed ratio to the number
of RBC units given.

Hypothermia, Tissue Oxygenation, and
2,3-DPG
Hypothermia as a complication of transfusion is discussed in Chapter 27. In hypovolemic shock, the underlying pathophysiologic defect is inadequate tissue
oxygenation. Oxygen supply to the tissues
is determined by many factors, the most
important of which are blood flow (perfusion) and hemoglobin concentration. The
level of 2,3-DPG decreases in stored RBCs,
and this decrease has been suggested as a
potential cause of poor tissue oxygenation after massive transfusion. Low 2,3-DPG

512

AABB Technical Manual

levels have not been shown to be detrimental to massively transfused patients,
although for infants undergoing exchange
transfusion, blood with near-normal
2,3-DPG levels is frequently requested.
Within 3 to 8 hours after transfusion, previously stored red cells regenerate 50% of
normal 2,3-DPG levels.98

Pharmacologic Alternatives
to Transfusion
Concern over the risks and limitations of
transfusion has led to examination of
pharmacologic alternatives. Such alternatives might: 1) stimulate increased production or release of blood elements that
otherwise would require replacement (eg,
erythropoietin); 2) substitute for a blood
component (eg, colloid solutions or oxygen-carrying solutions including chemically modified hemoglobins); or 3) alter
physiologic mechanisms to reduce the
need for replacement (eg, fibrinolytic inhibitors).99

Recombinant Growth Factors
Growth factors are low-molecular-weight
protein hormones that regulate hematopoiesis by specific interaction with receptors found on progenitor cells. The use of
growth factors to stimulate endogenous
blood cell production is an important alternative to the use of blood.100

need for transfusion in patients with endstage renal disease and is indicated for
the treatment of anemia in patients infected with human immunodeficiency virus. Intensive EPO treatment (40,000 units
weekly) reduced the transfusion requirement of ICU patients.101 It may also have a
role in treating anemia due to chronic disease or to receipt of other medications
that suppress the marrow.

Other Blood Cell Growth Factors
Granulocyte-macrophage colony-stimulating factor (GM-CSF) and G-CSF stimulate marrow production of granulocytes.100
G-CSF is approved for the treatment of
chemotherapy-induced neutropenia, for
patients undergoing peripheral blood
progenitor cell collection and therapy,
and for patients with chronic neutropenia.
The use of these stimulants decreases the
duration of neutropenia, increases tolerance to cytotoxic drugs, and decreases the
need for granulocyte transfusions. GMCSF is approved for use in patients undergoing autologous marrow transplantation. Another potential use for GM-CSF
and G-CSF is support of patients undergoing allogeneic marrow transplantation
or patients receiving antiviral agents that
suppress the marrow. Recombinant interleukin-11 is licensed for cancer patients
with thrombocytopenia, but other activators for thrombopoietin receptors have
been disappointing and none are available at this time.

Erythropoietin
Recombinant erythropoietin (EPO) is a
growth factor that stimulates RBC production.100 It has been approved for presurgical administration to increase preoperative hemoglobin and hematocrit levels;
typical dose regimens range from 300 to
600 U/kg by subcutaneous injection weekly.
The use of EPO has markedly reduced the

Oxygen Therapeutics (Carriers)
Stroma-free hemoglobin solution, in which
free hemoglobin has been separated from
cell membranes, has several characteristics that render it unsuitable as a blood
substitute, including a low p50, short circulation time, high oncotic pressure, and
vasopressor/nephrotoxic properties.102,103

Chapter 21: Blood Transfusion Practice

However, chemical modifications of hemoglobin solutions may successfully
overcome these disadvantages and such
products are in Phase III clinical trials at
the time of this writing. Patients in these
trials have survived very severe anemia
(residual cellular hemoglobin as low as 1
g/dL) without significant toxicity when
supported by these agents.104 Bovine hemoglobin is approved for veterinary use.
Hemoglobin produced by recombinant
DNA techniques has also been investigated. Finally, fluorocarbon products that
bind oxygen have been extensively investigated.103 One of the latter, Fluosol, was
approved by the FDA for use during percutaneous transluminal angioplasty, but
it is no longer available in the United
States.

DDAVP
DDAVP is a synthetic analogue of the hormone vasopressin that lacks significant
pressor activity.99,105 First used in the treatment of diabetes insipidus, DDAVP is also
useful in promoting hemostasis because
of its ability to cause the release of endogenous stores of high-molecular-weight
vWF from the vascular subendothelium
and the concomitant increase in Factor
VIII. Because of its effect on Factor VIII
and vWF, DDAVP is used as a hemostatic
agent in patients with mild-to-moderate
hemophilia A and in patients with some
von Willebrand syndromes. Because
platelet adhesion and the subsequent formation of a platelet plug depend upon
vWF, DDAVP may also be beneficial in a
wide variety of platelet function disorders, including uremia, cirrhosis, drug-induced platelet dysfunction (including aspirin), primary platelet disorders, and
myelodysplastic syndromes.99,105,106
DDAVP can be administered intravenously, subcutaneously, or intranasally. It is

513

usually given as a single injection (0.3 to 0.4
µg/kg) to treat bleeding or for prophylaxis
before a procedure. Doses are not usually
repeated within a 24- to 48-hour period because of tachyphylaxis (the loss of biologic
effect with repeated administration of an
agent) and the induction of water retention
and hyponatremia. Some patients experience facial flushing or mild hypotension,
but side effects are rare. Its effect on vWF
occurs within 30 minutes and lasts 4 to 6
hours.105

Fibrinolytic Inhibitors
Epsilon aminocaproic acid (EACA) and
tranexamic acid—synthetic analogues of
lysine—competitively inhibit fibrinolysis
by saturating the lysine binding sites at
which plasminogen and plasmin bind to
fibrinogen and fibrin. The drugs can be
used locally or systemically and can be
given orally. Aprotinin is a polypeptide
prepared from bovine lung that inhibits
proteinases including plasmin, kallikrein,
trypsin, and, to some extent, urokinase.
Thus, it has an antifibrinolytic action but
may also inhibit coagulation because
kallikrein activates Factor XII. Aprotinin is
used intravenously. Because it is a polypeptide, hypersensitivity reactions can
occur.99,107
Antifibrinolytic agents have been used
successfully in cardiac surgery, prostatectomy, and liver transplantation. EACA and
tranexamic acid can also be used locally at
sites where fibrinolysis contributes to
bleeding, as from mucosal lesions of the
mouth and gastrointestinal tract, and are of
benefit in the control of hemorrhage following dental extractions in patients with
hemophilia and in the control of gastrointestinal bleeding. EACA and tranexamic
acid may be helpful in controlling bleeding
due to severe thrombocytopenia. Systemic

514

AABB Technical Manual

administration of fibrinolytic inhibitors has
been associated with serious thrombotic
complications, including ureteral obstruction due to clot formation and thrombosis
of large arteries and veins. When used in
excessive doses, fibrinolytic inhibitors can
prolong the bleeding time. These drugs
should be employed by physicians with
experience in their use.
All three of these antifibrinolytic agents,
as well as DDAVP, have been used in an attempt to decrease blood use in cardiac surgery, and meta-analyses have shown a
decrease in the proportion of patients receiving allogeneic RBCs, the number of units
transfused,108,109 estimated blood losses,108
and the number of patients requiring re108,109
operation for bleeding.
Of the three
agents, a much larger data base exists for
aprotinin, which is the most frequently
used. DDAVP was not effective overall but
may be useful in patients taking aspirin.108
Of concern has been a trend toward increased thrombotic complications (myocardial infarction and graft thrombosis)
with aprotinin, but they do not appear to
be statistically significant.107,108 Unfortunately, very large studies would be required
to demonstrate whether the risk profile of
these agents is superior to that of blood,
particularly in view of the decreasing risks
of transfusion.

Oversight of Transfusion
Practice
Of the various institutions that regulate or
accredit aspects of transfusion, the Joint
Commission for Accreditation of Healthcare Organizations (JCAHO) has historically emphasized oversight of transfusion
practice as a requirement for accreditation. As part of its performance improvement standards, the JCAHO requires collection of data regarding the use of blood

and scores the institution on the appropriateness of the selected performance
measures and the size of the data sample.110 Moreover, the JCAHO standards require that the medical staff take the leadership role in measurement, assessment,
and improvement of clinical processes related to the use of blood and blood components. This assessment process must
include peer review, and its findings must
be communicated to involved staff members as well as being a part of the renewal
of their clinical privileges.
Typically, this function has been delegated to a medical staff committee, often a
dedicated “Transfusion Committee.” The
medical director of the transfusion service
should be a member of the committee. This
committee should review blood bank activities and statistics, blood ordering, and
transfusion practices and should have a
process to review records of patients transfused with blood or components. The committee should monitor significant developments in transfusion medicine that would
affect patients in the health-care institution
and take appropriate action regarding these
developments.
The College of American Pathologists
(CAP) laboratory accreditation program
also requires transfusion oversight. This is
mandated by its general standard on quality control and improvement, which states
that the blood bank director must evaluate
the appropriateness of any laboratory’s output in a multidisciplinary fashion.111 Moreover, the CAP accreditation checklist for
blood banks seeks documentation that “. . .
the transfusion service medical director actively participates in establishing criteria
and in reviewing cases not meeting transfu112
sion audit criteria.”
Finally, the AABB Standards requires that
there be a peer-review program that monitors appropriateness of use of blood components.59(p86)

Chapter 21: Blood Transfusion Practice

References
1. Welch HG, Meehan KR, Goodnough LT. Prudent strategies for elective red blood cell
transfusion. Ann Intern Med 1992;116:393402.
2. Wilkerson DK, Rosen AL, Gould SA, et al. Oxygen extraction ratio: A valid indicator of
myocardial metabolism in anemia. J Surg
Res 1987; 42:629-34.
3. Practice guidelines for blood component
therapy: Report by the American Society of
Anesthesiologists Task Force on Blood Component Therapy. Anesthesiology 1996;84:
732-47.
4. Laine E, Steadman R, Calhoun L, et al. Comparison of RBCs and FFP with whole blood
during liver transplant surgery. Transfusion
2003;43:322-7.
5. American Association of Blood Banks, America’s Blood Centers, American Red Cross. Circular of information for the use of human
blood and blood components. Bethesda,
MD: AABB, 2002.
6. Practice parameter for the use of red blood
cell transfusions: Developed by the Red
Blood Cell Administration Practice Guidelines Development Task Force of the College
of American Pathologists. Arch Pathol Lab
Med 1998; 122:130-8.
7. National Institutes of Health Consensus Development Conference. Perioperative red
blood cell transfusion. JAMA 1988;260:
2700-3.
8. Hébert PC, Schweitzer I, Calder L. Review of
the clinical practice literature on allogeneic
red blood cell transfusion. Can Med Assoc J
1997;156(Suppl):S9-26.
9. Goodnough LT, Brecher ME, Kanter MH,
AuBuchon JP. Transfusion medicine: First of
two parts. N Engl J Med 1999;340:438-47.
10. Weiskopf RB, Viele MK, Feiner J, et al. Human cardiovascular and metabolic response
to acute, severe isovolemic anemia. JAMA
1998;279:217-21.
11. Carson JL, Noveck H, Berlin JA, Gould SA.
Mortality and morbidity in patients with
very low postoperative Hb levels who decline
blood transfusion. Transfusion 2002;42:8128.
12. Vamvakas EC. Epidemiology of red blood cell
transfusion. Transfus Med Rev 1996;10:44-61.
13. Bracey AW, Radovancevic R, Riggs SA, et al.
Lowering the hemoglobin threshold for
transfusion in coronary artery bypass procedures: Effect on patient outcome. Transfusion 1999; 39:1070-7.
14. Johnson RG, Thurer RL, Kruskall MS, et al.
Comparison of two transfusion strategies

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

515

after elective operations for myocardial
revascularization. J Thorac Cardiovasc Surg
1992;104:307-14.
Carson JL, Terrin ML, Barton FB, et al. A pilot
randomized trial comparing symptomatic
vs. hemoglobin-level-driven red blood cell
transfusions following hip fracture. Transfusion 1998;38:522-9.
Blair SD, Janvrin SB, McMollum CN, et al. Effect of early blood transfusion on gastrointestinal hemorrhage. Br J Surg 1986;73:783-5.
Hébert PC, Wells G, Blajchman MA, et al. A
multicenter, randomized, controlled clinical
trial of transfusion requirements in critical
care. N Engl J Med 1999;340:409-17.
Hébert PC, Yetisir E, Martin C. Is a low transfusion threshold safe in critically ill patients
with cardiovascular diseases? Critical Care
Med 2001;29:227-34.
Wu WC, Rathore SS, Wang Y, et al. Blood
transfusion in elderly patients with acute
myocardial infarction. N Engl J Med 2001;
345:1230-6.
Elwood PC, Waters WR, Green WJW, et al.
Symptoms and circulating hemoglobin level.
J Chronic Dis 1969;21:615-28.
Canadian Erythropoietin Study Group. Association between recombinant human erythropoietin and quality of life and exercise capacity of patients receiving hemodialysis. Br
Med J 1990;300:573-8.
Parise LV, Smyth SS, Collor BS. Platelet morphology, biochemistry, and function. In:
Beutler E, Lichtman MA, Coller BS, et al. Will i a m s’ h e m a t o l o g y. 6 t h e d . Ne w Yo r k :
McGraw-Hill, 2001:1357-408.
Darcy MD, Kanterman RY, Kleinhoffer MA, et
al. Evaluation of coagulation tests as predictors of angiographic bleeding complications.
Radiology 1996;198:741-4.
Doerfler ME, Kaufman B, Goldenberg AS.
Central venous catheter placement in patients with disorders of hemostasis. Chest
1996;110:185-8.
McVay PA, Toy P. Lack of increased bleeding
after paracentesis and thoracentesis in patients with mild coagulopathy. Transfusion
1991;31:164-71.
Ewe K. Bleeding after liver biopsy does not
correlate with indices of peripheral coagulation. Dig Dis Sci 1981;26:388-93.
McVay PA, Toy P. Lack of increased bleeding
after liver biopsy in patients with mild
hemostatic abnormalities. Am J Clin Pathol
1990;94: 747-53.
Lind SE. Review: The bleeding time does not
predict surgical bleeding. Blood 1991;77:
2547-52.

516

AABB Technical Manual

29. Rodgers RP, Levin J. A critical appraisal of the
bleeding time. Semin Thromb Hemost 1990;
16:1-20.
30. Despotis GJ, Goodnough LT. Management
approaches to platelet-related microvascular
bleeding in cardiothoracic surger y. Ann
Thorac Surg 2000;70:S20-32.
31. Hanson SR, Slichter SJ. Platelet kinetics in
patients with bone marrow hypoplasia: Evidence for a fixed platelet requirement. Blood
1985;66:1105-9.
32. Aster RH. Effect of anticoagulant and ABO incompatibility on recovery of transfused human platelets. Blood 1965;26:732-43.
33. Heal JM, Masel D, Rowe JM, Blumberg N. Circulating immune complexes involving the
ABO system after platelet transfusion. Br J
Haematol 1993;85:566-72.
34. Benjamin RB, Antin JH. ABO-incompatible
bone marrow transplantation: The transfusion of incompatible plasma may exacerbate
regimen-related toxicity. Transfusion 1999;
39:1273-4.
35. Heal JM, Blumberg N. The second century of
ABO: And now for something completely different. Transfusion 1999;39:1155-9.
36. McManigal S, Sims KL. Intravascular hemolysis secondary to ABO incompatible platelet
products; an underrecognized transfusion reaction. Am J Clin Pathol 1999;111:202-6.
37. Norfolk DR, Ancliffe PJ, Contreras M, et al.
Synopsis of background papers (Consensus
Conference on Platelet Transfusion). Br J
Haematol 1998;101:609-14.
38. National Institutes of Health Consensus Development Conference. Platelet transfusion
therapy. Transfus Med Rev 1987;1:195-200.
39. Development Task Force of the College of
American Pathologists. Practice parameter
for the use of fresh frozen plasma, cryoprecipitate, and platelets. JAMA 1994;271:777-81.
40. Schiffer CA, Anderson KC, Bennett CL, et al.
Platelet transfusion for patients with cancer:
Clinical practice guidelines of the American
Society of Clinical Oncology. J Clin Oncol
2001;19:1519-38.
41. Gmur J, Burger J, Schanz U, et al. Safety of
stringent prophylactic platelet transfusion
policy for patients with acute leukemia. Lancet 1991;338:1224-6.
42. Wandt H, Frank M, Ehninger G, et al. Safety
and cost effectiveness of a 10 × 109/L trigger
for prophylactic platelet transfusions compared with the traditional 20 × 109/L trigger;
a prospective comparative trial in 105 patients with acute myeloid leukemia. Blood
1991;10:3601-6.
43. Rebulla P, Finazzi G, Morangoni F, et al. The
threshold of prophylactic platelet transfu-

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

sions in adults with acute myeloid leukemia.
N Engl J Med 1997;337:1870-5.
Murphy S, Litwin S, Herring LM, et al. Indications for platelet transfusion in children with
acute leukemia. Am J Hematol 1982;12:34756.
Reed RL, Ciavarella D, Heimbach DM, et al.
Prophylactic platelet administration during
massive transfusion; a prospective, randomized, double-blind clinical study. Ann Surg
1986;203:40-8.
Simon TL, Akl BF, Murphy W. Controlled trial
of routine administration of platelet concentrates in cardiopulmonary bypass surgery.
Ann Thorac Surg 1984;37:359-64.
Nguyen CM, Harrington RA. Glycoprotein
IIb/IIIa receptor antagonists; a comparative
review of their use in percutaneous coronary
intervention. Am J Cardiovasc Drugs 2003;3:
423-36.
McFarland JG. Matched apheresis platelets.
In: McLeod BC, Price TH, Weinstein R, eds.
Apheresis: Principles and practice. 2nd ed.
Bethesda, MD: AABB Press, 2003:199-220.
Kickler TS, Herman JH, eds. Current issues in
platelet transfusion therapy and platelet alloimmunity. Bethesda, MD: AABB Press, 1999.
Warkentin TE. Management of immune
thrombocytopenia. In: Simon TL, Dzik WH,
Snyder EL, et al, eds. Rossi’s principles of
transfusion medicine. 3rd ed. Baltimore, MD:
Lippincott Williams and Wilkins, 2002:36795.
Gordon LI, Kwaan HC, Rossi EC. Deleterious
effects of platelet transfusions and recovery
thrombocytosis in patients with thrombotic
microangiopathy. Semin Hematol 1987;24:
194-201.
Strauss RG. Granulocyte transfusion. In:
McLeod BC, Price TH, Weinstein R, eds.
Apheresis: Principles and practice. 2nd ed.
Bethesda, MD: AABB Press, 2003:237-52.
Price TH. The current prospects for neutrophil transfusions for the treatment of granulocytopenic infected patients. Transfus Med
Rev 2000;14:2-11.
Vamvakas EC, Pineda A. Determinants of the
efficacy of prophylactic granulocyte transfusions: A meta-analysis. J Clin Apheresis 1997;
12:74-81.
Gresens CJ, Paglieroni TG, Moss CB, et al.
WBC populations in thawed fresh frozen
plasma (abstract). Transfusion 1999;39(Suppl):
99S.
Stringham JC, Bull DA, Fuller TC, et al. Avoidance of cellular blood product transfusions
in LVAD recipients does not prevent HLA
allosensitization. J Heart Lung Transplant
1999;18(2):160-5.

Chapter 21: Blood Transfusion Practice

57. Food and Drug Administration. Memorandum: Recommendations and license requirements for leukocyte-reduced blood
products. (May 29, 1996) Rockville, MD:
CBER Office of Communication, Training,
and Manufacturers Assistance, 1996.
58. Food and Drug Administration. Draft guidance for industry: Prestorage leukocyte reduction with whole blood and blood components intended for transfusion. ( January 23,
2001) Rockville, MD: CBER Office of Communication, Training, and Manufacturers
Assistance, 2001.
59. Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.
60. Preiksaitis J. The cytomegalovirus-“safe”
blood product: Is leukoreduction equivalent
to antibody screening? Transfus Med Rev
2000;14:112-36.
61. The trial to reduce alloimmunization to platelets study group. Leukocyte reduction and
ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to
platelet transfusions. N Engl J Med 1997;337:
1861-9.
62. Vamvakas EC, Dzik WH, Blajchman MA. Deleterious effects of transfusion associated
immunomodulation: Appraisal of the evidence and recommendations for prevention.
In: Vamvakas EC, Blajchman MA, eds. Immunomodulatory effects of blood transfusion. Bethesda, MD: AABB Press, 1999:
253-85.
63. Krishnan LA, Brecher ME. Transfusion transmitted bacterial infection. Hematol Oncol
Clin North Am 1995;9:167-85.
64. Dzik WH, Anderson JK, O’Neill EM, et al. A
prospective, randomized clinical trial of universal WBC reduction. Transfusion 2002;42:
1114-22.
65. Vamvakas EC, Blajchman MA. Universal
WBC reduction: The case for and against.
Transfusion 2001;41:691-712.
66. Akahoshi M, Takanashi M, Masuda H, et al.
Case reports: A case of transfusion-associated graft-versus-host disease not prevented
by white cell-reduction filters. Transfusion
1992; 32:169-72.
67. Roberts HR, Monroe DM III, Hoffman M.
Molecular biology and biochemistry of the
coagulation factors and pathways of hemostasis. In: Beutler E, Lichtman MA, Coller BS,
et al. Williams’ hematology. 6th ed. New
York: McGraw-Hill, 2001:1409-34.
68. van Aken WG. Preparation of plasma derivatives. In: Simon TL, Dzik WH, Snyder EL, et
al, eds. Rossi’s principles of transfusion med-

69.

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

517

icine. 3rd ed. Baltimore, MD: Lippincott Williams and Wilkins, 2002:304-15.
The United States Pharmacopoeial Convention, Inc. Hemophilia management. Transfus
Med Rev 1998;12:128-40.
British Committee for Standards in Haematology, Working Party of the Blood Transfusion Task Force. Guidelines for the use of
fresh frozen plasma. Transfus Med 1992;2:
557-63.
Rock G, Shumak KH, Sutton DM, et al. Cryosupernatant as replacement fluid for plasma
exchange in thrombotic thrombocytopenic
purpura. Members of the Canadian Apheresis Group. Br J Haematol 1996;94: 383-6.
Spector I, Corn M, Ticktin HE. Effect of
plasma transfusions on the prothrombin
time and clotting factors in liver disease. N
Engl J Med 1966;275:1032-7.
Mannucci PM, Vicente V, Vianello L, et al.
Controlled trial of desmopressin in liver cirrhosis and other conditions associated with
a prolonged bleeding time. Blood 1986;67:
1148-53.
Murray DJ, Pennell BJ, Weinstein SL, Olson
JD. Packed red cells in acute blood loss: Dilutional coagulopathy as a cause of surgical
bleeding. Anesth Analg 1995;80:336-42.
National Institutes of Health Consensus Development Conference. Fresh frozen plasma;
indications and risks. JAMA 1985;253:551-3.
Weinstein R. Basic principles of therapeutic
blood exchange. In: McLeod BC, Price TH,
Weinstein R, eds. Apheresis: Principles and
practice. 2nd ed. Bethesda, MD: AABB Press,
2003:295-320.
Jaffee CJ, Atkinson JP, Gelfand JA, Frank MM.
Hereditary angioedema: The use of fresh
frozen plasma for prophylaxis in patients
u nd ergoing ora l su rger y. J Allergy Clin
Immunol 1975;545:386-93.
Ginsburg D. von Willebrand disease. In:
Beutler E, Lichtman MA, Coller BS, et al, eds.
Williams’ hematology. 6th ed. New York:
McGraw-Hill, 2001:1813-28.
Rousou J, Levitsky S, Gonzalez-Levin J, et al.
Randomized clinical trial of fibrin sealant in
patients undergoing resternotomy or reoperation after cardiac operations. J Thorac
Cardiovasc Surg 1989;97:194-203.
Streiff MB, Ness PM. Acquired FV inhibitors:
A needless iatrogenic complication of bovine
thrombin exposure. Transfusion 2002;42:
18-26.
Roberts HR, Hoffman M. Hemophilia A and
hemophilia B. In: Beutler E, Lichtman MA,
Coller BS, et al, eds. Williams’ hematology.
6th ed. New York: McGraw-Hill, 2001:163971.

518

AABB Technical Manual

82. Smith KJ. Factor IX concentrates: The new
products and their properties. Transfus Med
Rev 1992;6:124-36.
83. Nydegger UE, Mohacsi PJ. Immunoglobulins
in clinical medicine. In: Simon TL, Dzik WH,
Snyder EL, et al, eds. Rossi’s principles of
transfusion medicine. 3rd ed. Baltimore, MD:
Lippincott Williams and Wilkins, 2002:31632.
84. Kobayashi RH, Stiehm ER. Immunoglobulin
therapy. In: Petz LD, Swisher SN, Kleinman S,
et al, eds. Clinical practice of transfusion
medicine. 3rd ed. New York: Churchill Livingstone, 1995:985-1010.
85. G o o d n i g h t S W, G r i f f i n J H . He r e d i t a r y
thrombophilia. In: Beutler E, Lichtman MA,
Coller BS, et al, eds. Williams’ hematology.
6th ed. New York: McGraw-Hill, 2001:1697714.
86. Griffin JH. Control of coagulation reactions.
In: Beutler E, Lichtman MA, Coller BS, et al,
eds. Williams’ hematology. 6th ed. New York:
McGraw-Hill, 2001:1435-49.
87. Bucar SJ, Levy JH, Despotis GJ, et al. Uses of
antithrombin III concentrate in congenital
and acquired deficiency states. Transfusion
1998;38:481-98.
88. Friedman KE, Menitove JE. Preparation and
clinical use of plasma and plasma fractions.
In: Beutler E, Lichtman MA, Coller BS, et al,
eds. Williams’ hematology. 6th ed. New York:
McGraw-Hill, 2001:1917-34.
89. McClelland DBL. Safety of human albumin as
a constituent of biologic therapeutic products. Transfusion 1998;38:690-4.
90. Doweiko JP, Nompleggi DJ. The role of albumin in human physiology and pathophysiology. Part III: Albumin and disease states. J
Parenter Enteral Nutr 1991;15:476-83.
91. Vermeulen LC Jr., Ratko TA, Erstad BL, et al. A
paradigm for consensus. The University Hospital consortium guidelines for the use of albumin, nonprotein colloid, and crystalloid
solutions. Arch Intern Med 1995;155:373-9.
92. Cochrane Injuries Group Albumin Reviewers.
Human albumin administration in critically
ill patients: Systemic review of randomized
controlled trials. Br Med J 1998;317:235-46.
93. Vichinsky EP, Haberkern CM, Newmayer L, et
al. A comparison of conservative and aggressive transfusion regimens in the perioperative management of sickle cell disease. N
Engl J Med 1995;333:206-13.
94. Rosse W, Telen M, Ware R. Transfusion support for patients with sickle cell disease.
Bethesda, MD: AABB Press, 1998.
95. The National Institutes of Health, National
Heart, Lung and Blood Institute, Division of
Blood Diseases and Resources. The manage-

96.

97.

98.

99.
100.

101.

102.

103.

104.

105.

106.

107.

108.

ment of sickle cell disease. 4th ed. Bethesda,
MD: NHLBI, 2002. (Available at http://www.
nhlbi.nih.gov/health/prof/blood/sickle/
sc_mngt.pdf.)
Mollison PL, Engelfriet CP, Contreras M.
Blood transfusion in clinical medicine. 10th
ed. Oxford, England: Blackwell Scientific
Publications, 1998.
Ciavarella D, Reed RL, Counts RB, et al. Clotting factors and the risk of diffuse microvascular bleeding in the massively transfused
patient. Br J Haematol 1987;67:365-8.
Heaton A, Keegan T, Holme S, et al. In vivo regeneration of red cell 2,3-diphosphoglycerate
following transfusion of DPG-depleted AS-1,
AS-3 and CPDA-1 red cells. Br J Haematol
1989;71:131-6.
Mannucci PM. Drug therapy: Hemostatic
drugs. N Engl J Med 1998;339:245-53.
Kruskall MS. Biologic response modifiers—
hematopoietic growth factors. In: Petz LD,
Swisher SN, Kleinman S, et al, eds. Clinical
practice of transfusion medicine. 3rd ed. New
York: Churchill Livingstone, 1996:1023-39.
Corwin HL, Gettinger A, Pearl RG, et al. Efficacy of recombinant human erythropoietin
in critically ill patients: A randomized, controlled trial. JAMA 2002;288:2827-35.
Spence RK. Blood substitutes. In: Petz LD,
Swisher SN, Kleinman S, et al, eds. Clinical
practice of transfusion medicine. 3rd ed. New
York: Churchill Livingstone, 1996:967-84.
Stowell CP, Levin J, Spiess BD, Winslow RM.
Progress in the development of RBC substitutes. Transfusion 2001;41:287-99.
Gould S, Moore ED, Hoyt DB, et al. The lifesustaining capacity of human polymerized
hemoglobin when red cells might be unavailable. J Am Cell Surg 2002;195:445-52.
Schulman S. DDAVP, the multipotent drug in
patients with coagulopathies. Transfus Med
Rev 1991;5:132-44.
Shattil SJ, Abrams CS, Bennett JS. Acquired
qualitative platelet disorders due to diseases,
drugs, and foods. In: Beutler E, Lichtman MA,
Coller BS, et al, eds. Williams’ hematology.
6th ed. New York: McGraw-Hill, 2001:1583602.
Bachman F. Disorders of fibrinolysis and use
of antifibrinolytic agents. In: Beutler E, Lichtman MA, Coller BS, et al, eds. Williams’ hematology. 6th ed. New York: McGraw-Hill,
2001:1829-40.
Laupacis A, Fergusson D. Drugs to minimize
perioperative blood loss in cardiac surgery:
Meta-analysis using perioperative blood
transfusion as the outcome. Anesth Analg
1997;85:1258-67.

Chapter 21: Blood Transfusion Practice

109. Munoz JJ, Birkmeyer NJ, Birkmeyer JD, et al.
Is epsilon-aminocaproic acid as effective as
aprotinin in reducing bleeding with cardiac
surgery? A meta-analysis. Circulation 1999;
99:81-9.
110. Jo i n t Co m m i s s i o n f o r Ac c re d i t a t i o n o f
Healthcare Organizations. 2004 Comprehensive accreditation manual for hospitals,
standard PI.3.1.1 and MS.8.1.3 and MS.8.3.

519

Oakbrook, IL: Joint Commission Resources,
2004.
111. Hamlin WB. Requirements for accreditation
by the College of American Pathologists Laboratory Accreditation Program. Arch Pathol
Lab Med 1999;123:465-7.
112. CAP Transfusion Medicine Checklist. Revision date 12/31/2003. Northfield, IL: College
of American Pathologists, 2003.

Chapter 22: Administration of Blood and Components

Chapter 22

Administration of Blood and
Components

OOD TRANSFUSION PRACTICE
requires that comprehensive policies and procedures for blood administration be designed to prevent errors.
The development of these policies should
be a collaborative effort between the medical director of the transfusion service, the
directors of the clinical services, both nursing and medical, and all personnel involved
in blood administration. Policies and procedures must be accessible, periodically
reviewed for appropriateness, and monitored for compliance. In addition to blood
administration policies and procedures,
this chapter discusses pretransfusion preparation, issuing of blood components,
the equipment used in blood administration, and compatible intravenous solutions.

Pre-Issue Events
Patient Education and Consent
Patients who are aware of the steps involved in a transfusion will experience less
anxiety. This is important not only for an

adult but also for any child who has the
ability to understand the process. In the
latter situation, it is appropriate to educate the parents, so that they are better
prepared to support their child throughout the transfusion. The transfusionist
should explain how the transfusion will be
given, how long it will take, what the expected outcome is, what symptoms to report, and that vital signs will be taken. The
physician has a responsibility to explain
the benefits and risks of transfusion therapy as well as the alternatives in a fashion
that the patient can comprehend. Other
than in emergency situations, the patient
should be given an opportunity to ask
questions, and his or her informed choice
should be documented. State and local
laws govern the process of obtaining and
documenting the consent of the patient.
Some states have specific requirements
for blood transfusion consent. Institutions should be careful to ensure that
their individual processes and procedures
comply with applicable laws.
521

522

AABB Technical Manual

Individual institutions have different requirements for obtaining and documenting
this interaction, as well as different policies
about how often it is necessary. Some facilities require the use of a formal consent
form, which provides information in understandable language, signed by the patient. Others expect the physicians to make
a note in the medical record stating that the
risks of, and alternatives to, blood transfusions were explained and that the patient
consented. If a patient is unable to give
consent, a responsible family member
should be asked. If no family member is
available or if the emergency need for
transfusion leaves no time for consent, it is
prudent to note this in the medical record.1-3

Prescription and Special Instructions
There must be documentation of the order by the physician for the blood component(s).4 Although a telephone order may
be acceptable during urgent situations, this
must be followed by a written request.
Special instructions should be indicated
regarding the transfusion relating to the:
■
Component—eg, washed, irradiated,
leukocyte-reduced, cytomegalovirus
negative
■
Patient—eg, premedication, timing
■
Process—flow rates, rate of infusion,
use of a blood warmer or electromechanical pump
■
Need for emergency release

vice must strive to make every effort to
ensure that the component is ready when
needed, but not so early that it expires before administration.
Medical and nursing staffs need to be
aware of special requirements for preparation and to understand that these times
cannot be significantly shortened, even in
urgent situations. Close communication is
required. Some examples of pretransfusion
processing procedures are given in Table
22-1.

Patient Considerations
Patients with a history of recurrent allergic transfusion reactions may benefit from
premedication with antihistamines or by
slowing the rate of transfusion. Routine
premedication with antipyretics should
be discouraged because delaying a rise in
temperature may mask one sign of a
hemolytic reaction and partly because
they may be ineffective.5-7
Antipyretics typically do not mask other
clinical features of hemolysis, such as
changes in blood pressure, pulse, or respiration. Premedication orders should be
carefully timed with the anticipated administration of the unit. Medication ordered intravenously may be given immediately before the start of the transfusion, but orally
administered drugs need to be given 30 to
60 minutes before the start of the transfusion.

Process Considerations
Component Considerations
Some components require special preparation before release for transfusion. Because these steps are time-consuming and
may significantly shorten the shelf life of
the component, preparation should be
carefully coordinated with the anticipated
time of transfusion. The transfusion ser-

Blood warmers and electromechanical
pumps need to be available if required for
the transfusion.

Emergency Release
Blood may be released without completing pretransfusion testing if it is urgently
needed for a patient’s survival, provided
that: 1) the records properly document

Chapter 22: Administration of Blood and Components

523

Table 22-1. Component Preparation Times
Component

Minimum Time*

Shelf Life

RBCs: saline-washed

45 minutes

24 hours

RBCs: thawed-deglycerolized

75 minutes

24 hours or 2 weeks†

Fresh Frozen Plasma: thawed

30 minutes

24 hours

Thawed Plasma

—

5 days

Platelets: pooled

15 minutes

4 hours

CRYO: thawed (single unit)

15 minutes

6 hours

CRYO: pooled

15 minutes

4 hours

*Will vary with institutional procedures.
†
Depends on the method used.
RBCs = Red Blood Cells; CRYO = Cryoprecipitated AHF.

the emergency request and 2) the issued
units are of an ABO group unlikely to
cause immediate harm to the recipient.

Venous Access
To avoid any delay in transfusion and potential wastage of blood components, venous access should be established before
the component is issued. If a pre-existing
line is to be used, it should be checked for
patency; signs of infiltration, inflammation, or infection; and the compatibility of
any intravenous solutions (see below). Many
venous access devices can be used for
blood component transfusion. Selection
depends on the location, size, and integrity of the patient’s veins; the type of medication or solution to be infused; the type
of component to be transfused; the volume and timing of the administration; the
possibility of interactions among parenteral solutions; and expected duration of
intravenous therapy.
The lumen of needles or catheters used
for blood transfusion should be large
enough to allow appropriate flow rates

without damaging the vein. There are no
strict guidelines limiting the size of the
catheter or needles used for transfusion. An
18-gauge catheter provides good flow rates
for cellular components without excessive
discomfort to the patient, but patients with
small veins require much smaller catheters.
High-pressure flow through needles or
catheters with a small lumen may damage
red cells8-10 unless the transfusion compo10
nent is sufficiently diluted. Undiluted
preparations of red cells flow very slowly
through a 23-gauge needle, but dilution
with saline to increase the flow rate may
cause unwanted volume expansion. Even
in patients with cardiac disease or volume
expansion, transfusions should be able to
be given safely within 4 hours. For rare patients unable to tolerate a transfusion within 4 hours, local policy should be developed
regarding whether to split units or to discard the unused portion. Specific models of
infusion pumps have been approved for
use in blood transfusion. These pumps
maintain a constant delivery of blood, and
studies have indicated no significant evidence of hemolysis as the needle size varies.11

524

AABB Technical Manual

Central venous catheters are used for
medium- and long-term therapy or for the
administration of solutions potentially toxic
to a peripheral vein to allow the dilution
achieved with high-volume blood flow.
Some catheters are placed via special introducing needles and guide-wires; others are
surgically implanted. Catheters with a
multilumen design have separate infusion
ports for each lumen, permitting the simultaneous infusion of fluids without intermixture in the infusion line, thereby avoiding
the potential for hemolysis from incompatible fluids.

Need for Compatibility Testing
The transfusion service personnel determine what pretransfusion testing is required. Compatibility testing must be performed for transfusion of Whole Blood,
components with a clinically significant
red cell content, and all red cell components. For test and specimen requirements, refer to Chapter 18. Compatibility
testing other than ABO and Rh typing is
not required for platelet and plasma components, but most facilities require that
the recipient’s ABO and D types be known
(on file) before such components are selected for issue. Whenever possible, plasmacontaining components should be compatible with the patient’s red cells (see
Chapter 21).

Blood Issue and
Transportation
Delivering Blood to the Patient Area
Institutions should define blood pick-up
and delivery policies and appropriate
training programs for the staff assigned to
these functions. Blood is not routinely
dispensed from the controlled environment of the blood bank until all testing is

completed, the patient is properly prepared, and the transfusionist is ready to
begin the procedure. There must be a
mechanism to identify the intended recipient and the requested component at
the time of issue. It is optimal to identify
the transporter.
Transfusion service personnel will review identifying information, inspect the
appearance of the component before release, and ensure there is a system to maintain proper storage temperature during
transport. The safest practice is to issue one
unit to one patient at a time. For patients
who are rapidly bleeding or who have multiple venous access sites, multiple units
may be issued to a single patient. It is not
recommended that blood for two or more
patients be issued simultaneously to one
transporter because this could increase the
chance of transfusion error. However, logistical considerations may make this impractical. The transporter should transport the
blood to the intended site of transfusion
without delay—preferably to the transfusionist. It is preferable to place the unit of
blood in a protective container that would
contain any spillage in the event of inadvertent breakage during transport.
The responsibility for accurately identifying a transfusion component rests with both
the transfusion service personnel who issue
the blood and the transfusionist who receives it. Before a unit of blood is issued,
transfusion service personnel complete the
following steps:
1.
The records that identify the intended recipient and the requested
component are reviewed.
2.
The identifiers (Standards requires
at least two) of the intended recipient, the ABO and D type of the recipient, the component unit number,
the ABO and D type of the donor
unit, and the interpretation of compatibility tests (if performed) are re-

Chapter 22: Administration of Blood and Components

corded on a transfusion form for
each unit. This form, or a copy, becomes a part of the patient’s permanent medical record after completion of the transfusion. In some
institutions, the transfusion form is
attached to the unit and, therefore,
serves as the tie tag that is required
and described below. This form typically will have fields to identify the
transfusionist and co-identifier (if required) and other information, such
as pre- and posttransfusion vital signs,
amount of blood given, whether a reaction occurred, etc.
A tie tag or label with the name and
identification number of the intended recipient, the component unit
number, and the interpretation of
compatibility tests (if performed) must
be securely attached to the blood
container.
The appearance of the unit is checked
before issue and a record is made of
this inspection.
The expiration date (and time if applicable) is checked to ensure that the
unit is suitable for transfusion.
The name of the person issuing the
blood and the date and time of issue
are recorded. Recording the name of
the transporter to whom the blood is
issued is optimal.

Delay in Starting the Transfusion
Ideally, blood should be requested from
the blood bank only at the time when it is
intended to be administered. If the transfusion cannot be initiated promptly, the
blood should be returned to the blood
bank for storage, unless the transfusion to
the originally intended recipient can be
completed within 4 hours. It should not
be left at room temperature or stored in
an unmonitored refrigerator. Units re-

525

turned to the blood bank after a period
outside monitored refrigeration will be
unsuitable for reissue if the sterility of the
container is compromised or if the temperature has risen to 10 C or above, which
is generally considered to happen in less
than 30 minutes. If the units have been
kept in suitable conditions, such as iced
coolers that have been validated for several hours of storage, longer periods are
acceptable. Requirements for blood collected intraoperatively differ. See Table
5-8. Units that have been entered (punctured) after release from the blood bank
cannot be accepted into general inventory for later reissue.

Pre-Administration Events
Identifying the Recipient and Donor Unit
Accurate identification of the transfusion
component and the intended recipient
may be the single most important step
in ensuring transfusion safety.12-15 Most
hemolytic transfusion reactions and
deaths occur because of inadvertent administration of ABO-incompatible red
cells.14-15 Plasma and platelets are also capable of causing serious transfusion reactions.16 Identification and labeling of donor blood are discussed in Chapter 7;
procedures to identify the patient’s specimen used for compatibility testing are
discussed in Chapter 18. The most important steps in safe transfusion administration are clerical and occur when the
transfusion service issues blood for a specific patient and when the blood is administered.
The transfusionist who administers the
blood represents the last point at which
identification errors can be detected before
transfusion of the component is initiated.
The transfusionist must check all identifying information immediately before begin-

526

AABB Technical Manual

ning the transfusion and record that this information has been checked and found to
be correct, typically, on the transfusion
form. Any discrepancy must be resolved
before the transfusion is started. It is common practice in many institutions that a
second person (co-identifier) confirms the
identity of the blood unit and of the patient.
Some institutions may require that the
transfusionist check for documentation of
patient consent before blood is given. It is
also common practice to check the ABO
and D type as written in the transfusion
form with a record of ABO and D type in
the patients’ chart. The following information, however, must be reviewed and found
to be correct:
1.
Physician’s order. The nature of the
blood or component should be
checked against the physician’s written order to verify that the correct
component and dose (number of
units) are being given. All identification attached to the container must
remain attached until the transfusion has been completed.
2.
Recipient identification. The patient’s
identifiers on the patient’s identification band must be identical with the
identifiers attached to the unit. It is desirable to ask the patient to state his or
her name, if capable of so doing, because the information on the identification band may be in error.17
3.
Unit identification. The unit identification number on the blood container, the transfusion form, and the
tie tag attached to the unit (if not the
same as the latter) must agree.
4.
ABO and D. The ABO and D type on
the primary label of the donor unit
must agree with those recorded on
the transfusion form. The recipient’s
ABO and D type must be recorded
on the transfusion form. The patient’s type and the type of the com-

ponent may not be identical (see
Chapter 21), but the information on
the transfusion form and that on the
container label must be the same.
5.
Expiration. The expiration date and
time of the component should be
verified as acceptable.
6.
Compatibility. The interpretation of
compatibility testing (if performed)
must be recorded on the transfusion
form and on the tag attached to the
unit (if not the same). If blood was
issued before compatibility tests were
completed, this must be conspicuously indicated.
In certain clinical situations, such as the
operating room (OR), emergency room, or
in the outpatient setting, an identifying
band may not be attached to all patients at
the time of transfusion. Wristbands are frequently removed in the OR for arterial line
insertions and identity confirmation may
not be possible in this manner. Furthermore, a co-identifier confirming identity,
although always desirable, may not function for all circumstances in this setting.
Institutions should address such clinical
situations, and site-specific transfusion
protocols should be developed.18

Starting the Transfusion
After checking all the identifying information, the transfusionist (and co-identifier)
must indicate in the medical record that
the identification was correct (such as by
signing the transfusion form) to document who started the transfusion and to
record the date and time. Vital signs
should be taken and recorded, if not done
previously. A record of the date and time
of transfusion, the name and volume of
the component, and vital signs may be required on other parts of the medical record, such as intake/output records, anes-

Chapter 22: Administration of Blood and Components

thesia records, or intensive care flow
sheets, depending on the institution’s policy. This may suffice for documentation
purposes.
Several reports have documented the occurrence of errors at the point of blood administration.19-22 In particular, Baele and
colleagues19 studied the charts and records
of 808 patients who received 3485 units of
blood over a period of 15 months, to determine if there had been errors in blood administration. They detected 165 errors occurring after blood units had left the blood
bank, 15 of which were considered to be
major. Seven of the major errors involved
patient misidentification that resulted in
blood being given to patients for whom it
was not intended, constituting 0.74% of patients and 0.2% of units. One error resulted
in an ABO-incompatible hemolytic reaction
that was not reported to the blood bank.
Eight other major errors occurred in four
patients (0.5%), including the administration of five allogeneic units to a patient for
whom autologous blood was available, and
the transfusion of one anemic patient
whose doctor had ordered only a crossmatch. The remaining 150 errors included
misrecording (n = 61), mislabeling (n = 6),
and failure to adequately document the
transfusion (n = 83).
Both mechanical barrier systems23 and
electronic means of patient and product
identifications are marketed to supplement
(but not substitute for) the paper identification process. In electronic systems, a bar
code or radio frequency identifiers are attached to the blood component (and transfusion form) that, when scanned/read, will
match the patient’s information on the
identification band.24 It is likely that these
electronic means will find more widespread
use in the future. Empiric experience with
medication administration, however, suggests that unanticipated side effects of such
procedures may need consideration. 25

527

Compliance with institutional blood administration policies requires a Quality
Assurance/Continuous Improvement program in which continued monitoring and
re-education of staff occur when variance
with procedures is observed. Such an approach has been initiated in some institutions, resulting in improvement in transfusion practice. 2 6 Direct observation of
27
28
administration or educational videos
may also be useful.

Administration
Infusion Sets
Any blood component must be administered through a filter designed to retain
blood clots and particles potentially harmful to the recipient.4,29(p48) All filters and infusion devices must be used according to
the manufacturer’s directions.

Standard Sets
Standard blood infusion sets have inline
filters (pore size: 170 to 260 microns), drip
chambers, and tubing in a variety of configurations. Sets should be primed according to the manufacturer’s directions,
using either the component itself or a solution compatible with blood (see the section on Compatible IV Solutions). For optimal flow rates and performance, filters
should be fully wetted. Drip chambers
should be half-filled to allow observation
of blood flow.
Many institutions have a policy of changing sets after every transfusion or of limiting their use to several units or several
hours in order to reduce the risks of bacterial contamination. A reasonable time limit
is 4 hours; this is consistent with the 4-hour
outdate that the Food and Drug Administration (FDA) places on blood in an open
system held at room temperature. Most

528

AABB Technical Manual

standard filters are designed to filter 2 to 4
units of blood, but if the first unit required 4
hours for infusion, the filter should not be
reused. The filter traps cell aggregates, cellular debris, and coagulated proteins, resulting in a high protein concentration at
the filter surface. The high protein milieu
and room temperature conditions promote
growth of any bacteria that might be present. Accumulated material also slows the
30
rate of flow.

Special Sets
High flow sets for rapid transfusion have
large filter surface areas, large-bore tubing, and may have an inline hand pump.
Sets designed for rapid infusion devices
also may have “prefilters” to retain particles over 300 microns in diameter and to
extend the life of standard blood filters
“downstream.” Gravity-drip sets for the
administration of platelets and cryoprecipitate have small drip chamber/filter areas, shorter tubing, and smaller priming
volumes. Syringe-push sets for component administration have the smallest
priming volumes and an inline blood filter that may be inconspicuous.

Microaggregate Filters
Microaggregate filters are designed for the
transfusion of red cells. Microaggregates
(smaller than 170 microns in size) pass
through standard blood filters. Screen- or
depth-type filters have an effective pore
size of 20 to 40 microns and trap the
microaggregates composed of degenerating platelets, leukocytes, and fibrin strands
that form in blood after 5 or more days of
refrigerated storage.
Microaggregate filters may be used for
other components if this use is mentioned
in the manufacturer’s instructions; how-

ever, the large volume required for priming
causes a significant portion of these components to be lost if the set is not flushed
with saline afterward. Depth-type microaggregate filters, or any filters capable of removing leukocytes, must not be used for
the transfusion of granulocyte concentrates.29(p48) Hemolysis of red cells has been
30
reported with microaggregate filters.

Leukocyte Reduction Filters
Special “third-generation” blood filters
can reduce the number of leukocytes in
red cell or platelet components to less
than 5 × 106, a level that reduces the risk of
HLA alloimmunization and the transmission of cytomegalovirus as well as the incidence of febrile nonhemolytic transfusion reactions31-35 (see Chapters 8, 21, and
27). These filters contain multiple layers
of synthetic nonwoven fibers that selectively retain leukocytes but allow red cells
or platelets to pass, depending on the filter type. Selectivity is based on cell size,
surface tension characteristics, the differences in surface charge, density of the
blood cells, and, possibly, cell-to-cell interactions and cell activation/adhesion
properties.36 Because filters for red cells
and filters for platelets do not use the
same technology for leukocyte removal
and may have strict priming and flow rate
requirements, they must be used only
with their intended component and only
according to the manufacturer’s directions.37
The use of these filters at the bedside is
more complex than the use of standard infusion sets. The filters are expensive and
can be ineffective or may clog, if improp38-40
erly used. Those designed only for gravity infusion should not be used with infusion pumps or applied pressure. A quality
control program that measures the effectiveness of leukocyte reduction is impor-

Chapter 22: Administration of Blood and Components

tant, but impractical, at the bedside; therefore, adherence to proper protocol is very
important.

Blood Warmers
It is desirable for the medical staff of the
transfusion service to participate in the
assessment and selection of transfusion
equipment and ensure that such items
are included in the facility’s quality assurance program. The performance of devices such as blood warmers or infusion
pumps must be validated before the
equipment is used and must be monitored regularly throughout the facility to
identify malfunctions and ensure appropriate use. This characteristically requires
cooperation among personnel of several
hospital departments, including transfusion medicine, nursing, anesthesiology,
quality assurance, and clinical engineering.
Patients who receive blood or plasma at
rates faster than 100 mL/minute for 30
minutes are at increased risk for cardiac arrest unless the blood is warmed.41 Rapid infusion of large volumes of cold blood can
lower the temperature of the sinoatrial
node to below 30 C, at which point an arrhythmia can occur.
Transfusions at such rapid rates generally occur only in the OR or trauma settings.
There is no evidence that patients receiving
1 to 3 units of blood over several hours have
a comparable risk for arrhythmias; therefore, routine warming of blood is not recommended. 4 2 Several types of blood
warmers are available: thermostatically
controlled waterbaths; dry heat devices
with electric warming plates; and high-volume countercurrent heat exchange with water jackets.43 Blood warming devices must
not raise the temperature of blood to a level
that causes hemolysis.29(p6)

529

Devices should have a visible thermometer and, ideally, an audible alarm that
sounds before the manufacturer’s designated temperature limit is exceeded. The
standard operating procedure for warming
blood should include guidelines on performing temperature and alarm checks,
and instructions on what action to take
when warmers are out of range or the alarm
activates.44 Conventional microwave ovens
and microwave devices for thawing plasma
are not designed for warming other blood
components and can damage red cells.

Electromechanical Infusion Devices
Mechanical pumps that deliver infusions
at a controlled rate are useful, especially
for very slow rates of transfusion used for
pediatric, neonatal, and selected adult
patients. Some pumps use a mechanical
screw drive to advance the plunger of a
syringe filled with blood; others use roller
pumps or other forms of pressure applied
to the infusion tubing. Although some can
be used with standard blood administration sets, many require special plastic
disposables or tubing supplied by the
manufacturer. Blood filters can be added
to the required setups upstream of the
pumps.
The manufacturer should be consulted
before blood is administered with an infusion pump designed for crystalloid or
colloid solutions. Many induce hemolysis,
but of a magnitude that does not adversely
affect the patient.43 Red cells in components
with high hematocrit and high viscosity are
more likely to be hemolyzed when infused
under pressure than red cells in Whole
Blood or red cell components prepared in a
manner that reduces viscosity, such as additive solutions.45 Platelets and granulocytes
appear to sustain no adverse effects when
infused with a pumping device.46,47 Proper

530

AABB Technical Manual

training of personnel and appropriate policies for maintenance and quality control
should reduce the chances of damage to
transfused components.

Pressure Devices
Urgent transfusion situations may require
flow rates faster than gravity can provide.
The simplest method to speed infusion is
to use an administration set with an inline
pump that the transfusionist squeezes by
hand. Pressure bags specially designed as
compression devices are also available.
These devices operate much like blood
pressure cuffs except that they completely
encase the blood bag and apply pressure
more evenly to the bag’s surface. Such devices should be carefully monitored during use because pressures greater than
300 mm Hg may cause the seams of the
blood bag to rupture or leak and air embolism is a concern. Large-bore needles
are traditionally recommended for venous
access when the use of external pressure
is anticipated, but recent data question
this practice.11 Manually forcing red cells
through a small-gauge line has been
shown to cause hemolysis.45
Devices for intraoperative and postoperative blood collection are discussed in
Chapter 5.

Compatible IV Solutions
AABB Standards for Blood Banks and
29(p48)
Transfusion Services
and the Circular
of Information for the Use of Human
Blood and Blood Components48 are explicit
in stating that medications must not be
added to blood or components. If red cells
require dilution to reduce their viscosity
or if a component needs to be rinsed from
the blood bag or tubing, normal saline
(0.9% sodium chloride injection, USP)
can be used. Red cells prepared with an

additive solution (AS) ordinarily do not
require dilution. These red cell components have a hematocrit of approximately
60%. Other solutions intended for intravenous use may be added to blood or components or may come into contact with
blood in an administration set only if they
have been approved for this use by the
FDA or if there is documentation to show
that their addition to blood is safe and ef29(p6),48
Calcium-free, isotonic
ficacious.
electrolyte solutions that meet the above
requirements also may be used, but they
usually are more expensive than saline
and offer little benefit in routine transfusion. Lactated Ringer’s solution, 5% dextrose, and hypotonic sodium chloride solutions should not be added to blood.
Dextrose solution may cause red cells to
clump in the tubing and, more important,
to swell and hemolyze as dextrose and associated water diffuse from the medium
into the cells. Lactated Ringer’s solution
contains enough ionized calcium (3
mEq/L) to overcome the chelating agents
in anticoagulant-preservative or additive
solutions, which results in clot develop49-51
ment.

Patient Care During Transfusion
The transfusionist should either remain
with, or be in a position to closely observe,
the patient for at least the first 15 minutes
of the infusion. The transfusion should be
started slowly at a rate of approximately 2
mL/minute except during urgent restoration of blood volume. Catastrophic reactions from acute hemolysis, anaphylaxis,
transfusion-related acute lung injury, or
bacterial contamination can become apparent after a very small volume enters
the patient’s circulation (see Chapter 27).
After the first 15 minutes, some institutions record vital signs, but this is unneces-

Chapter 22: Administration of Blood and Components

sary if the patient’s condition is satisfactory.
The rate of infusion can be increased to
that specified in the order or to be consistent with institutional practice (approximately 4 mL/minute). The desirable rate of
infusion depends upon the patient’s blood
volume, cardiac status, and hemodynamic
condition. No experimental or clinical data
exist to support a specific time restriction;
however, the Circular of Information48 gives
4 hours as the maximum duration for an
infusion. Maximum time should not be
confused with recommended time. Most
RBC units are transfused within 1 to 2
hours, whereas platelet or plasma transfusions are commonly administered over a
shorter period (30 to 60 minutes). However,
there is no physiologic reason to administer
compatible red cells more slowly than
plasma or platelets and rapid infusion of
these products may increase the risk of adverse events. If rapid transfusion is needed,
blood can be infused as rapidly as the patient’s circulatory system will tolerate and
the type of vascular access will allow.
If it is anticipated that an infusion time
of longer than 4 hours may be required, the
physician covering the transfusion service
should be notified to assess the specific
clinical situation. Administration rates are
calculated by counting the drops per minute in the drip chamber and dividing this
number by the “drop/mL” rating of the infusion system. Blood may flow more slowly
than desired as a result of obstruction of
the filter or when there is excessive viscosity of the component. Steps to investigate
and correct the problem include the following:
■
Elevate the blood container to increase hydrostatic pressure.
■
Check the patency of the needle.
■
Examine the filter of the administration set for excessive debris.
■
Consider the addition of 50 to 100
mL of saline to a preparation of red

531

cells, if there is an order permitting
such addition.
Clinical personnel should continue to
observe the patient periodically throughout
the transfusion (eg, every 30 minutes) and
up to an hour after completion.

Action for Suspected Reactions
Most transfusions proceed without complication, but when adverse reactions do
occur, medical and nursing staff must be
prepared to deal with them immediately.
Different types of reactions, their etiology,
symptoms, treatment, and prevention are
discussed in Chapter 27. Because severity
can vary significantly and symptoms are
not specific, all transfusions must be carefully monitored and stopped as soon as a
reaction is suspected. It may be helpful to
summarize common symptoms and the
immediate steps to take on the transfusion form that accompanies the unit. This
eliminates the need to search for instructions and helps standardize patient care
in an urgent situation.

Post-Administration Events
After each unit of blood has been infused,
personnel should measure vital signs; record the time, the volume, and the component given; record the patient’s condition; and record the identity of the person
who stopped the transfusion (if not the
transfusionist), made the observations,
and measured and recorded vital signs.
Many transfusion services require that a
copy of the completed transfusion form
be returned to the laboratory to document the unit’s disposition for laboratory
records. The empty blood bag need not be
returned after uncomplicated transfusions, but bags, tubing, and attached solutions should be returned to the transfusion service if a complication occurs.

532

AABB Technical Manual

Some transfusion services choose to have
all bags returned following all transfusions in order to investigate reactions that
may not be evident at the time of transfusion. Proper biohazard precautions
should be used in the handling of entered
containers and used administration sets.
The patient should remain under observation after the transfusion has been
completed, if this is practical. Because not
all severe reactions are immediately apparent, all patients who receive transfusions in outpatient or home care settings,
or their caretakers, must be given clearly
written instructions outlining posttransfusion care, a description of the signs and
symptoms of acute and delayed reactions,
and the appropriate action to take if such
are noted.

References
1.

2.
3.
4.

Quality Assurance
The process of blood administration should
begin and end with patient safety in
mind, starting with the generation of an
appropriate order; continuing through
collection of the patient’s pretransfusion
specimen, preparation and delivery of the
unit, identification of the unit with the recipient, and selection and proper use of
equipment; and concluding with patient
care during the transfusion and maintenance of appropriate records. Policies,
procedures, training, and assessment are
all critical to this process and must be
monitored as parts of blood usage review.
Periodic auditing of the blood administration process and forms should be performed in order to identify patterns of nonconformance. Errors, regardless of clinical
outcome, should be subjected to root-cause
analysis because such errors are often indicative of systematic problems.

10.

11.

12.

13.

14.

15.

16.

Sazama K. Practical issues in informed consent for transfusion. Am J Clin Pathol 1997;
107:572-4.
Holland PV. Consent for transfusion: Is it informed? Transfus Med Rev 1997;11:274-85.
Williams FG. Consent for transfusion: A duty
of care. Br Med J 1997;315:380-1.
Co d e o f Fe d e r a l Re g u l a t i o n s. 4 2 C F R
482.23(c)(2). Washington, DC: US Government Printing Office, 2004 (revised annually).
Wang SE, Lara PN, Lee-Ow A, et al. Acetaminophen and diphenhydramine as premedication for platelet transfusions: A prospective randomized double-blind
placebo-controlled trial. Am J Hematol
2002;70: 191-4.
Agostini JV, Leo-Summers LS, Inouye SK.
Cognitive and other adverse effects of
diphenhydramine use in hospitalized older
patients. Arch Intern Med 2001;161:2091-7.
Patterson BJ, Freedman J, Blanchette V, et al.
Effect of pre-medication guidelines and
leukoreduction on the rate of febrile nonhemolytic platelet transfusions. Transfus Med
2000;10:199-206.
Wilcox GJ, Barnes A, Modanlou H. Does
transfusion using a syringe infusion pump
and small gauge needle cause hemolysis?
Transfusion 1981;21:750-1.
Herrera AJ, Corless J. Blood transfusions: Effect of speed of infusion and of needle gauge
on hemolysis. J Pediatr 1981;99:757-8.
de la Roche MR, Gauthier L. Rapid transfusion of packed red blood cells: Effects of dilution, pressure, and catheter size. Ann Emerg
Med 1993;22:1551-5.
Frelich R, Ellis MH. The effect of external
pressure, catheter gauge, and storage time on
hemolysis in RBC transfusion. Transfusion
2001;41:799-802.
AuBuchon JP, Kruskall MS. Transfusion
safety: Realizing efforts with risks. Transfusion 1997;37:1211-15.
McClelland DBL, Phillips P. Errors in blood
transfusion in Britain: Survey of hospital
haematology departments. Br Med J 1994;
308:1205-6.
Linden JV, Wagner K, Voytovich AE, Sheehan
J. Transfusion errors in New York State: An
analysis of 10 years’ experience. Transfusion
2000;40:1207-13.
Sazama K. Reports of 355 transfusion-associated deaths: 1976-1985. Transfusion 1990:30:
583-90.
McManigal S, Simms KL. Intravascular
hemolysis secondary to ABO incompatible

Chapter 22: Administration of Blood and Components

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

platelet products. Am J Clin Pathol 1999;11:
202-6.
Renner SW, Howanitz PJ, Bachner P. Wrist
band identification error reporting in 712
hospitals. Arch Pathol Lab Med 1993;117:
573-7.
Vicki C, Bower J. Blood administration in
perioperative settings. AORN J 1997;66:13343.
Baele PL, De Bruyere M, Deneys V, et al. Bedside transfusion errors. Vox Sang 1994;66:11721.
Murphy MF, Atterbury CLJ, Chapman JF, et al.
The administration of blood and blood components and the management of transfused
patients. Transfus Med 1999;9:227-38.
Ibojie J, Urbaniak SJ. Comparing near misses
with actual mistransfusion events: A more accurate reflection of transfusion error. Br J
Haematol 2000;108:458-60.
Galloway M, Woods R, Whitehead S, et al. An
audit of error rates in a UK district hospital
transfusion laboratory. Transfus Med 1999;9:
199-203.
Wenz B, Burns ER. Improvement in transfusion safety using a new blood unit and and
patient identification system as part of safe
transfusion practice. Transfusion 1991;31:
401-3.
Jensen NJ, Crosson JT. An automated system
for bedside verification of the match between
patient identification and blood unit identification. Transfusion 1996;36:216-21.
Patterson ES, Cook RI, Render ML. Improving
patient safety by identifying side effects from
introducing bar coding in medication administration. J Am Med Inform Assoc 2002;9:
540-3.
Shulman IA, Lohr K, Derdiarian A, Picukaric
JM. Monitoring transfusionist practices: A
strategy for improving transfusion safety.
Transfusion 1994;34:11-15.
Whitsett CF, Robichaux MG. Assessment of
blood administration procedures: Problems
identified by direct observation and administrative incident reporting. Transfusion 2001;
41:581-6.
Broods JP, Combest TG. In-service training
with videotape is useful in teaching transfusion medicine principles. Transfusion 1996;
36:739-42.
Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.
Schmidt WF, Kim HC, Tomassini N, Schwartz
E. Red blood cell destruction caused by a micro-pore blood filter. JAMA 1982;248:1629-32.
Stack G, Pomper GJ. Febrile allergic and nonimmune transfusion reactions. In: Simon TL,

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

533

Dzik WH, Stowell CP, et al, eds. Principles of
transfusion medicine. 3rd ed. Baltimore, MD:
Williams and Wilkins, 2002:831-51.
Novotny VM, van Doorn R, Witvliet MD, et al.
Occurrence of allogeneic HLA and non-HLA
antibodies after transfusion of prestorage filtered platelets and red blood cells: A prospective study. Blood 1995;85:1736-41.
Brand A, Claas FH, Voogt PJ, et al. Alloimmunization after leukocyte-depleted multiple random donor platelet transfusions. Vox
Sang 1998;54:160-6.
Bowden RA, Slichter SJ, Sayers M, et al. A
c o m p a r i s o n o f l e u k o c y t e - red u c e d a n d
cytomegalovirus (CMV ) seronegative blood
products for the prevention of transfusionassociated CMV infection after marrow transplant. Blood 1995;86:3598-603.
Narvios AB, Przepiorka K, Tarrand J, et al.
Transfusion support using filtered unscreened blood products for cytomegalovirus
negative allogeneic marrow transplant recipients. Bone Marrow Transplant 1998;22:575-7.
Buril A, Beugeling T, Feijen J, van Aken WG.
The mechanisms of leukocyte removal by filtration. Transfus Med Rev 1995;9:145-66.
Dzik WH. Leukoreduced blood components:
Laboratory and clinical aspects. In: Simon
TL, Dzik WH, Stowell CP, et al, eds. Principles
of transfusion medicine. 3rd ed. Baltimore,
MD: Williams and Wilkins, 2002:270-87.
Sprogre-Jakobsen U, Saetre AM, Georgsen J.
Preparation of white cell-reduced red cells by
filtration: Comparison of a bedside filter and
two blood bank filter systems. Transfusion
1995;35:421-6.
Ledent E, Berlin G. Inadequate white cell reduction by bedside filtration of red cell concentrates. Transfusion 1994;34:765-8.
Kao KJ, Hudson S, Orsini LA, et al. Effect of in
vitro storage time of platelet concentrates on
clogging of white cell reduction filters. Transfusion 1994;34:740-1.
Boyan CP, Howland WS. Cardiac arrest and
temperature of bank blood. JAMA 1963;183:
58-60.
Calhoun L. Blood product preparation and
administration. In: Petz LD, Swisher SN,
Kleinman S, eds. Clinical practice of transfusion medicine. 3rd ed. New York: Churchill
Livingstone, 1996:305-33.
Iserson KV, Huestis DW. Blood warming: Current applications and techniques. Transfusion 1991;31:558-71.
Uhl L, Pacini D, Kruskall MS. A comparative
study of blood warmer performance. Anesthesiology 1992;77:1022-8.
Burch KJ, Phelps SJ, Constance TD. Effect of
an infusion device on the integrity of whole

534

46.

47.

48.

AABB Technical Manual

blood and packed red cells. Am J Hosp Pharm
1991;48:92-7.
Snyder EL, Ferri PM, Smith EO, Ezekowitz MD.
Use of electromechanical infusion pump for
transfusion of platelet concentrates. Transfusion 1984;24:524-7.
Snyder EL, Malech HL, Ferri PM, et al. In vitro
function of granulocyte concentrates following passage through an electromechanical infusion pump. Transfusion 1986;26:141-4.
American Association of Blood Banks, America’s Blood Centers, American Red Cross. Circular of information for the use of human

49.

50.

51.

blood and blood components. Bethesda, MD:
AABB, 2002.
Ryden SE, Oberman HA. Compatibility of
common intravenous solutions with CPD
blood. Transfusion 1975;15:250-5.
Dickson DN, Gregory MA. Compatibility of
blood with solutions containing calcium. S
Afr Med J 1980;57:785-7.
Strautz RL, Nelson JM, Meyer EA, Shulman
IA. Compatibility of ADSOL-stored red cells
with intravenous solutions. Am J Emerg Med
1989;7:162-4.

Chapter 23: Perinatal Issues in Transfusion Practice

Chapter 23

Perinatal Issues in
Transfusion Practice

REGNANCY PRESENTS SPECIAL
immunohematologic problems for
the transfusion service. The mother
may exhibit alloimmunization to antigens
on fetal cells, and the fetus may be affected
by maternal antibodies provoked by previous pregnancies, by previous or present
transfusions, or by the ongoing pregnancy.
This chapter discusses hemolytic disease
of the fetus and newborn (HDFN) and neonatal alloimmune thrombocytopenia (NAIT)—
the two primary immunohematologic
concerns during the perinatal period. Also
included is a brief discussion of neonatal
thrombocytopenia secondary to maternal
idiopathic thrombocytopenic purpura.

Hemolytic Disease of the
Fetus and Newborn
In HDFN, fetal red cells become coated
with IgG alloantibody of maternal origin,

directed against a paternally inherited antigen present on the fetal cells that is
absent from maternal cells. The IgGcoated cells may undergo accelerated destruction both before and after birth, but
the severity of the disease can vary from
serologic abnormalities detected in an
asymptomatic infant to intrauterine death.

Pathophysiology
Accelerated red cell destruction stimulates
increased production of red cells, many of
which enter the circulation prematurely as
nucleated cells, hence the term “erythroblastosis fetalis.” Severely affected fetuses
may develop generalized edema, called
“hydrops fetalis.” In HDFN resulting from
anti-D, erythropoiesis in the fetal liver
may be so extensive that portal circulation
is disrupted and albumin synthesis impaired, thereby reducing plasma colloid
osmotic pressure. The severe anemia may
cause cardiovascular failure, tissue hypo535

536

AABB Technical Manual

xia, and death in utero. Intrauterine transfusion may be lifesaving in these circumstances. If live-born, the severely affected
infant exhibits profound anemia and
heart failure.1 Less severely affected infants continue to experience accelerated
red cell destruction, which generates
large quantities of bilirubin. Unlike HDFN
due to anti-D, HDFN due to anti-K1 results from suppression of fetal erythropoiesis in addition to causing peripheral red
2
cell destruction.
Before birth severs the communication
between maternal and fetal circulation, fetal bilirubin is processed by the mother’s
liver. At birth, the infant’s immature liver is
incapable of conjugating the amount of bilirubin that results from destruction of antibody-coated red cells. Unconjugated bilirubin is toxic to the developing central
nervous system (CNS), causing brain damage referred to as “kernicterus.” For the
live-born infant with HDFN and rising levels of unconjugated bilirubin, kernicterus
may pose a greater clinical danger than the
consequences of anemia.3 Prematurity, acidosis, hypoxia, and hypoalbuminemia increase the risk of CNS damage. Decisions
about undertaking exchange transfusion
are based primarily on the bilirubin level,
the rate of bilirubin accumulation, and, to a
lesser degree, on the severity of the anemia.
Recently, the American Academy of Pediatrics has published guidelines aimed toward
preventing and managing hyperbilirubinemia in infants ≥35 weeks of gestation.4

Mechanisms of Maternal Immunization
HDFN is often classified into three categories, on the basis of the specificity of the
causative IgG antibody. In descending order of potential severity, they are:
1.
D hemolytic disease caused by anti-D
alone or, less often, in combination
with anti-C or anti-E. (The Rh blood

group is discussed in greater detail
in Chapter 14.)
2.
“Other” hemolytic disease caused by
antibodies against other antigens in
the Rh system or against antigens in
other systems; anti-c and anti-K1 are
most often implicated.5
3.
ABO HDFN caused by anti-A,B in a
group O woman or by isolated anti-A
or anti-B.
In all but ABO HDFN, maternal antibodies reflect alloimmunization by pregnancy
or transfusion. Rising titers of antibody can
be documented, at least in the first affected
pregnancy, and the infant may be symptomatic at birth as a result of effects on the
fetus in utero. In contrast, ABO fetomaternal incompatibility cannot be diagnosed
during pregnancy and the infant is rarely
symptomatic at birth.

Pregnancy as the Immunizing Stimulus
Pregnancy causes immunization when fetal red cells, possessing a paternal antigen
foreign to the mother, enter the maternal
circulation as a result of fetomaternal hemorrhage (FMH). FMH occurs in the vast
majority of pregnancies, usually during the
third trimester and during delivery.6 Delivery is the most common immunizing
event, but fetal red cells can also enter the
mother’s circulation after amniocentesis,
spontaneous or induced abortion, chorionic villus sampling, cordocentesis, rupture of an ectopic pregnancy, and blunt
trauma to the abdomen.
Immunogenic Specificities. The antigen
that most frequently induces immunization
is D, but, in theory, any red cell antigen
present on fetal cells and absent from the
mother can stimulate antibody production.
One retrospective study determined that
there was a 0.24% prevalence of production
of clinically significant antibodies other
than anti-D during pregnancy. Because

Chapter 23: Perinatal Issues in Transfusion Practice

other red cell antigens are less immunogenic than D, sensitization is more likely to
result from exposure to a large volume of
red cells, such as during blood transfusion.
Immunization to D, on the other hand, can
occur with volumes of fetal blood less than
0.1 mL.7
Frequency of Immunization. The probability of immunization to D correlates with
the volume of D-positive red cells entering
the D-negative mother’s circulation.6 The
overall incidence of D sensitization in untreated D-negative mothers of D-positive
infants is about 16%; 1.5% to 2% become
sensitized at the time of their first delivery,
an additional 7% become sensitized within
6 months of the delivery, and the final 7%
become sensitized during the second affected pregnancy.8 The sensitization during
the second affected pregnancy probably reflects primary immunization during the
first D-positive pregnancy and delivery that
happened without production of detectable
levels of antibody. The small numbers of
D-positive fetal red cells entering the maternal circulation early during the second
affected pregnancy constitute a secondary
stimulus sufficient to elicit overt production
of IgG anti-D. In susceptible women not
immunized after two D-positive pregnancies, later pregnancies may be affected but
with diminished frequency. The incidence
of the more common genotypes in D-positive individuals can be found in Table 14-4.
This can be used to get a general idea of the
likelihood of how often an infant with a
D-positive father and D-negative mother
will express the D antigen.
Once immunization has occurred, successive D-positive pregnancies often manifest HDFN of increasing severity, particularly between the first and second affected
pregnancies. After the second affected
pregnancy, the history is predictive of outcome, although, in rare instances, some
women have a stable or diminishing pat-

537

tern of clinical disease in subsequent pregnancies.
Effect of ABO Incompatibility. Rh immunization of untreated D-negative women
occurs less frequently after delivery of an
ABO-incompatible D-positive infant than
when the fetal cells are ABO-compatible
with the mother. ABO incompatibility between mother and fetus has a substantial
but not absolute protective effect against
maternal immunization by virtue of the increased rate of red cell destruction by anti-A
or anti-B. The rate of immunization is decreased from 16% to between 1.5% and 2%.7

Transfusion as the Immunizing Stimulus
It is extremely important to avoid transfusing D-positive whole blood or red cells
to D-negative females of childbearing
potential because anti-D stimulated by
transfusion characteristically causes severe
HDFN in subsequent pregnancies with a
D-positive fetus. Red cells present in platelet or granulocyte concentrates can constitute an immunizing stimulus; if components from D-positive donors are
necessary for young D-negative female
recipients, Rh immunoprophylaxis should
be considered. This is discussed further in
Chapter 21.
The risk of immunization to a red cell
antigen other than D after an allogeneic red
cell transfusion has been estimated to be
1% to 2.5% in the general hospital population.7 This will endanger the fetus only if the
antibody is IgG and directed against an antigen that is also present on the fetal red
cells. For a couple planning to have children, the woman should not be transfused
with red cells from her sexual partner or his
blood relatives. This form of directed donation increases the risk that the mother will
be immunized to paternal red cell, leukocyte, and/or platelet antigens, which could
cause alloimmune cytopenias in future

538

AABB Technical Manual

children who share the same paternal antigens. Programs using parents as directed
donors for their sick newborns deserve special consideration because of these unique
issues in the face of strong parental desires.9,10

ABO Antibodies
The IgG antibodies that cause ABO HDFN
nearly always occur in the mother’s circulation without a history of prior exposure
to human red cells. ABO HDFN can occur
in any pregnancy, including the first. It is
restricted almost entirely to group A or B
infants born to group O mothers because
group O individuals make the IgG antibody, anti-A,B. Group A or B mothers with
an A- or B-incompatible fetus predominantly produce IgM antibody, with only
small amounts of IgG antibody capable of
crossing the placenta.

Prenatal Evaluation

Maternal History
Information about previous pregnancies or
blood transfusions is essential in evaluating fetal risk. Invasive tests, which carry
risk to the fetus, should be performed
only for pregnancies in which the fetus is
at risk for HDFN, by history and/or serologic testing. For a woman with a history
of an infant with hydrops fetalis due to
anti-D, there is a 90% or more chance of a
subsequent fetus being similarly affected.5
In contrast, during the first sensitized
pregnancy, the risk of a hydropic fetus is
8% to 10%. Experience with other alloantibodies has not been as extensive as with
anti-D; in some series, anti-c and anti-K1
were by far the most common causes of
severe HDFN, other than anti-D.5,11

Serologic Studies
Alloantibodies capable of causing HDFN
can be detected during pregnancy. Initial

studies should be performed on all pregnant women as early in pregnancy as
possible; they should include tests for
ABO and D, and a screen for unexpected
red cell antibodies.12 If a woman’s red cells
are not directly agglutinated by anti-D,
testing for weak D is not required. When
testing for weak D is not performed,
women with weak D will be labeled as D
negative, although they are in fact D positive; the only potential negative outcome
is that these women will receive unnecessary Rh Immune Globulin (RhIG). Women
with some partial D phenotypes, such as
DVI, will also most likely type as D negative
in direct tests, and, in the absence of weak
D testing, these women are also candidates for RhIG antenatal prophylaxis.
With the application of molecular techniques, knowledge of the RHD gene is
evolving. Not all weak D red cells are the result of a decreased expression of the D antigen, but, rather, some have altered RhD
proteins. Consequently, these patients are
at risk for immunization when exposed to
the D antigen, explaining formation of
anti-D in some patients classified as weak
D. As more is learned about the D antigen,
the distinction between partial D and weak
D is blurring.13,14
Whether or not prophylaxis would be
successful in the setting of partial D remains
unknown. The appropriate dose is also subject to speculation. As a result, some practitioners administer RhIG to these women,
whereas others consider it unnecessary.
Very rarely, a mother with partial D antigen
produces anti-D as a result of pregnancy.
If weak D testing is performed and the
test is clearly positive, the woman should
be regarded as D positive. If testing for
weak D is not performed, women whose
red cells do not react in direct tests with
anti-D can be considered candidates for
RhIG prophylaxis. Some laboratories continue to do weak D testing to avoid confu-

Chapter 23: Perinatal Issues in Transfusion Practice

sion in the interpretation of the FMH screen
during postpartum testing.12 Chapter 14
contains a more complete discussion of the
Rh blood group.
A woman should be classified as D positive if the test for either D or weak D is positive. If a D-negative woman has a negative
initial antibody screen, the test can be repeated at 28 weeks’ gestation before administration of RhIG to detect immunization that might have occurred before 28 weeks,
in accordance with AABB recommendations.
Because the incidence of immunization
during this period of pregnancy is extremely
low, the American College of Obstetricians
and Gynecologists (ACOG) points out that
no data exist that support the cost-effective15
ness of this practice. Repeat antibody
screening of D-positive women may be recommended if there is a history of clinically
significant red cell antibodies associated
with HDFN, previous blood transfusion, or
trauma to the abdomen.
Antibody Specificity. All positive screens
for red cell antibodies require identification
of the antibody.12 The mere presence of an
antibody, however, does not indicate that
HDFN will occur. Non-red-cell-stimulated
IgM antibodies, notably anti-Lea and anti-I,
are relatively common during pregnancy but
do not cross the placenta. In addition, the
fetal red cells may lack the antigen corresponding to the mother’s antibody; the likelihood of fetal involvement can often be
predicted by typing the father’s red cell antigens.16 The laboratory report on prenatal
antibody studies should include sufficient
information to aid the clinician in determining the clinical significance of the identified antibody.
Typing the Fetus. The fetal D type can be
established by using the polymerase chain
reaction (PCR) to amplify DNA obtained
from amniotic fluid, chorionic villus samples, or by serologic typing of fetal blood
17
obtained by cordocentesis. Chorionic villus

539

sampling is discouraged because it causes
FMH and has been associated with more
severe HDFN. Amniotic fluid samples are
recommended over cordocentesis because
cordocentesis has a fourfold or higher rate
of perinatal loss over amniocentesis.18 The
use of molecular techniques can help detect variations in the RHD gene that might
go undetected using serology alone. It is
preferable that both paternal and maternal
blood samples accompany the fetal samples.19 Fetal DNA typing is also available for
a
b 20
21
22
23
Jk /Jk , K1/K2, c, and E/e antigens. A
more recent development in fetal RhD typing involves the isolation of free fetal DNA
in the maternal serum. Although not routinely available in the United States at this
time, this will likely replace amniocentesis
for fetal genotyping in the near future.24

Maternal Antibody Titer
Antibody titrations can help in decisions
about the performance and the timing of
invasive procedures, especially if the antibody is anti-D. The antibody titer should
be established in the first trimester to
serve as a baseline, and the specimen
should be frozen for future comparisons
(see Method 5.3).7 Because invasive tests
will not be undertaken before 16 to 18
weeks’ gestation, no further titration is indicated until this time. The true significance of an antibody titer in maternal serum is controversial because some studies
have shown poor correlation between the
level of the titer and effects on the fetus.
For antibodies other than anti-D, critical
titers have not been identified, although a
critical titer similar to that used in cases
of anti-D alloimmunization is often utilized.25 These techniques continue to be
performed because they represent a noninvasive way to try to assess the presence
and severity of alloimmunization. When
performed, it is important that successive

540

AABB Technical Manual

titrations use the same methods and test
cells of the same red cell phenotype. Testing previously frozen serum samples in
parallel with a current specimen minimizes the possibility that changes in the
titer result from differences in technique.
The critical titer for anti-D (the level below which HDFN and hydrops fetalis are
considered so unlikely that no further invasive procedures will be undertaken)
should be selected at each facility and is
usually 16 or 32 in the antihuman globulin phase.26,27 Follow-up testing is recom26
mended for any titer greater than 8. The
critical titer for anti-K1 may be lower than
anti-D, typically a value of 8.25,28 Currently,
it is not recommended that gel technology be used for prenatal antibody titration because of the lack of data showing a
correlation between gel and tube agglutination titers.12

Other Measures of HDFN Severity
Numerous laboratory procedures have been
investigated to improve the accuracy of
predicting the severity of hemolysis.29 The
antibody titer discussed above is not always reliable, nor is the serial change in
titer. Functional assays, including measures
of adherence, phagocytosis, antibody-dependent cytotoxicity, and chemiluminescence have been investigated, but their use
has been limited and remains controversial. These procedures are usually performed in referral centers and may be useful when additional information is required
to manage difficult and complex cases.

Amniotic Fluid Analysis
A good index of intrauterine hemolysis
and fetal well-being is the level of bilirubin pigment found in amniotic fluid obtained by amniocentesis. Amniocentesis
is usually performed in alloimmunized
women who have a history of previously

affected pregnancies or have an antibody
titer at or above the critical titer.28 Because
fetal anemia secondary to K1 alloimmunization is not always associated with elevated levels of bilirubin in amniotic fluid,
it has been recommended that fetal blood
sampling be used instead of serial amniocentesis when anti-K1 is detected in a
pregnant woman.2
Amniotic fluid is obtained by inserting a
long needle through the mother’s abdominal wall and uterus into the uterine cavity
under continuous ultrasound guidance.
The aspirated fluid is scanned spectrophotometrically at wavelengths of 350 to 700
nm. Peak absorbance of bilirubin is at 450
nm. An increase in optical density from the
projected baseline at 450 nm (∆OD450) is a
measure of the concentration of bile pigments.30,31 The ∆OD450 value is plotted on a
graph against the estimated length of gestation, because bile pigment concentration
has different clinical significance at different gestational ages. Liley’s system31 (Fig
23-1) of predicting the severity of fetal disease based on the ∆OD450 has been used for
decades. It delineates three zones to estimate severity of disease: a top zone (zone 3)
indicates severe disease, the bottom zone
(zone 1) indicates mild or no disease, and
mid-zone (zone 2) values require repeat determination to establish a trend. This
method is applicable to pregnancies from
27 weeks through term. Queenan et al32
have proposed a system for managing
D-immunized pregnancies based on the
∆OD450 from as early as 14 weeks’ gestation.
They identified four zones (Fig 23-2), with
early invasive intervention recommended if
∆OD450 values fall in the highest zone. With
both systems, the severity of HDFN is more
accurately predicted with serial ∆OD450
measurements than with a single observation, to evaluate whether readings are falling, rising, or stable. In general, the higher
the pigment concentration, the more se-

Chapter 23: Perinatal Issues in Transfusion Practice

541

Figure 23-1. Liley graph for collecting data from amniotic fluid studies. Intrauterine transfusion should
be done if the ∆OD 450 value is in the top zone before 32 weeks’ gestation. After 34 weeks, top zone values indicate immediate delivery. Either intrauterine transfusion or immediate delivery may be indicated for top zone ∆OD 450 between 32 and 34 weeks, depending on studies of fetal maturity. Modified
from Liley.31

vere the intrauterine hemolysis. It is important to perform an ultrasound to establish
the correct gestational age so that the test
can be interpreted and the course of therapy for a particular ∆OD450 will be appro18
priate. Alternatively, a ∆OD450 value in the
upper mid-zone of the Liley curve indicates
the need for fetal blood sampling.
Amniocentesis, particularly if the needle
goes through the placenta, or cordocentesis
may cause FMH, which can boost the titer
of existing red cell alloantibody, thereby increasing the severity of HDFN, or inducing
18
immunization to additional antigens.
Therefore, when amniocentesis or cordocentesis is performed for any reason on a
D-negative woman who does not have
anti-D, Rh immunoprophylaxis should be
given.

Percutaneous Umbilical Blood Sampling
In the early 1980s, the use of sophisticated
ultrasound equipment made it feasible to
direct a needle into an umbilical blood
vessel, preferably the vein at its insertion
into the placenta, and obtain a fetal blood
sample. Percutaneous umbilical blood
sampling (PUBS, or cordocentesis) allows
direct measurement of hematologic and
biochemical variables. Determination of the
fetal hematocrit provides an accurate assessment of the severity of fetal hemolytic
18
disease. It is important to verify that the
sample has been obtained from the fetus.
Fetal and maternal red cells can be distinguished because of differences in red cell
size and red cell phenotyping, as well as
33
by the presence of fetal hemoglobin.

542

AABB Technical Manual

Figure 23-2. Amniotic fluid OD 450 management zones. (Reproduced with permission from Queenan et
al.32 )

The fetal mortality of intrauterine fetal
blood sampling has been reported to be 1%
to 2%,34 and the procedure carries a high
risk of FMH. Its use is recommended only
for certain circumstances, such as when serial amniotic fluid determinations indicate
severe HDFN, when hydrops is present,
when the D titer is high or rising, or when
HDFN occurred in previous pregnancies. In
addition to diagnosis, PUBS allows treatment of the affected fetus.

Doppler Flow Studies
Because fetal anemia results in increased
cardiac output, several investigators have
measured various blood velocities in fetal
vessels using Doppler ultrasonography to
determine the clinical status of the fetus
in a noninvasive manner.18,35 Recent stud-

ies have found good correlation between
middle cerebral artery (MCA) peak velocity, fetal hemoglobin, and ∆OD450 read36
ings. Many centers routinely use an MCA
peak systolic velocity value of greater than
1.5 multiples of the median to proceed
with cordocentesis to determine if the fetus is anemic. In such centers, amniocentesis is performed only after 35 weeks’
gestation when MCA Doppler is associated with a high false-positive rate for the
diagnosis of fetal anemia (Moise K, personal communication).

Suppression of Maternal
Alloimmunization
Several approaches to suppress maternal
alloimmunization have been attempted,
two of which have limited clinical benefit

Chapter 23: Perinatal Issues in Transfusion Practice

in reducing maternal antibody levels: intensive plasma exchange and the administration of immunoglobulin (intravenous)
(IGIV).5,18 Plasma exchange can reduce antibody levels by as much as 75%. Unfortunately, rebound usually follows because
the IgG antibody is mostly extravascular
and antigen exposure may be ongoing.
Plasma exchange has been proposed as a
way to delay the need for fetal intervention, particularly when there is a previous
pregnancy complicated by early hydrops.37
In this setting, plasma exchange can delay
the need for more invasive procedures until the second trimester. The AABB and
the American Society for Apheresis (ASFA)
categorize plasma exchange as treatment
Category III because its efficacy and safety
have not been proven for this indication
(see Chapter 6). With the increasing safety
of intrauterine transfusion through ultrasound guidance and the decreasing incidence of HDFN due to anti-D, the use of
plasma exchange as a treatment modality
has declined.
IGIV infusion has also been shown to
stabilize anti-D titers, with best results obtained when started before 28 weeks’ gestation and when the fetus is not hydropic.38 In
a small study assessing the efficacy of IGIV,
it was found to be well tolerated and there
was a decrease in hemolysis.39 The mechanism of IGIV effect is not clear, although it
may work by saturating placental Fc receptors and inhibiting the transplacental transfer of maternal antibody or by suppressing
ingestion of IgG-coated red cells by the fetal
reticuloendothelial system. An alternative
explanation is that the introduction of
anti-idiotype antibodies modifies maternal
antibody production. IGIV has also been
used with plasma exchange to reduce the
antibody rebound that has been seen following plasma exchange.18 Until larger studies assessing safety and efficacy can be per-

543

formed, intrauterine transfusion remains
the mainstay of standard therapy.

Intrauterine Transfusion
Intrauterine transfusion can be performed
by the intraperitoneal route (IPT) or the
direct intravascular approach (IVT) by the
umbilical vein. In many instances, IVT is
the procedure of choice, but there may be
problems of access that make IPT preferable; a combination may also be used to
minimize peaks and troughs of fetal hematocrit between procedures. Intrauterine
transfusion is seldom feasible before the
20th week of gestation; once initiated,
transfusions are usually administered periodically until delivery. It is important
that blood is available and ready at the
time of the first diagnostic, and subsequent, cordocenteses. If fetal anemia is
detected, an intrauterine transfusion can
be performed at once, minimizing fetal
risks. The interval between transfusions
depends on the presence or absence of
hydrops, the gestational age, and the
amount of blood infused. Good outcomes
are achieved over 80% of the time and
94% of nonhydropic fetuses survive.40 Because intrauterine transfusion carries a 1%
to 2% risk of perinatal loss, it should be
performed only after careful clinical evaluation.18,34,41,42 Other perinatal conditions
that have been treated with intrauterine
transfusion include parvovirus infection,
large FMH, and alpha thalassemia.43

Techniques
IPT is performed through a needle passed,
with ultrasonographic monitoring, through
the mother’s abdominal wall into the
abdominal cavity of the fetus. Transfused
red cells enter the fetal circulation through
lymphatic channels that drain the peritoneal cavity. In IVT, the umbilical vein is
penetrated under ultrasound guidance,

544

AABB Technical Manual

and a blood sample is taken to verify positioning in the fetal vasculature. Injection
of saline can also confirm correct placement because it can be visualized by ultrasound. Blood is infused directly, as either a simple transfusion or as a partial
exchange transfusion. IVT can be particularly valuable for very severe cases of
HDFN associated with hydrops fetalis. In
hydropic infants, red cells administered
by IPT are not efficiently absorbed.

Selection of Red Cells
The red cells used should be group O,
D-negative, or negative for the antigen
corresponding to the mother’s antibody if
the specificity is not anti-D. Blood for
intrauterine transfusion should be irradiated (see Chapter 27), and should be cytomegalovirus (CMV)-reduced-risk. It may
also be desirable to transfuse blood that is
known to lack hemoglobin S in order to
transfuse red cells with maximal oxygentransporting capacity, in the setting of low
oxygen tension. For optimal survival of the
transfused cells, blood used for intrauterine transfusion should be drawn as recently as possible, generally less than 7
days old.
The hematocrit of the RBCs prepared for
exchange transfusion is usually high to
minimize the chance of volume overload in
the fetus. Washed, irradiated maternal
blood has also been used for intrauterine
44
transfusion. To remove the offending antibody, the red cells are washed and resuspended in saline to a final hematocrit
between 75% and 85%. Washed or deglycerolized preparations have been used as a
means to remove plasma, anticoagulant/
preservative solutions, and excess electrolytes that might accumulate during prolonged storage. Blood for intrauterine
transfusions and all blood and cellular
components subsequently transfused in

the neonatal period should be irradiated to
prevent transfusion-associated graft-vs-host
disease because the fetus is considered im45,46
munologically naïve and tolerant.

Volume Administered
The volume transfused varies with the
technique used as well as the fetal size,
initial hematocrit, and gestational age.
For IPT, a volume calculated by the formula V = (gestation in weeks – 20) × 10 mL
appears to be well tolerated by the fetus.
The volume of red cells transfused by IVT
can be calculated by the following formula.47
Fetoplacental volume (mL) =
ultrasound estimated fetal weight
(g) × 0.14
Volume to transfuse (mL) =
Fetoplacental volume ×
(Hct after IVT – Hct before IVT)
Hct of donor cells
where Hct = hematocrit
Transfusion is repeated on the basis of
an estimated decline in fetal hematocrit of
approximately 1% per day in an effort to
maintain the fetal hematocrit in the range
of 27% to 30%.48

Postpartum Evaluation
It may be desirable to collect a sample of
cord blood, preferably by cannulation of
an umbilical vessel at delivery, from newborns where there is a risk of HDFN (eg,
Rh-positive infants born to Rh-negative
mothers, type A and B infants born to
type O mothers). This sample should be
identified as cord blood and labeled in
the delivery suite with the mother’s name,
the date, and two unique forms of identification for the infant (eg, name and medical record number).

Chapter 23: Perinatal Issues in Transfusion Practice

In cases of suspected HDFN, samples of
both cord and maternal blood should be
tested. When the mother is known to have
antibodies capable of causing HDFN, the
hemoglobin or hematocrit and the bilirubin level of cord blood should also be determined. If the mother is D negative and
the infant D positive, the mother’s blood
should be tested to determine the volume
of FMH, as discussed later. Testing cord
blood may present some special problems,
which are described below.

ABO Testing
ABO testing on newborns relies entirely on
red cell typing because ABO antibodies in
cord serum are nearly always of maternal
origin and are IgG. However, in the investigation of possible HDFN due to ABO incompatibility, cord serum should be tested
for antiglobulin-reactive ABO antibodies.
Also, if the infant will receive non-group-O
red cells, testing must be taken to the
antiglobulin phase.49(p42)

D Testing
Newborns who have had successful intrauterine transfusions often type at birth as
D negative or very weakly positive because over 90% of their circulating red
cells may be those of the donors. The ABO
and direct antiglobulin tests may also give
misleading results. If the infant’s red cells
are heavily coated with IgG antibodies, tests
with anti-D may give either false-positive
or false-negative results (see Chapter 14).

Antiglobulin Testing
The direct antiglobulin test (DAT) is usually strongly positive in HDFN resulting
from anti-D or antibodies to other blood
groups; reactions are much weaker or

545

even negative in HDFN resulting from
ABO antibodies. However, the strength of
the DAT does not correlate with the severity of hemolysis, especially in ABO HDFN.
Infants who have received an intrauterine
transfusion may have a weakly positive
DAT with a mixed-field pattern of agglutination. If the DAT on cord cells is positive,
the antibody can be eluted from the red
cells and tested for specificity. It is not
necessary to make and test an eluate if
the maternal serum has been shown to
contain a single red cell antibody. All clinically significant red cell antibodies in the
maternal serum must be respected. (See
the section on Selection of Blood, later in
this chapter.) If the DAT is positive and
the maternal antibody screen is negative,
investigation should turn toward ABO antibodies or HDFN caused by an antibody
directed against a low-incidence antigen
not present on reagent red cells.
Evaluation of ABO Antibodies. ABO
HDFN may be suspected on clinical
grounds even though the DAT is negative.
Testing the eluate from the cord cells
against A1 and B red cells should establish
the diagnosis of ABO HDFN. Cord blood or
peripheral blood serum should be tested by
an indirect antiglobulin technique against
A1, B, and O red cells. The presence of anti-A,
anti-B, or anti-A,B confirms the potential
for ABO HDFN. It is often possible to elute
anti-A and/or anti-B from the infant’s red
cells despite a negative DAT, but this step is
not necessary for the presumptive diagnosis. In the rare cases of ABO HDFN that require transfusion, D-compatible group O
blood should be transfused, whether or not
the diagnosis has been serologically confirmed. Nonimmune causes of hyperbilirubinemia and hemolysis should still be considered in an infant with a negative DAT
before concluding that it is due to ABO
HDFN because other hematologic disorders might be present.50

546

AABB Technical Manual

Antibodies to Low-Incidence Antigens.
If ABO HDFN is ruled out, antibody against
a low-incidence red cell antigen should be
suspected. Testing an eluate or maternal
serum against the father’s red cells with an
antiglobulin technique may provide an answer. Maternal serum must be ABO compatible, if it is used. If either or both of these
tests are positive, it indicates that the infant
has an antigen of paternal origin that the
mother lacks, causing her to make an IgG
antibody directed against this antigen. Unless the mother has been exposed to red cells
from the father or his blood relations, transfusion would be an unlikely immunizing
event for a low-incidence antigen. Because
there should be no difficulty in obtaining
compatible blood, diagnostic studies can
be performed after initial clinical concerns
have been resolved. If the DAT is positive
and all attempts to characterize a coating
red cell antibody are consistently negative,
causes of a false-positive DAT should be
considered (see Chapter 20).

Exchange Transfusion
Exchange transfusion for HDFN achieves
several desired effects, including:
1.
Removal of antibody-coated fetal red
cells.
2.
Removal of maternal antibody.
3.
Removal of bilirubin.
4.
Replacement of red cells, thereby
treating anemia.
The red cells used for replacement must
be compatible with the causative antibody.
Fresh Frozen Plasma is frequently used to
reconstitute whole blood because it provides coagulation factors. Platelet values
should also be monitored and transfusion
used as necessary. Plasma frozen within 24
hours and thawed plasma can be used too,
with the understanding that there might be
decreases in the activity of the labile
clotting Factors V and VIII.

Selection of Blood
In most cases, the mother’s serum is used
for crossmatching and the red cells selected for transfusion are compatible with
her ABO antibodies as well as any additional antibody(ies) responsible for the
hemolytic process. Group O red cells resuspended in AB plasma are commonly
used. In ABO HDFN, the red cells used for
exchange must be group O. If the antibody is anti-D, the red cells must be D
negative, but not every exchange transfusion requires group O RBCs. If mother
and infant are ABO-identical, group-specific red cells or whole blood can be used.
If the implicated antibody is not anti-D,
D-positive red cells may be given to a
D-positive infant.
Maternal serum or plasma is the specimen of choice for crossmatching in exchange transfusion; it is available in large
quantities, decreases the volume of blood
taken from the infant, has the red cell antibody present in high concentration, and
can be analyzed accurately and completely
before delivery. On the other hand, use of
maternal serum may be problematic if it
contains antibodies directed against antigens not present on the infant’s red cells
because of other sources of sensitization, or
if it contains IgM antibodies that have not
crossed the placenta. These additional
antibodies could complicate the serologic
picture.
If maternal blood is not available or is
unsuitable for crossmatching, the infant’s
serum and/or, preferably, an eluate from
the infant’s red cells can be used for crossmatching. The concentration of antibody in
the infant’s serum may be low, especially if
most of the molecules are bound to the red
cells. Use of the eluate or serum, or of both
together, may be indicated if attempts to
obtain a maternal specimen would delay
therapy. Blood used for exchange transfu-

Chapter 23: Perinatal Issues in Transfusion Practice

sion should be irradiated. Typically, a volume of twice the infant’s blood volume is
used for exchange.51
2.

Subsequent Transfusion
Bilirubin may reaccumulate rapidly after
a successful exchange transfusion despite
appropriate phototherapy. This occurs
because most bilirubin in extravascular
fluid will reequilibrate by entering the
intravascular space and also because residual antibody-coated cells continue to
hemolyze. If rising bilirubin levels make a
second or third exchange transfusion necessary, the same considerations of red cell
selection and crossmatching apply.
Infants who have undergone intrauterine transfusion need to be followed closely
after birth because intrauterine transfusion
suppresses fetal erythropoiesis. Weekly
hematocrit and reticulocyte counts should
be performed on the neonate for a 1- to
3-month period.18 Many of these infants
will subsequently develop anemia and
need to be supported with red cell transfusion until their own production begins, as
evidenced by reticulocytosis and age-appropriate hemoglobin levels.18,52

Antibody Against a High-Incidence Antigen
Rarely, the mother’s antibody reacts with
a high-incidence antigen and no compatible blood is available. If this problem is
recognized and identified before delivery,
the mother’s siblings can be evaluated for
compatibility and suitability, or compatible donors can be sought through a rare
donor file. Maternal red cells can also be
collected and frozen. Any products collected
from blood relatives must be irradiated. If
this very rare event is not recognized until
after delivery, three choices are available:
1.
Collect blood from the mother, if the
obstetrician agrees. Remove as much
plasma as possible, preferably by sa-

547

line washing, and resuspend the red
cells in compatible plasma to the desired hematocrit.
If time permits, test the mother’s siblings or other close relatives for
compatibility and eligibility.
Use incompatible donor blood for
the exchange transfusion if the clinical situation is sufficiently urgent.
The exchange will reduce the bilirubin load, the most heavily antibody-coated cells, and the number
of unbound antibody molecules.
However, residual antibody will attach to the transfused cells, and one
or more additional exchanges will
probably be needed as bilirubin accumulates.

Rh Immune Globulin
RhIG is a concentrate of predominantly
IgG anti-D derived from pools of human
plasma. A full dose of anti-D (300 µg, 1500
IU, or the actual content of a “dose” as indicated by the individual manufacturer53)
is sufficient to counteract the immunizing
effects of 15 mL of D-positive red cells;
this corresponds to approximately 30 mL
of fetal whole blood. RhIG is available in a
reduced dose, approximately 50 µg, which
is protective for up to 2.5 mL of D-positive
fetal red cells. This dose can be used for
first-trimester abortion or miscarriage, when
the total blood volume of the fetus is less
than 2.5 mL. However, because of fears of
miscalculating the length of pregnancy
and concerns of inadvertently mixing up
inventory resulting in undertreatment, a
full dose is usually administered and
these low doses are frequently not stocked.
The protective effect of RhIG on D-negative individuals exposed to D-positive
cells probably results from interference
with antigen recognition in the induction
phase of primary immunization.54

548

AABB Technical Manual

RhIG is available in two formulations: 1)
for intramuscular (IM) injection only and 2)
for either IM or intravenous (IV) administration. The dose of the intravenous preparation is expressed in international units (5
IU is equivalent to 1 µg), with 1500 IU (300
µg) neutralizing 17 mL of D-positive red
cells, according to the package insert.

Antepartum Administration
Widespread postpartum use of Rh immunoprophylaxis has reduced pregnancy-associated immunization to the D antigen to
1% to 2%.8 This risk is further decreased to
0.1% if RhIG is also given antepartum at
28 weeks of gestation.7 The ACOG recommends antepartum RhIG prophylaxis at
28 weeks of gestation, based on the observation that, of women who develop anti-D
during pregnancy, 92% do so at or after 28
weeks.7,15
Blood obtained before injection of RhIG
should be tested for ABO and D. A D-negative woman who has antibodies other than
anti-D (eg, anti-G) is still a candidate for
anti-D immunoprophylaxis. When the
mother receives RhIG during pregnancy,
the infant may be born with a positive DAT,
but without hemolysis. The mother’s serum
will often exhibit anti-D reactivity. There
must be good communication between the
patient’s physician and the blood bank staff
at the institution where delivery takes
place, to ensure correct interpretation of
laboratory tests made at the time of delivery. The half-life of an injected dose of RhIG,
in the absence of significant FMH, is approximately 21 days. Therefore, of 300 µg of
anti-D given at 28 weeks, 20-30 µg could remain at the time of delivery 12 weeks later.
Some practitioners will administer another
dose of RhIG if delivery is delayed beyond
40 weeks.18 Anti-D can be detected in the
maternal circulation for as long as 6 months.

Postpartum Administration
Cord blood from infants born to D-negative mothers should be tested for the D
antigen. A D-negative woman with a
D-positive infant should receive one full
dose of RhIG within 72 hours of delivery,
unless she is known to be alloimmunized
to D previously. The presence of residual
anti-D from antepartum RhIG does not
indicate ongoing protection.
Active vs Passive Antibody. In-vitro clues
can help distinguish passively administered
RhIG from the anti-D formed as a result of
active alloimmunization. Passively acquired
anti-D is entirely IgG; if a woman’s anti-D is
saline-reactive or can be completely or partially inactivated by treating the serum with
2-mercaptoethanol or dithiothreitol, it has
an IgM component and probably represents active immunization. Passively acquired anti-D rarely achieves an antiglobulin titer above 4, so a high-titered
antibody or rising antibody titer is likely to
indicate active immunization. It is desirable
to obtain confirmation from the physician’s
records, but RhIG should always be given
when doubt cannot easily be resolved. It
should also be given if there is any problem
determining the Rh type.
Postpartum Evaluation. A sample of the
mother’s blood should be drawn, preferably
within 1 hour after delivery, and evaluated
for FMH of a quantity greater than that for
which 300 µg RhIG is immunosuppressive.
If the screening test demonstrates the presence of fetal cells, the extent of FMH must
be determined so that an appropriate dose
49(pp48,49),55
of RhIG can be administered.
Postpartum RhIG should be given within
72 hours of delivery. If prophylaxis is delayed, the likelihood that alloimmunization
will be prevented decreases. Despite the decrease seen, the ACOG recommends that
treatment still be administered because
some studies have found partial protection

Chapter 23: Perinatal Issues in Transfusion Practice

has occurred as late as 13 days after exposure and, possibly, as late as 28 days.15
The following women are not candidates
for RhIG:
1.
The D-negative woman whose infant
is D-negative.
2.
Any D-positive woman. Very rare cases
of HDFN have been reported in infants whose mothers had a weak/partial D phenotype, but routine RhIG
prophylaxis is not routinely recommended for women of the weak/
partial D phenotype.12
3.
A D-negative woman known to be
immunized to D.

Other Indications for RhIG
RhIG should be given to a D-negative woman
after any obstetric event that might allow
fetal cells to enter the mother’s circulation: spontaneous or therapeutic abortion, ectopic pregnancy, amniocentesis,
chorionic villus sampling, molar pregnancy, cordocentesis, antepartum hemorrhage, blunt abdominal trauma, or fetal
death.55 As mentioned earlier, at 12 weeks
of gestation or earlier, a 50 µg dose of RhIG
would be adequate to protect against the
small fetal blood volume during the first
trimester. From 13 weeks’ gestation until
term, a full dose of RhIG should be given.
At <20 weeks, the fetal blood volume is
rarely more than 30mL,56 small enough
that a single dose of 300 µg Rh immune
globulin will be sufficient for prophylaxis
for any FMH. Therefore, it is not necessary to quantitate fetal red cells in the maternal circulation before 20 weeks of gestation.57

Amniocentesis
Amniocentesis can cause FMH and consequent Rh immunization. The D-negative
woman who has amniocentesis at 16 to 18
weeks’ gestation for genetic analysis should

549

receive a full dose of RhIG at that time, a
second full dose at 28 weeks of gestation,
and the usual postpartum dose if the infant is D positive. If a nonimmunized
D-negative woman undergoes amniocentesis for any reason in the second or third
trimester, a full dose of RhIG is indicated.
If the procedure is repeated more than 21
days later, an additional full dose should
18
be given. If amniocentesis is performed
to assess fetal maturity, and if delivery is
expected within 48 hours of the procedure, RhIG can be withheld until the infant is born and confirmed to be D positive. If more than 48 hours will elapse,
RhIG should be given following amniocentesis. If delivery occurs within 21 days
thereafter and there is no evidence of a
15
massive FMH, additional RhIG may not
be essential, but prudent management
suggests repeat RhIG administration at
delivery.

Screening for Large-Volume FMH
Postpartum administration of RhIG may
not prevent immunization if the quantity
of D-positive fetal red cells entering the
mother’s circulation exceeds the immunosuppressive capacity of RhIG. One 300-µg
dose protects against 15 mL of D-positive
red cells or 30 mL of fetal blood. Only 0.3%
of pregnancies are estimated to sustain
FMH greater than 30 mL, but large FMH
is an important and preventable cause of
7
failed immunoprophylaxis. The ACOG
recommends postpartum testing for large
15
FMH only for high-risk pregnancies, but
58
Ness and colleagues showed that testing
based only on the ACOG criteria would
miss 50% of mothers exposed to large-volume FMH. AABB Standards for Blood
Banks and Transfusion Services requires
examination of a postpartum specimen
from all D-negative women at risk of im-

550

AABB Technical Manual

munization, to detect the presence of FMH
49(p49)
that requires more than one dose of RhIG.
In rare cases, a massive FMH can cause
fetal death and infuse enough Rh-positive
cells into the maternal circulation to simulate a weak D phenotype in an Rh-negative patient. 15,59 Unless recognized and
treated with an adequate dose of RhIG, this
will likely lead to alloimmunization.
“Microscopic Weak D.” In the past, some
workers looked for D-positive red cells in
the mother’s D-negative blood by examining the antiglobulin phase of the test for D
microscopically (“microscopic weak D
test”); mixed-field reactivity indicated a
substantial admixture with D-positive cells.
This procedure should not be used to identify large FMH, however, because of its lack
of reliability.60
The Rosette Test. The rosette test demonstrates small numbers of D-positive cells
in a D-negative suspension. The suspension is incubated with an anti-D reagent of
human origin, and antibody molecules attach to sites on D-positive cells in the suspension. Indicator D-positive cells are
added, which react with antibody molecules bound to the surface of the already-present D-positive cells and form visible agglutinates (rosettes) around them
(see Method 5.1). This method will detect
FMHs of approximately 10 mL,60 a sensitivity that provides a desirable margin of
safety for a screening test. Weak D-positive
cells do not react as strongly in the rosette
procedure as normal D-positive cells. If the
newborn has a weak D phenotype, FMH
can be evaluated by the Kleihauer-Betke
acid-elution test (see below), which identifies fetal hemoglobin, not a surface antigen.
In all cases, the rosette test gives only qualitative results and a positive result must be
followed by a quantitative test, such as an
acid-elution procedure. Other tests that can
be used to detect and/or quantify FMH are
flow cytometry, gel agglutination, and the

enzyme-linked antiglobulin test (ELAT,
which is in limited use). Each of these
methods has various advantages and disadvantages that must be evaluated by each institution. The use of nucleic acid amplification techniques, designed to detect very
small amounts of fetal cells, remains a re61-63
search endeavor.

Quantifying FMH
Historically, quantification of FMH has
been achieved by the Kleihauer-Betke
acid-elution test, which relies on the differences between fetal and adult hemoglobin in resistance to acid elution (see
Method 5.2). Results are reported as a percentage of fetal cells, but the precision
and accuracy of the procedure may be
poor. Because 300 µg of RhIG will protect
against FMH of 30 mL of D-positive fetal
blood, the number of doses of RhIG required is determined by dividing the estimated volume of fetal blood present by 30.
For example:
1.
Kleihauer-Betke test results reported
as 1.3%
2.
(1.3/100) × 5000 mL* = 65 mL of fetal
blood
3.
65/(30 mL per dose) = 2.2 doses of
RhIG required
* = mother’s arbitrarily assigned blood volume

Because quantification by this procedure
is inherently inaccurate and because the
consequences of undertreatment can be
serious, it is desirable to provide a safety
margin in calculating RhIG dosage. One
approach is as follows:
1.
When the number to the right of the
decimal point is less than 5, round
down and add one dose of RhIG (example: If the calculation comes to
2.2 doses, give 3 doses).

Chapter 23: Perinatal Issues in Transfusion Practice

When the number to the right of the
decimal point is 5 or greater, round
up to the next number and add one
dose of RhIG (example: If the calculation comes to 2.8 doses, give 4
doses). (See Table 23-1).
Not more than five doses of RhIG should
be injected intramuscularly at one time. For
larger quantities, injections can be spaced
over a 72-hour period for the patient’s comfort; an optimal time sequence has not
been established. The intravenous preparation of RhIG can be used when higher
doses are required. According to the package insert, a maximum dose of 300 IU
should be given at each injection, every 8
hours, until the total calculated dose has
been administered.

Neonatal Immune
Thrombocytopenia
Maternal IgG antibodies to platelets can
cross the placenta and cause severe antenatal and neonatal thrombocytopenia. Two
categories of immune thrombocytopenia
are recognized, and the distinction between them is therapeutically important.

551

Neonatal Alloimmune Thrombocytopenia
The mechanism of NAIT is similar to that
of HDFN. Fetal platelets, expressing a paternal antigen absent from the mother’s
cells, may enter the mother’s circulation
during gestation or delivery. If she becomes
immunized, the maternal IgG antibody
crosses the placenta and causes fetal and
neonatal thrombocytopenia. The maternal
platelet count remains normal. The incidence of NAIT is approximately 1 in 1500
to 2000 live births.64,65 NAIT is the cause of
the majority of cases of intracranial hemorrhage due to thrombocytopenia, greater
than all other etiologies of thrombocytopenia combined.66
Unlike HDFN, NAIT often affects firstborn
children, with about 50% to 60% of cases
occurring in a woman’s first child. The
thrombocytopenia is self-limiting, normally
resolving in 2 to 3 weeks. NAIT varies in severity from mild thrombocytopenia with no
clinical signs to overt clinical bleeding. The
incidence of intracranial hemorrhage has
been reported as 10% to 30%, with approximately half occurring in utero.65,67 Recurrence in subsequent pregnancies is frequent, with equal or increasing severity, so

Table 23-1. RhIG Dosage for Massive Fetomaternal Hemorrhage, Based on the Acid
Elution Test
Dose
% Fetal Cells

Vials of RhIG to Inject

In g

In IU

0.3 - 0.8

600

3000

0.9 - 1.4

900

4500

1.5 - 2.0

1200

6000

2.1 - 2.5

1500

7500

Notes:
1. Based on a maternal blood volume of 5000 mL.
2. 1 vial of 300 µg (1500 IU) is needed for each 15 mL fetal red cells or 30 mL fetal whole blood.

552

AABB Technical Manual

a woman known to be alloimmunized must
receive skilled antenatal attention.

Serologic Testing
Serologic diagnosis should be sought in a
woman whose infant has had NAIT if further pregnancies are planned. Several
platelet-specific antigen systems have
been associated with NAIT, with HPA-1a
antigen (PlA1) accounting for the vast majority of cases in Caucasians.64 Pregnancy,
rather than transfusion, is the usual immunizing event. Approximately 2% of the
population is HPA-1a negative; approximately 10% of HPA-1a-negative women
with HPA-1a-positive infants become im68
munized. Some studies have shown an
association between developing anti-HPA1a and possessing the HLA phenotype
DRw52a.66,68 Chapter 16 contains more information about platelet antigens. Although antibodies to HLA Class I antigens
are frequently encountered in pregnancy,
and platelets express Class I antigens, this
is a rare cause of NAIT, and the true role of
HLA antibodies in this setting remains
controversial.69,70
Any family with a history of an infant
born with a platelet count of <50,000/µL
should be evaluated. Ideally, serologic testing uses maternal serum and maternal and
paternal whole blood from which platelets
are isolated. Maternal serum is screened for
both platelet-nonspecific and platelet-specific antibodies against paternal cells, as
well as panels of phenotyped platelets. Maternal and paternal platelet typing can be
performed using serologic and/or molecular typing methods. Some clinicians have
proposed screening pregnant women for
HPA-1a antigen because it is the most commonly implicated antigen causing incompatibility in Caucasians. Because only 10%
of HPA-1a-negative women are truly at risk
for forming antibody, and, of those, only

about 33% will have neonates with clinically important thrombocytopenia, this has
not been widely adopted. In addition, the
cost and logistics of performing platelet antigen typing are impediments to broad implementation.65,71

Prenatal Considerations
With knowledge of antibody specificity
and gene frequencies, the likelihood of
subsequent offspring being affected can
be predicted (see Table 16-1). The recognized platelet-specific antigens occur in
diallelic systems, so typing the father’s
platelets indicates zygosity. If the father is
homozygous for the expression of the antigen, there is no need to determine the
fetal antigen status because all offspring
will be affected. If the father is heterozygous for the expression of the antigen,
then there is a 50% chance that subsequent offspring will have the offending
antigen. In an at-risk pregnancy, the genotype of the fetus (and by inference the
platelet phenotype) can be determined by
DNA typing on fetal cells obtained by amniocentesis.
When the risk of NAIT is high, a fetal
blood sample for platelet count determination can be obtained by cordocentesis as
early as 20 weeks’ gestation. Because
cordocentesis carries a risk of serious
bleeding in a thrombocytopenic fetus,
compatible platelets must be available at
the time of the procedure and are often infused if the platelet count is low. Some institutions will infuse platelets during the
procedure, before knowing the platelet
count, because of the risk of bleeding during the cannulation itself. When the fetus
is found to be thrombocytopenic, the
mother is often given infusions of IGIV in
weekly doses of 1 g/kg, with or without steroids, until delivery.64-67,72 Alternatively, some
would recommend empiric treatment with

Chapter 23: Perinatal Issues in Transfusion Practice

IGIV in cases of a homozygous paternal genotype for the specific platelet antigen or
in situations when PCR performed on
amniotic fluid reveals that the fetus carries
that platelet antigen. Many centers will proceed with elective cesarean section instead
of cordocentesis near term to determine
the fetal platelet count.
Sources of Platelets. Maternal platelets
are often prepared for use at cordocentesis
or delivery. The mother will undergo required testing for infectious disease markers. Prior administration of high-dose IGIV
may cause false-positive immunoassays;
therefore, it is desirable to test the mother
before initiating IGIV therapy. Those with
confirmed positive results (eg, hepatitis C
virus, which is transmitted more efficiently
by transfusion than perinatally) should not
be used as a source of platelets because
these results are more likely to represent
maternal infection. Of note, pregnancy itself can also cause false-positive results on
serologic infectious disease tests.
Platelets can be collected either from the
mother or from another donor whose
platelets lack the corresponding antigen
and whose plasma is compatible with the
fetal red cells. If maternal platelets are used,
the antibody-containing plasma should be
removed or reduced and the platelets resuspended in compatible plasma or saline
with reduced volume (see Method 6.15). All
components for intrauterine transfusion must
be irradiated (see Chapter 27) and should
be CMV-reduced risk.73
Scheduling Therapy. Various strategies
have been used in the management of fetal
thrombocytopenia. Although weekly platelet transfusions have been used in the past,
the inherent risk of repeated cordocentesis
makes the administration of IGIV to the
mother the preferred treatment in the
United States. Practice is different in Europe, where weekly platelet transfusions are
still performed. Platelet transfusion and re-

553

peated cordocentesis are reserved for patients when noninvasive forms of therapy
are not effective. Another approach is administration of a single platelet transfusion
just before delivery if cordocentesis reveals
severe thrombocytopenia. This approach is
usually reserved for pregnancies at extreme
risk for intracranial hemorrhage.

Management After Delivery
Platelet counts can continue to decrease
after birth and should be monitored. For
patients at increased risk of bleeding due
to severe thrombocytopenia, compatible
platelets should be given prophylactically.
If compatible platelets are not available,
the use of high-dose IGIV should be considered, but response to this treatment is
variable. In patients who do respond, platelet counts usually start increasing within 24 to 48 hours, although it may take
longer in some patients. 64 Because response is slow, the neonate with an urgent
need for transfusion and no available
compatible platelets can receive platelets
from random donors, frequently resulting
in an adequate response. For patients requiring platelet transfusion, giving concurrent IGIV can accelerate the recovery
of the patient’s own platelets and shorten
the period of transfusion dependency. If
the patient has mild thrombocytopenia
without bleeding, it can be managed
without specific therapeutic intervention.

Thrombocytopenia Secondary to
Maternal ITP
Infants born to mothers with active idiopathic (immune) thrombocytopenic purpura
(ITP) are often not profoundly thrombocytopenic and have a smaller risk of hem69,74
The
orrhage than infants with NAIT.
antibody in ITP is usually IgG, which
readily crosses the placenta. Occasionally,
delivery of a severely thrombocytopenic

554

AABB Technical Manual

infant has led to the diagnosis of previously unsuspected ITP in a moderately affected mother (postpartum platelet count
75,000-100,000/µL). Such cases of mild ITP
should be distinguished from gestational
thrombocytopenia, in which a mother
with no history of autoimmune thrombocytopenia has a platelet count less than
150,000/µL. In cases of maternal ITP, the
risk of severe fetal thrombocytopenia
(usually defined as a platelet count less
than 50,000/µL) is 7% to 10%.74,75 The risk
of intracranial hemorrhage in infants
born to mothers with ITP is low (≤1%),
with only a few cases reported in the literature. This is lower than the rate in NAIT
because, infants born to mothers with ITP
are generally born with higher platelet
counts and their platelet function is not
impaired, as it seems to be in NAIT. Routine fetal platelet assessment is not recommended and cesarean section is re74,76
served for obstetric indications only.
The antibody in ITP has broad reactivity
against platelets. If the infant has a high
concentration of antibody, there will be
uniformly short survival of platelets from
random donors, from the mother, or from
other family members. Responses do sometimes occur, and, in the presence of hemorrhage, platelet transfusions will be used as
emergency therapy.69 IGIV therapy may also
be effective for severe autoimmune thrombocytopenia.66,72,74,76

7.
8.

10.

11.

12.

13.

14.

15.

16.

References
1.

Goodstein M. Neonatal red cell transfusion.
In: Herman JH, Manno CS, eds. Pediatric
transfusion therapy. Bethesda, MD: AABB
Press, 2002:39-91.
Vaughn JI, Manning M, Warwick RM, et al. Inhibition of erythroid progenitor cells by antiKell antibodies in fetal alloimmune anemia.
N Engl J Med 1998;338:798-803.

17.

18.

Dennery PA, Seidman DS, Stevenson DK.
Neonatal hyperbilirubinemia. N Engl J Med
2001;344:581-90.
American Academy of Pediatrics Subcommittee on Hyperbilirubinemia. Management of
hyperbilirubinemia in the newborn infant 35
or more weeks of gestation. Clinical practice
guideline. Pediatrics 2004;114:297-316.
Bowman JM. Intrauterine and neonatal
transfusion. In: Anderson KC, Ness PM, eds.
Scientific basis of transfusion medicine: Implications for clinical practice. Philadelphia:
WB Saunders, 2000:307-20.
Bowman JM. Treatment options for the fetus
with alloimmune hemolytic disease. Transfus
Med Rev 1990;4:191-207.
Bowman JM. The prevention of Rh immunization. Transfus Med Rev 1988;2:129-50.
Bowman JM. Controversies in Rh prophylaxis. Who needs Rh immune globulin and
when should it be given? Am J Obstet Gynecol
1985;151:289-94.
Strauss RG, Burmeister LE, Johnson K, et al.
Randomized trial assessing the feasibility and
safety of biologic parents as RBC donors for
their preterm infants. Transfusion 2000;40:450-6.
Elbert C, Strauss RG, Barrett F, et al. Biological
mothers may be dangerous blood donors for
their neonates. Acta Hematol 1991;85:189-91.
Geifman-Holtzman O, Wojtowycz M, Kosmas
K, Artal R. Female alloimmunization with antibodies known to cause hemolytic disease.
Obstet Gynecol 1997;89:272-5.
Judd WJ. Practice guidelines for prenatal and
perinatal immunohematology, revisited.
Transfusion 2001;41:1445-52.
Legler TJ, Maas JH, Köhler M, et al. RHD sequencing: A new tool for decision making on
transfusion therapy and provision of Rh prophylaxis. Transfus Med 2001;11:383-8.
Wagner FF, Frohmajer A, Ladewig B, et al. Weak
D alleles express distinct phenotypes. Blood
2000;95:2699-708.
Prevention of Rh D alloimmunization. ACOG
Practice Bulletin Number 4. Washington, DC:
American College of Obstetricians and Gynecologists, May 1999.
Kanter MH. Derivation of new mathematic
formulas for determining whether a D-positive father is heterozygous or homozygous for
the D antigen. Am J Obstet Gynecol 1992;166:
61-3.
Bennett PR, Le Van Kim C, Colon Y, et al. Prenatal determination of fetal RhD type by DNA
amplification. N Engl J Med 1993;329:607-10.
Moise KJ. Management of Rhesus alloimmunization in pregnancy. Obstet Gynecol
2002;100:600-11.

Chapter 23: Perinatal Issues in Transfusion Practice

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

Singleton BK, Green CA, Avent ND, et al. The
presence of an RHD pseudogene containing a
37 base pair duplication and a nonsense mutation in Africans with the Rh D-negative
blood group phenotype. Blood 2000;95:12-8.
Hessner MJ, Pircon RA, Luhm RA. Development of an allele specific polymerase chain
reaction assay of prenatal genotyping of Jka
and Jkb of the Kidd blood group system (abstract). Am J Obstet Gynecol 1998;178(Suppl):
52S.
Lee S, Bennett PR, Overton T, et al. Prenatal
diagnosis of Kell blood group genotypes:
KEL1 and KEL2. Am J Obstet Gynecol 1996;
175:445-9.
Le Van Kim C, Mouro I, Brossard Y, et al. PCRbased determination of the Rhc and RhE status of the fetus at risk for Rhc and RhE
hemolytic disease. Br J Haematol 1994;88:
193-5.
Spence WC, Potter P, Maddalena A, et al. DNAbased prenatal determination of the RhEe genotype. Obstet Gynecol 1995;86:670-2.
Ra n d e n I , Ha u g e R , K j e l d s e n - K r a g h J ,
Fagerhol MK. Prenatal genotyping of RHD
and SRY using maternal blood. Vox Sang
2003;85:300-6.
Issitt PD, Anstee DJ. Applied blood group serology. 4th ed. Durham, NC: Montgomery
Scientific Publications, 1998:1067-9.
Management of isoimmunization in pregnancy. ACOG Educational Bulletin Number
227. Washington, DC: American College of
Obstetricians and Gynecologists, 1996.
Gottvall T, Hilden JO. Concentration of anti-D
antibodies in Rh(D) alloimmunized pregnant
women as a predictor of anemia and/or
hyperbilirubinemia in their newborn infants.
Acta Obstet Gynecol Scand 1997;76:733-8.
Bowman JM, Pollock JM, Manning FA, et al.
Maternal Kell blood group alloimmunization.
Obstet Gynecol 1992;79:239-44.
Hadley AG. Laboratory assays for predicting
the severity of haemolytic disease of the fetus
and newborn. Transpl Immunol 2002;10:1918.
Hume HA. Fetal and neonatal transfusion
therapy. In: Pomphilon DH, ed. Modern
transfusion medicine. Boca Raton, FL: CRC
Press, 1995:193-215.
Liley AW. Liquor amnii analysis in the management of the pregnancy complicated by
rhesus sensitization. Am J Obstet Gynecol
1961;82:1359-70.
Queenan JT, Tomai TP, Ural SH, King JC. Deviation in amniotic fluid optical density at a
wavelength of 450 nm in Rh-immunized
pregnancies from 14 to 40 weeks’ gestation: A

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

555

proposal for clinical management. Am J
Obstet Gynecol 1993;168:1370-6.
Steiner EA, Judd WJ, Oberman HA, et al. Percutaneous umbilical blood sampling and
umbilical vein transfusions: Rapid serologic
differentiation of fetal blood from maternal
blood. Transfusion 1990;30:104-8.
Ludomirsky A. Intrauterine fetal blood sampling—a multicenter registry evaluation of
7462 procedures between 1987-1991 (abstract). Am J Obstet Gynecol 1993;168:318.
Mari G, Deter RL, Carpenter FL, et al. Noninvasive diagnosis by Doppler ultrasonography of fetal anemia due to maternal
red-cell alloimmunization. N Engl J Med
2000;342:9-14.
Nishie EN, Brizot ML, Liao AW, et al. A comparison between middle cerebral artery peak
systolic velocity and amniotic fluid optical
density at 450 nm in the prediction of fetal
anemia. Am J Obstet Gynecol 2003;188:214-9.
Quillen K, Berkman EM. Introduction to therapeutic apheresis. In: McLeod BC, Price TH,
Weinstein R, eds. Apheresis: Principles and
practice. 2nd ed. Bethesda, MD: AABB Press,
2003:49-69.
Margulies M, Voto LS, Mathet E, Margulies M.
High dose intravenous IgG for the treatment
of severe Rhesus alloimmunization. Vox Sang
1991;61:181-9.
Ulm B, Kirchner G, Svolba G, et al. Immunoglobulin administration to fetuses with anemia due to alloimmunization to D. Transfusion 1999;39:1235-8.
Ghidini A, Sepulveda W, Lockwood CJ,
Romero R. Complications of fetal blood sampling. Am J Obstet Gynecol 1993;168:1339-44.
Weiner CP, Okamura K. Diagnostic fetal blood
sampling-technique related losses. Fetal
Diagn Ther 1996;11:169-75.
Schumacher B, Moise KJ Jr. Fetal transfusion
for red blood alloimunization in pregnancy
(review). Obstet Gynecol 1996;88:137-50.
Skupski DW, Wolf CF, Bussel JB. Fetal transfusion therapy. Obstet Gynecol Surg 1996;51:18192.
Gonsoulin WJ, Moise KJ, Milam JD, et al. Serial maternal blood donations for intrauterine transfusion. Obstet Gynecol 1990;75:
158-62.
Sanders MR, Graeber JE. Posttransfusion
graft-versus-host disease in infancy. J Pediatr
1990;117:159-63.
Linden JV, Pisciotto PT. Transfusion-associated graft-versus-host disease and blood irradiation. Transfus Med Rev 1992;6:116-23.
Mandelbrot L, Daffos F, Forestier F, et al. Assessment of fetal blood volume for computer-

556

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

AABB Technical Manual

assisted management of in utero transfusion.
Fetal Ther 1988;3:60-6.
Moise KJ, Carpenter RJ, Kirshon B, et al. Comparison of four types of intrauterine transfusion: Effect on fetal hematocrit. Fetal Ther
1989;4:126-37.
Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.
Herschel M, Karrison T, Wen M, et al. Isoimmunization is unlikely to be the cause of
hemolysis in ABO-incompatible but direct
antiglobulin test-negative neonates. Pediatrics 2002;110:127-30.
Koenig JM. Evaluation and treatment of
erythroblastosis fetalis in the neonate. In:
Christensen RD, ed. Hematologic problems
of the neonate. Philadelphia: WB Saunders
Company, 2000:185-207.
Saade GR, Moise KJ, Belfort MA, et al. Fetal
and neonatal hematologic parameters in red
cell alloimmunization: Predicting the need
for late neonatal transfusions. Fetal Diagn
Ther 1993;8:161-4.
Moise KJ, Brecher ME. Package insert for
Rhesus immune globulin (letter). Obstet
Gynecol 2004;103:998-9.
Mollison PL, Engelfriet CP, Contreras M.
Blood transfusion in clinical medicine. 10th
ed. Oxford, England: Blackwell Scientific Publications, 1997.
Hartwell EA. Use of Rh immune globulin.
ASCP Practice Parameter. Am J Clin Pathol
1998;110:281-92.
Nicolaides KH, Clewell WH, Rodeck CH. Measurement of human fetoplacental blood volume in erythroblastosis fetalis. Am J Obstet
Gynecol 1987;157:50-3.
Vengelen-Tyler V, Telen MJ. Collected questions and answers. 5th ed. Bethesda, MD:
AABB, 1997:52-3.
Ness PM, Baldwin ML, Niebyl JR. Clinical
high risk designation does not predict excess
fetal maternal hemorrhage. Am J Obstet
Gynecol 1987;156:154-8.
Owen J, Stedman CH, Tucker TL. Comparison
of predelivery versus postdelivery KleihauerBetke stains in cases of fetal death. Am J
Obstet Gynecol 1989;161:663-6.
Sebring ES. Fetomaternal hemorrhage—incidence and methods of detection and quantitation. In: Garratty G, ed. Hemolytic disease
of the newborn. Arlington, VA: AABB, 1984:87-117.
Duguid JKM, Bromilow IM. Laboratory measurement of fetomaternal hemorrhage and
its clinical relevance. Transfus Med Rev 1999;
13:43-8.
Riley JZ, Ness PM, Taddie SJ, et al. The detection and quantitation of fetal-maternal hem-

63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.
74.

75.

76.

orrhage using an enzyme-linked antiglobulin
test (ELAT). Transfusion 1982;22:472-4.
Bayliss KM, Kueck BD, Johnson ST, et al. Detecting fetomaternal hemorrhage: A comparison of five methods. Transfusion 1991;31:303-7.
Uhrynowska M, Maslanka K, Zupanska B.
Neonatal thrombocytopenia: Incidence,
serological and clinical observations. Am J
Perinatol 1997;14:415-18.
Williamson LM, Hackett G, Rennie J, et al.
The natural history of fetomaternal alloimmunization to the platelet-specific antigen
HPA-1a (PlA1, Zwa) as determined by antenatal screening. Blood 1998;92:2280-7.
Bussel JB. Alloimmune thrombocytopenia in
the fetus and newborn. Semin Thromb
Hemost 2001;27:245-52.
Johnson JA, Ryan G, al-Musa A, et al. Prenatal
diagnosis and management of neonatal alloimmune thrombocytopenia. Semin Perinatol
1997;21:45-52.
Waters AW, Murphy M, Hambley H, Nicolaides K. Management of alloimmune thrombocytopenia in the fetus and neonate. In: Nance
SJ, ed. Clinical and basic science aspects of
immunohematology. Arlington, VA: AABB,
1991:155-77.
Blanchette VS, Kuhne T, Hume H, Hellman J.
Platelet transfusion therapy in newborn infants. Transfus Med Rev 1995;9:215-30.
Maes LY, Gautreaux M, Southgate WM, Lazarchick J. Evidence of neonatal alloimmune
thrombocytopenia mediated by anti-HLA antibodies (abstract). Transfusion 2001;41
(Suppl):20S.
Murphy MF, Williamson LM, Urbaniak SJ. Antenatal screening for fetomaternal alloimmune thrombocytopenia; should we be
doing it? Vox Sang 2002;83(Suppl 1):409-16.
Gaddipati S, Berkowitz RL, Lembet AA, et al.
Initial fetal platelet counts predict the response to intravenous gammaglobulin therapy in fetuses that are affected by PLA1 incompatibility. Obstet Gynecol 2001;185:976-80.
Leukocyte reduction. Association Bulletin
99-7. Bethesda, MD: AABB, 1999.
Bussel JB. Immune thrombocytopenia in
pregnancy: Autoimmune and alloimmune. J
Reprod Immunol 1997;37:35-61.
Payne SD, Resnik R, Moore TR, et al. Maternal
characteristics and risk of severe neonatal
thrombocytopenia and intracranial hemorrhage
in pregnancies complicated by autoimmune
thrombocytopenia. Am J Obstet Gynecol
1997;177:149-55.
Blanchette VS, Kirby MA, Turner C. Role of intravenous immunoglobulin G in autoimmune hematologic disorders. Semin Hematol
1992;29:72-82.

Chapter 24: Neonatal and Pediatric Transfusion Practice

Chapter 24

Neonatal and Pediatric
Transfusion Practice

ANY PHYSIOLOGIC CHANGES
accompany the transitions from
fetus to neonate, neonate to
infant, and throughout childhood. Hematologic values, blood volume, and physiologic responses to stresses such as hypovolemia and hypoxia vary widely. The
most rapid changes occur during early infancy. Consequently, discussions of pediatric transfusion are usually divided into
two periods: neonates from birth through
4 months, and older infants (>4 months)
and children. Some concerns in neonatal
transfusion practice overlap with those of
the perinatal period and are discussed in
Chapter 23.
Advances in medical care now permit
the survival of extremely premature neonates. Blood providers must be capable of
furnishing blood components that are tailored to satisfy the specific needs of very
low birthweight (VLBW <1500 g) and extremely low birthweight (ELBW <1000 g)

patients, whose small blood volumes and
impaired or immature organ functions provide little margin for safety. Ill neonates are
more likely than hospitalized patients of
any other age group to receive red cell
transfusions.1 Advances in critical-care neonatology, such as surfactant therapy, nitric
oxide therapy, use of high-frequency ventilators, and adherence to transfusion practice guidelines, have diminished the number of blood transfusions given; most are
now given to infants with birthweights less
than 1000 g.1 The doses of various components used for simple, small-volume transfusions are given in Table 24-1.2

Fetal and Neonatal
Erythropoiesis
The predominant sites of hematopoiesis
in the developing embryo shift from the
wall of the yolk sac to the liver to the mar557

558

AABB Technical Manual

Table 24-1. Volumes for Simple, Small-Volume Transfusions of Neonates
Component

Volume

Estimated Change

RBC

10-15 mL/kg

Hemoglobin ↑ 2-3 g/dL

Platelet

5-10 mL/kg

Platelet ↑ 50,000-100,000/µL

Granulocyte

≥1 × 10 neutrophils/kg in
volume of 10-15 mL/kg

Repeat until clinical response

FFP

10-15 mL/kg

Factor activity ↑ 15-20%

Cryoprecipitate

1-2 units/10 kg

↑ 60-100 mg/dL fibrinogen (infant)
↑ 5-10 mg/dL fibrinogen (larger child)

Adapted with permission from Roseff.

row in the first 24 weeks. Hematopoiesis
is regulated by gradually increasing erythropoietin (EPO) levels stimulated by low
oxygen tensions during intrauterine life.
Fetal red cells, rich in hemoglobin F, are
well adapted to low intrauterine oxygen
tensions. The high oxygen affinity of fetal
hemoglobin enhances transfer of oxygen
from maternal erythrocytes to fetal erythrocytes throughout pregnancy.
The “switch” from fetal to adult hemoglobin begins at about 32 weeks’ gestation;
at birth, 60% to 80% of the total hemoglobin is hemoglobin F. Preterm neonates,
therefore, are born with higher levels of fetal hemoglobin than those born at term.
The mean cord hemoglobin of healthy term
neonates is 16.9 ± 1.6 g/dL, and that of
preterm neonates is 15.9 ± 2.4 g/dL.4 Hemoglobin concentration gradually falls in
the first few weeks of life. This has been
called “physiologic anemia of infancy” in
term newborns and “physiologic anemia of
prematurity” in preterm newborns. The
anemia is considered physiologic because
it is self-limited, is usually well tolerated,
and is not associated with any deleterious
effects to the infant. Erythropoietic activity
diminishes secondary to an increase in pulmonary blood flow and a rise in arterial
pO2, as well as the increase in red cell con-

tent of 2,3-diphosphoglycerate (2,3-DPG)
and hemoglobin A, which enhance the release of oxygen to the tissues. As tissue oxygenation improves, levels of EPO decline
and erythropoiesis diminishes. This, along
with decreased survival of fetal red cells
and expansion of the blood volume due to
rapid growth, causes the hemoglobin concentration to decline. The rate of decline is
dependent on gestational age at birth; hemoglobin may drop to as low as 8.0 g/dL at
4 to 8 weeks of age in preterm infants with
birthweights of 1000 to 1500 g, and 7.0 g/dL
in neonates with birthweights less than
1000 g.3
Despite hemoglobin levels that would
indicate anemia in older children and
adults, the normally developing infant usually maintains adequate tissue oxygenation.
Physiologic anemia requires treatment only
if the degree or timing of the anemia causes
symptoms in the patient.

Unique Aspects of Neonatal
Physiology
Infant Size and Blood Volume
Full-term newborns have a blood volume
of approximately 85 mL/kg; preterm low

Chapter 24: Neonatal and Pediatric Transfusion Practice

birthweight newborns have an average
blood volume of 100 mL/kg. As survival
rates continue to improve for infants
weighing 1000 g or less at birth, blood
banks are being asked to provide blood
components for patients whose total
blood volume is less than 100 mL on a
more frequent basis. The need for frequent laboratory tests has made replacement of iatrogenic blood loss the most
common indication for transfusion of low
birthweight preterm neonates. However,
the previous practice of replacing blood
mL for mL is giving way to replacement as
needed in order to maintain a target hematocrit in certain clinical situations.1
Newborns do not compensate for hypovolemia as well as adults. After 10% volume
depletion in a newborn, left ventricular
stroke volume is diminished without increasing heart rate. To maintain systemic
blood pressure, peripheral vascular resistance increases, and this, combined with a
decreased cardiac output, results in poor
tissue perfusion, low tissue oxygenation,
and metabolic acidosis.5

Erythropoietin Response
Erythropoietin response in newborns differs from that in adults and older children. In older children and adults, oxygen
sensors in the kidney recognize diminished oxygen delivery and release EPO
into the circulation. In the fetus, the oxygen sensor that stimulates EPO production is believed to be the liver, which appears to be programmed for the hypoxic
intrauterine environment.
This hyporesponsiveness to hypoxia protects the fetus from becoming polycythemic in utero. Eventually, EPO production shifts from the liver to the kidneys, a
developmental change thought to be regu-

559

lated based on the time of conception, not
birth, and possibly not beginning until
term. The most premature infants produce
the least amount of EPO for any degree of
anemia; this may reflect the absence of the
developmental shift of erythropoietin production from the liver to the kidneys.6 Sick
preterm neonates who receive many transfusions shortly after birth have reduced circulating levels of fetal hemoglobin. Circulating EPO levels are lower, for a given
hematocrit, in preterm neonates with
higher proportions of hemoglobin A relative to hemoglobin F, which favors release
6
of oxygen to the tissues. Erythroid progenitor cells in the hypoproliferative marrow of
these preterm infants show normal intrinsic sensitivity to EPO. Clinical trials of recombinant human erythropoietin (rHuEPO)
in premature neonates show that the number of transfusions and severity of anemia
7
can be lessened. Adverse effects of rHuEPO
in this age group are also different from
those seen in older children and adults and
include transient, reversible neutropenia.
Since the adherence to strict transfusion
guidelines and the decrease in phlebotomy
in VLBW infants, the ultimate role of
rHuEPO in the management of “anemia of
prematurity” has remained unclear. A recent multicenter study in Europe showed
decreased need for transfusion when administering EPO to ELBW infants within 3
to 5 days of life (early EPO administration)
and continuing for 9 weeks.8 The mean
number of transfusions dropped from 2.66
to 1.86, with a reduction in donor exposures from 2 to 1. Questions regarding optimal dosing, route of administration, and
use of supplemental iron remain to be answered.1,7-10 In any event, with the tremendous strides made in decreasing donor exposure in transfused newborns by using
restrictive transfusion practices alone, the
role of EPO for this purpose may no longer
be relevant.

560

AABB Technical Manual

Cold Stress
Hypothermia in the newborn causes exaggerated effects, including increased
metabolic rate, hypoglycemia, metabolic
acidosis, and a tendency toward apneic
episodes that may lead to hypoxia, hypotension, and cardiac arrest. Blood used for
exchange transfusion should be warmed
because blood at room temperature may
decrease a newborn’s core temperature by
0.7 to 2.5 C. The usual method is to use an
inline warmer. Blood, either large or small
volumes, should not be warmed under
a radiant heater because of the risk of
hemolysis in an unmonitored apparatus.
When transfusions are given to infants
undergoing phototherapy, the tubing
should be positioned to minimize exposure to the phototherapy light in order to
prevent hemolysis.11

Immunologic Status
Infants have an immature and inexperienced cellular and humoral immune system.
Antibodies present derive almost entirely
from the maternal circulation. Transplacental transfer of immunoglobulin and
other proteins is independent of molecular size; IgG (150 kD) is transferred much
more readily than albumin (64 kD). In humans, maternal IgM does not reach the
fetus and IgA is not readily transferred, although low levels have been found in the
newborn.
All four subclasses of IgG are transported
across the placenta, but the rate varies between individual mother-fetus pairs. Early
in pregnancy (approximately 12 weeks), IgG
probably passes from mother to fetus by
diffusion, and concentration in fetal serum
is low for all subgroups.12 Between 20 and
33 weeks of gestation, fetal IgG levels rise
markedly, apparently because of maturation of a selective transport system that involves, in part, specific protein receptors on

the membrane of placental cells. IgG1, the
predominant subclass in maternal blood,
crosses the placenta first and is transported
in greatest quantity. Cord blood has higher
antibody concentrations than maternal
blood. Catabolism of IgG occurs more
slowly in the fetus than in the mother, so
that transplacental maternal antibody is
conserved during the neonatal period.
A fetus exposed to an infectious process
in utero or an infant exposed shortly after
birth may produce small amounts of IgM
detectable by sensitive techniques, but unexpected red cell alloantibodies of either
IgG or IgM class are rarely formed during
the neonatal period. The mechanisms
responsible for the lack of alloantibody production in the neonate are not clearly understood and are most likely multifactorial,
including deficient T helper function, enhanced T suppressor activity, and poor antigen-presenting cell function.13
The cellular immune response is critical
to the occurrence of transfusion-associated
graft-vs-host disease (TA-GVHD). In the
newborn, TA-GVHD has been reported
most often in the clinical setting of confirmed or suspected congenital immunodeficiency. It is recommended that infants
with suspected and/or documented T-cell
immunodeficiency receive irradiated blood
components. The majority of TA-GVHD cases
reported in nonimmunocompromised hosts
have occurred in infants who received intrauterine transfusion followed by postnatal
exchange transfusion.14 A proposed explanation is that lymphocytes given during
intrauterine transfusion could induce host
tolerance, impairing rejection of lymphocytes
given in the subsequent exchange transfusions. There have also been rare cases of
TA-GVHD reported in association with extreme prematurity, neonatal alloimmune
thrombocytopenia, and the use of extracorporeal membrane oxygenation (ECMO).14,15
Neonates present with TA-GVHD after a

Chapter 24: Neonatal and Pediatric Transfusion Practice

longer latent period than adults, with fever
occurring at an average of 28 days after
exposure, rather than 10 days for immunocompetent adults. There may be several
risk factors other than the immune status of
the recipient that predispose to TA-GVHD,
such as the number and viability of lymphocytes in the transfused component and
donor-recipient HLA compatibility. The
true incidence of TA-GVHD in the neonatal
setting is not known. However, on the basis
of data from Japan, it appears that the incidence of reported TA-GVHD is far lower
than would be expected.15 The postulated
mechanism for this apparent decreased
susceptibility of newborns to TA-GVHD is
thought to be extrathymic and/or thymic
semitolerance of allogeneic donor T lymphocytes. As for all patients, directed-donor
units from biologic relatives must be irradiated.15,16 There are no data to support the
practice of universal irradiation of blood
transfused to all infants or children.

Metabolic Problems
Acidosis or hypocalcemia may occur after
large-volume whole blood or plasma
transfusion because the immature liver of
the newborn metabolizes citrate inefficiently. Immature kidneys have reduced
glomerular filtration rate and concentrating ability, and newborns may have difficulty excreting excess potassium, acid,
and/or calcium.

561

days in extended storage medium would
deliver 2 mL of plasma containing only
0.1 mmol/L of potassium. This is much
less than the daily potassium requirement
of 2 to 3 mmol/L for a 1-kg patient.17 However, serum potassium may, rise rapidly
after infusion of large volumes of red cells
in such circumstances as surgery, exchange transfusion, or extracorporeal circulation, depending upon the plasma
potassium levels in the blood and manipulation of the blood component. 18,19 Of
interest, a unit of Red Blood Cells (RBCs)
preserved in AS-1 will deliver less extracellular potassium compared to the
amount in RBCs stored in CPDA-1.16,20 In
stored irradiated blood, the problem of
potassium leak is potentiated; for selected
patients, it may be desirable to wash irradiated cells, if they have subsequently
been stored >24 hours. 19 There are increasing anecdotal reports of infants who
receive either older RBC units or units irradiated more than 1 day before transfusion having severe adverse effects (eg,
cardiac arrest, death) after transfusion of
these products into central lines or intracardiac lines.21,22
The untoward consequences of washing
blood, such as reducing its shelf life and the
possible introduction of bacteria, must be
considered. It is preferable to perform irradiation as close to the time of administration as possible, obviating the concern for
high levels of potassium in the transfused
product.

Potassium
Although potassium levels increase rapidly
in the plasma of stored red cells, smallvolume, simple transfusions administered
slowly have little effect on serum potassium concentration in newborns. It has
been calculated that transfusion of 10
mL/kg of red cells (hematocrit 80%) obtained from a unit of blood stored for 42

2,3-Diphosphoglycerate
Neonates with respiratory distress syndrome or septic shock have decreased
levels of intracellular red cell 2,3-DPG.
Alkalosis and hypothermia may further
increase the oxygen affinity of hemoglobin, shifting the dissociation curve to the
left and making oxygen even less available

562

AABB Technical Manual

to the tissues. Arterial oxygenation may
be further compromised by respiratory
distress syndrome or other pulmonary
disease. Mechanisms that compensate for
hypoxia in adults, such as increased heart
rate, are limited in newborns. If a large
proportion of an infant’s blood volume has
come from transfusion of 2,3-DPG-depleted blood, this may cause problems
that would not affect older children or
adults. Because 2,3-DPG levels decline
rapidly after the first week of storage, the
freshest blood conveniently available (up
to 14 days) should be used for exchange
transfusion in newborns. For small-volume
transfusions, the medical necessity for
fresh blood has never been demonstrated
and arguments have been raised to suggest it is unnecessary.1,16,19

Cytomegalovirus Infection
Perinatal infection with cytomegalovirus
(CMV) may occur, acquired either in utero
or during the birth process. Neonates can
be infected during breast-feeding or by
close contact with mothers or nursery
personnel. CMV can also be transmitted
by transfusion, although the risk from the
current blood supply is small.23,24
CMV infection in newborns has extremely variable manifestations, ranging
from asymptomatic seroconversion to death.
Studies of CMV in neonatal transfusion recipients reveal the following observations:
1.
The overall risk of symptomatic posttransfusion CMV infection seems to
be inversely related to the seropositivity rate in the community. Although many adults are positive for
CMV antibodies, the rate of symptomatic CMV infection in newborns
is low.
2.
Symptomatic CMV infection during
the neonatal period is uncommon in

children born to seropositive mothers.25
The risk of symptomatic posttransfusion infection is high in multitransfused preterm infants weighing
less than 1200 g who are born to
seronegative mothers.17,26
The risk of acquiring CMV infection
is directly proportional to the cumulative number of donor exposures
incurred via transfusion.
Cytomegalovirus in blood is associated with leukocytes. The risk of virus transmission can be reduced by
transfusing CMV-reduced-risk blood
from seronegative donors or by using leukocyte-reduced components.
Although deglycerolized and washed
red cells also have a reduced risk of
CMV infection, leukocyte reduction
by filtration is the technique of
choice.16,27-30

Red Cell Transfusions in
Infants Less than 4 Months
of Age
RBCs are the component most often
transfused during the neonatal period.
Many of the physiologic considerations
mentioned above directly affect decisions
regarding indications for transfusion, selection, and administration of red cell
components, as well as the requirements
for compatibility testing.

Compatibility Testing
Because the neonate and young infant are
immunologically immature, alloimmunization to red cell antigens is rare during
the neonatal period. A study of 90 neonates who received 1269 transfusions from
different donors found no instances of
antibody production even with use of

Chapter 24: Neonatal and Pediatric Transfusion Practice

very sensitive detection techniques. Other
investigators confirm the relative infrequency of alloantibodies directed against
13,28
red cell, as well as HLA, antigens.
Because alloimmunization is extremely
rare and repeated testing increases iatrogenic blood loss, AABB Standards for Blood
Banks and Transfusion Services32(p42) requires
only limited pretransfusion serologic testing for infants under 4 months old. Initial
testing must include ABO and D typing of
red cells and a screen for red cell antibodies, using either serum or plasma, from the
mother or the infant.
During any one hospitalization, compatibility testing and repeat ABO and D typing
may be omitted, provided that the screen
for red cell antibodies is negative; that all
red cells transfused are group O or ABOidentical or ABO-compatible; and that red
cells are either D negative or the same D
type as the patient. It is unnecessary to test
the infant’s serum for anti-A and/or anti-B
as a component of blood typing. Before giving non-group-O red cells, the neonate’s serum must be checked for passively acquired maternal anti-A or anti-B and must
include the antiglobulin phase. If the antibody is present, ABO-compatible red cells
lacking the corresponding A or B antigen
must be used until the antibody is no longer detected. In this setting, it is not necessary to perform crossmatches. If an unexpected non-ABO red cell antibody is
detected in the infant’s specimen or the
mother’s serum contains a clinically significant red cell antibody, the infant should be
given either RBC units tested and found to
lack the corresponding antigen(s) or units
compatible by antiglobulin crossmatch.
This practice should continue for as long as
maternal antibody persists in the infant’s
blood. The institution’s policy will determine how frequently to recheck the screen
for red cell antibodies; once a negative result is obtained, subsequent crossmatches

563

and/or provision of blood lacking the target
antigen are unnecessary. It is important to
avoid transfusion of any component that
may transfer unexpected antibody or ABOincompatible antibodies to the infant.

Indications for Red Cell Transfusion
Certain events in the perinatal period cause
anemia, for which the benefits of red cell
transfusion are unquestioned. These include spontaneous fetomaternal or fetoplacental hemorrhage, twin-twin transfusion, obstetric accidents, and internal
hemorrhage. A venous hemoglobin of less
than 13 g/dL in the first 24 hours of life indicates significant anemia.33 For severely
anemic neonates with congestive heart
failure, it may be necessary to remove aliquots of their dilute blood and transfuse
concentrated red cells. This “partial exchange” transfusion will prevent intravascular volume overload. Most red cell
transfusions in the neonatal period, however, are given either to replace iatrogenic
blood loss or to treat the physiologic decline in hemoglobin (anemia of prematurity) when it complicates clinical
problems.
Because tissue demand for oxygen cannot be measured directly and because so
many variables determine oxygen availability, no universally accepted criteria exist for
transfusion of preterm or term neonates.
Despite the widespread use of micromethods for laboratory tests and growing
use of bedside or noninvasive monitoring
devices, infants still sustain significant cumulative blood loss from laboratory sampling. In a sick neonate, red cell replacement
is usually considered when approximately
10% of the blood volume has been removed. The decision to transfuse a newborn for anemia should include evaluation
of the hemoglobin levels expected for the
patient’s age and clinical status, as well as

564

AABB Technical Manual

the amount of blood loss over time. Transfusion may be more aggressive in the infant
in respiratory distress who is hypoxic and
more vulnerable to cerebral hemorrhage.
Considerable controversy surrounds the
correlation of the “signs of anemia” in the
preterm infant (tachycardia, tachypnea,
bradycardia, recurrent apnea, and poor
weight gain) with response to red cell transfusions.1 When red cells are transfused, they
are usually given in small volumes of 10 to
15 mL/kg (or less if the infant cannot tolerate this volume). The hematocrit of the red
cell component transfused will depend on
the anticoagulant/preservative used and
how the original unit is processed to provide small component transfusions for neonates. A transfusion of 10 mL/kg of red cells
adjusted to a hematocrit greater than 80%
just before release for transfusion should
raise the hemoglobin concentration by approximately 3 g/dL. A transfusion of 10
mL/kg of red cells in additive solution,
which have a hematocrit of approximately
65%, will result in a posttransfusion hemoglobin increment less than 3 g/dL.

Red Cell Components Used for Neonatal
Transfusion
The small-volume requirements of transfusion to neonatal recipients make it possible to prepare several aliquots from a
single donor unit, thus limiting donor exposure and decreasing donor-related risks.
Several technical approaches are available to realize this advantage and to minimize wastage.34

Aliquoting for Small-Volume Transfusion
A multiple-pack system is a common technique for providing small-volume red cell
transfusions.34,35 Quad packs, where a single unit of Whole Blood is collected into a
bag with four integrally attached containers, can be used to increase the number of

transfusions an infant can receive from
one donor. Because the original seal remains intact, each container has the expiration date of the original unit. With use
of a sterile connecting device, multiple
bags called pedi-packs or specially designed syringe systems can be integrally
attached to a unit of RBCs after component preparation. This maintains a closed
system and further increases the number
of small-volume transfusions obtained
from a single donor. Sterile connecting
devices can be used to prepare small
aliquots for transfusion in the blood bank.
If aliquots are prepared by entering the
bag through a port, the unit and the
aliquot are assigned a 24-hour shelf life, if
refrigerated.
Each aliquot must be fully labeled as it is
prepared, including the time it outdates.
The origin and disposition of each aliquot
must be recorded. Using these techniques,
a recipient can receive multiple small-volume transfusions from a single donation
until the expiration of the original unit,
thereby reducing donor exposure.34,36 The
disadvantage of any method that creates
aliquots from a single donation is that any
undetected, transmissible pathogen that
might be present in the primary unit can be
disseminated to multiple recipients.
In order to prevent waste, many transfusion services assign a unit of RBCs to one or
more infants based on their weight. As an
example, 1 or 2 lower birthweight infants
may be assigned to one unit because they
will most likely require the greatest number
of transfusions. On the other hand, four
larger infants may be assigned to one unit
because their transfusion needs will not be
as great.37,38

Red Cells with Additive Solution
RBCs used for pediatric transfusions were
traditionally stored in CPDA-1.35 Additive

Chapter 24: Neonatal and Pediatric Transfusion Practice

solutions (AS) used as anticoagulants/
preservatives contain additional adenine
and dextrose and some contain mannitol.
There has been concern about the potential side effects of these additives because
large amounts of adenine and mannitol
have been associated with renal toxicity.
Mannitol is also a potent diuretic and, because of its effect on the fluid dynamics in
preterm infants, may cause unacceptable
fluctuations in cerebral blood flow. The
different constituents of these solutions
are found in Chapter 8. However, when
the dose of transfused red cells is small (5
to 15 mL/kg), the recipient is exposed to
relatively small amounts of the preservative solutions. Clinical studies comparing
red cells stored in AS-1 and AS-3 solutions
have shown no apparent detrimental effects in neonates receiving simple transfusions, and, after adjustment for the
lower hematocrit of the component, they
are as effective as CPDA-1 cells in increasing hemoglobin. Furthermore, the additional sugars present have been shown to
benefit glucose homeostasis in comparison with CPDA-1.39 Studies on the safety
of AS-3-preserved red cells in neonates have
been published.38,40 Because the components of AS-5 are the same as those found
in the other additive solutions, it is considered acceptable for use in neonates.
Using theoretical calculations in a vari41
ety of clinical situations, Luban et al demonstrated that red cells preserved in
extended-storage media present no substantive risks when used for small-volume
transfusions. For preterm infants with severe hepatic or renal insufficiency, however,
removing the additive-containing plasma
may be beneficial. This is particularly important if there will be multiple transfusions that could have a cumulative effect.
The safety of red cells stored in additive solutions and used for massive transfusions,
such as cardiac surgery or exchange trans-

565

fusion, has not been studied. Concern still
remains regarding the use of blood stored
in additive solutions being used outside the
setting of simple, small-volume transfusion.40,41 However, with extensive anecdotal
use in large transfusion services, there have
not been reports of deleterious effects from
the infusion of additive solutions.42,43
Whether or not placental/umbilical cord
blood will become an acceptable form of
neonatal transfusion remains to be seen.
Although use of this type of “autologous”
blood would eliminate some infectious disease risks, questions regarding quantity,
quality, and sterility raise concerns about
its safety and efficacy.18

Transfusion Administration
Vascular access is often difficult in the tiny
newborn and in any infant requiring
long-term or repeated intravenous infusions. Within a short time after birth, the
umbilical artery may be cannulated. Transfusion through a needle as small as 25gauge or a vascular catheter as small as
24-gauge has been shown to cause little
hemolysis and to be safe when constant
flow rates are used. Transfusion through
smaller gauge catheters has not been
thoroughly evaluated.
It is not usually necessary to warm smallvolume transfusions that are given slowly,
but it is important to be able to control the
volume and rate of infusion. Constant-rate
electromechanical syringe delivery pumps
provide this control and cause minimal
hemolysis, even when used with inline leukocyte reduction filters.44,45
The length of the plastic tubing used can
add significantly to the volume required for
transfusion. Infusion sets identified as suitable for platelets or components have less
dead space than standard sets because they
have short tubing and a small 170-micron
filter. Pediatric microaggregate filters (20-

566

AABB Technical Manual

or 40-micron) are often used for their small
priming volume, not for the removal of
microaggregates. Hemolysis may occur
when stored blood is given by negative
pressure filtration through these filters.46
Administration rates for RBCs have not
been extensively studied, nor are there
standard practices; rates of transfusion as
well as devices used vary with institutions.
The rate of administration of blood and
blood components in neonates and infants
should be individualized on the basis of the
patient’s clinical needs. Theoretical concerns regarding rapid changes in intravascular volume and electrolyte changes in
these small, labile patients have focused on
an increased risk for intracranial hemorrhage. This has not been clearly demonstrated. For simple RBC transfusions, transfusing products over 2 to 4 hours is usually
adequate. When there is an urgent need because of shock or severe bleeding, infusion
should be as rapid as possible. Products
can be transfused safely using a variety of
devices. It is important that mechanical
systems be tested and validated for use
with blood and blood components.

Exchange Transfusion for
Hyperbilirubinemia
The fetal liver has limited capacity to conjugate bilirubin. In utero, unconjugated
bilirubin crosses the placenta for excretion through the mother’s hepatobiliary
system. After birth, transient mild hyperbilirubinemia normally occurs during the
first week of life and is referred to as “physiologic jaundice.” Liver function is less mature, and jaundice worsens in premature
neonates. When the level of unconjugated
bilirubin is excessive, bilirubin may cross
the blood-brain barrier and concentrate
in the basal ganglia and cerebellum; the
resulting damage to the central nervous
system (CNS) is called kernicterus. Photo-

therapy with fluorescent blue lights is the
most common treatment for hyperbilirubinemia; exchange transfusion is reserved
for phototherapy failures. The most common reason, however, for an exchange to
be performed in a neonate is to correct
hyperbilirubinemia.
Pathologic processes that may result in
excessively high unconjugated bilirubin
levels in neonates include immune-mediated hemolysis, nonimmune hemolysis,
bile excretion defects and impaired albumin binding. Exchange transfusion removes unconjugated bilirubin and provides
additional albumin to bind residual bilirubin. If hyperbilirubinemia is due to antibody-mediated hemolysis, exchange transfusion is of additional benefit by removing
free antibody and antibody-coated red cells
while providing antigen-negative red cells
that will survive normally.
Exchange transfusion should be performed before bilirubin rises to levels at
which CNS damage occurs. Several factors
affect the threshold for toxicity. CNS damage occurs at lower levels if there is prematurity, decreased albumin binding capacity, or the presence of such complicating
conditions as sepsis, hypoxia, acidosis, hypothermia, or hypoglycemia. In full-term
infants, kernicterus rarely develops at indirect bilirubin levels less than 25 mg/dL, but,
in sick VLBW infants, kernicterus has oc47
curred at levels as low as 8 to 12 mg/dL.
The rate at which bilirubin rises is more
predictive of imminent need for exchange
transfusion than the absolute level attained.
Neonates with severe anemia and a rapid
rise in bilibrubin, despite phototherapy, require exchange transfusion. A two-volume
exchange transfusion removes approximately 70% to 90% of circulating erythrocytes and about 25% of the total bilirubin.
Because of reequilibration between the
extravascular tissue and plasma bilirubin,
levels may again rise, resulting in the need

Chapter 24: Neonatal and Pediatric Transfusion Practice

for a second exchange transfusion. Indications for repeat exchange are similar to
those for the initial exchange.
The American Academy of Pediatrics recently released new guidelines for the management of newborn infants born ≥35
weeks of gestation with hyperbilirubinemia. It is hoped that raising awareness
about the potential for hyperbilirubinemia
in this patient group will reduce its frequency and provide a framework for optimal treatment, to include the use of phototherapy, exchange transfusion, and Immune
49
Globulin, Intravenous (IGIV).

Exchange Transfusion for Other Causes
The safety and efficacy of exchange transfusion in the neonatal period for other indications should be evaluated on a caseby-case basis, using guidelines in published literature. Treatment categories for
disorders based on proven vs theoretical
benefits have been recommended (see
Table 6-1). The treatment of disseminated
intravascular coagulation (DIC) using exchange transfusion has yielded variable
results, perhaps because only the sickest
infants have been selected to receive this
therapy. The most important aspect of
therapy for neonatal DIC is to treat the
underlying disease.
Exchange transfusion is occasionally
used to remove other toxins, such as drugs
or chemicals given to the mother near the
time of delivery, drugs given in toxic doses
to the neonate/infant, or substances such
as ammonia that accumulate in the newborn because of prematurity or inherited
metabolic diseases.50,51

Technique of Exchange Transfusion

Choice of Components
Red cells are resuspended in compatible
thawed Fresh Frozen Plasma (FFP) for exchange transfusion. If AS-RBC units are

567

used, depending on the clinical situation,
some institutions would choose to remove
the additive-containing plasma, to reduce
the volume transfused. As discussed earlier, washing components may not be
necessary or desirable. Many transfusion
services use red cells that have been
screened and found to lack hemoglobin S
for exchange transfusion, to avoid the
possibility of intravascular sickling.
The glucose load administered during
exchange transfusion can be extremely
high. This stimulates the infant to secrete
insulin, which may lead to rebound hypoglycemia. It is important to monitor blood
glucose levels for the first few hours after
the procedure.
Because unconjugated bilirubin binds to
albumin, albumin is frequently used to increase intravascular binding. With additional albumin in the circulation, bilirubin
from the extravascular space diffuses out to
the intravascular space. This, in turn, increases the total quantity of bilirubin removed during the exchange. There have
been conflicting results, however, about the
efficacy of administering albumin either
before or during exchange to enhance bilirubin removal. A study that compared 15
hyperbilirubinemic neonates given albumin with 27 who received none found similar efficiency of bilirubin removal in both
groups.52 Infusing albumin raises the colloid
osmotic pressure and increases intravascular volume. Therefore, it should be given
cautiously, if at all, to neonates or infants
who are severely anemic, have increased
central venous pressure, or are in renal or
congestive heart failure.
Exchange transfusion may cause dilutional thrombocytopenia and/or coagulopathy that require transfusion of platelets
and/or other components containing coagulation factors. Platelet counts and coagulation parameters should be monitored after exchange transfusion.

568

AABB Technical Manual

Volume and Hematocrit
An exchange transfusion equal to twice the
patient’s blood volume is typically recommended for newborns; rarely is more than
one full unit of donor blood required. In
practice, the volume calculated for exchange is an estimate. The final hematocrit of transfused blood should be approximately 40% to 50%, with sufficient plasma
to provide clotting factors, if needed. In
the unusual event that the infant’s condition demands a high postexchange hematocrit, a small-volume transfusion of red cells
can be given after the exchange, or units
with a higher hematocrit used for exchange. It is important to keep the blood
mixed during the exchange; if it settles in
the container, the final aliquots will not
have the intended hematocrit. The infant’s hematocrit and bilirubin level
should be measured using the last aliquot
removed in the exchange.

Vascular Access
Exchange transfusions in the newborn
period are usually accomplished via catheters in the umbilical vessels. Catheterization is easiest within hours of birth, but it
may be possible to achieve vascular access at this site for several days. The catheters should be radio-opaque to facilitate
radiographic monitoring during and after
placement. If umbilical catheters are not
available for exchange transfusion, small
central venous or saphenous catheters
may be used.

Methods Used
Two methods of exchange transfusion are
in common use. In the isovolumetric
method, there is vascular access through
two catheters of identical size. Withdrawal
and infusion occur simultaneously, regulated by a single peristaltic pump. The
umbilical artery is usually used for with-

drawal and the umbilical vein for infusion.
The manual push-pull technique can be
accomplished through a single vascular access. A three-way stopcock joins the unit of
blood, the patient, and an extension tube
that leads to the graduated discard container. An inline blood warmer and a standard blood filter should be incorporated in
the administration set. The maximum volume of each withdrawal and infusion will
depend on the infant’s size and hemodynamic status. The rate at which exchange
transfusion occurs may alter the infant’s
hemodynamic status. It is important to
maintain careful records during an exchange transfusion. The procedure should
take place over 1 to 1.5 hours.

Transfusion of Other
Components
Although the percentage of VLBW and
ELBW infants being transfused has decreased significantly since the 1980s, between 61% and 94% of these neonatal patients can be expected to receive multiple
red cell transfusions. The smallest patients will receive the greatest number of
transfusions. It is estimated that a much
lower percentage of infants receive other
components.1,9,53,54

Platelet Transfusion
The normal platelet count of newborns is
similar to that of adults. A platelet count
less than 150,000/µL in a full-term or premature infant is abnormal. Approximately
20% of infants in neonatal intensive care
units have mild-to-moderate thrombocytopenia, which is the most common
hemostatic abnormality in the sick in55
fant. Neonatal thrombocytopenia may
result from impaired production or in-

Chapter 24: Neonatal and Pediatric Transfusion Practice

creased destruction of platelets, abnormal
distribution, or a dilutional effect secondary to massive transfusion such as exchange transfusion. Increased destruction
is the most common cause; it may be associated with a multitude of conditions and
is usually transient. Neonatal alloimmune
thrombocytopenia is discussed in Chapter 23.

Indications
Platelet transfusion is indicated in neonates and young infants with platelet
counts below 50,000/µL who are experi55
encing bleeding. The use of prophylactic
platelet transfusions in the newborn remains controversial. In thrombocytopenic adults, the risk of severe bleeding is
rare unless the platelet count is less than
56
10,000/µL. Conversely, preterm neonates
and infants with other complicating illnesses may bleed at higher platelet counts.
Factors contributing to this increased risk
of bleeding include a quantitatively lower
concentration of plasma coagulation factors; circulation of an anticoagulant that
enhances inhibition of thrombin; intrinsic or extrinsic platelet dysfunction; and
57
increased vascular fragility. Of major
concern is intraventricular hemorrhage,
which occurs in up to 40% of preterm neonates in the first 72 hours. Although prophylactic platelet transfusions increase
platelet counts and shorten the bleeding
time in these infants, the incidence or extent of intraventricular hemorrhage is not
57
reduced. Because of the apparent lack of
clinical benefit, controversy exists about
the use of platelet transfusion in this setting, as well as the selection of an optimal
dose. After a platelet transfusion, a posttransfusion platelet count soon after
transfusion can be used to evaluate survival in the circulation but may not pre-

569

dict hemostatic efficacy. Repeated platelet
transfusions, without an appropriate rise,
may not be beneficial.

Platelet Components
A platelet dose of 5 to 10 mL/kg body
weight should raise the platelet count of
an average full-term newborn by 50,000
to 100,000/µL, depending on the platelet
concentration in the component used.17,57
The platelet component should be group
specific, if possible, and should not contain clinically significant unexpected red
cell antibodies. Transfusion of ABO-incompatible plasma is more dangerous in
infants than in adults because of their
very small blood volumes. If it is necessary to give a platelet unit that contains
incompatible plasma (due to antibodies
in the ABO or other blood groups), plasma
can be removed (see Method 6.15) and
the platelets resuspended in saline. FFP
can be substituted as the resuspending
medium if the patient also requires clotting factors, but it carries the risk of infectious disease transmission. Routine centrifugation of platelets to reduce the
volume of transfusion is not necessary.53,55
If platelets have been volume reduced
and placed in a syringe, the pH declines
rapidly, a potential problem for an already
ill, acidotic patient.58 Therefore, if there is
a need to reduce the volume of platelets,
it should be done just before transfusion
and the component must be infused
within 4 hours, if done in an open system.

Granulocyte Transfusion
Neonates are more susceptible to severe
bacterial infection than older children because of both the quantitative and qualitative defects of neutrophil (polymorphonuclear cell or PMN) function and, in the

570

AABB Technical Manual

absence of pathogen-specific maternal
antibody, to deficiency of humoral immunity. Group B streptococcus is the most
frequent cause of early-onset neonatal
sepsis, and, despite improvement in
antimicrobial therapy and intensive care,
it is still associated with a high mortality
rate. Controversy surrounds several issues
in granulocyte transfusions for neonates,
including dose, neutrophil level at which
to transfuse, and efficacy as compared to
59
other forms of therapy. Because it appears that efficacy is related to dose, the
smaller blood volume of infants and
young children may result in these patients having a better response to this
form of therapy. A meta-analysis published in 1996 concluded that granulocyte
9
doses greater than 1 × 10 PMN/kg re60
sulted in a better clinical response.
Granulocyte concentrates prepared by
apheresis are more desirable than those
prepared from buffy coats because they
17,60
Products colproduce a higher yield.
lected from donors on a regimen of
granulocyte colony-stimulating factor
(G-CSF) and steroid mobilization can
yield higher numbers of granulocytes
than those collected from an unstimulated donor. There have been some encouraging observations on the use of IGIV in
the treatment of early neonatal sepsis, although conflicting data result in a lack of
59,61-64
Preliminary studconsensus on its use.
ies of hematopoietic growth factors (eg,
G-CSF) show promise in the treatment of
overwhelming bacterial infection in the
59,61,65,66
For adults and larger chilnewborn.
10
dren, a minimum dose of 1 × 10 PMN/kg
60,67,68
is recommended.

Indications
Although the precise role of granulocyte
transfusion for neonatal sepsis is unclear,
certain clinical situations exist in which

granulocyte transfusion may be considered as an adjunct to antibiotic therapy.
Candidates for possible granulocyte
transfusion are infants with strong evidence of bacterial septicemia, an absolute
neutrophil count below 3000/µL, and a
diminished marrow storage pool, such
that less than 7% of their nucleated cells
in the marrow are granulocytes at the
stage of metamyelocytes or more mature
forms.18,69

Granulocyte Components
Granulocytes are harvested by standard
apheresis techniques. For infants, a dose
of 10 to 15 mL/kg is recommended, which
is about 1 × 109 to 2 × 109 PMN/kg.17,60 Because neonatal neutrophil function is often abnormal, the use of granulocyte
transfusions in this age group may be
beneficial.17,60,67,70-72 Administration should
be continued daily until an adequate
white cell mass is achieved or the patient
has clinically improved. Based on the fact
that granulocyte concentrates contain
large numbers of lymphocytes, there is
general consensus that all granulocyte
transfusions should be irradiated to prevent graft-vs-host disease. For granulocyte transfusions to neonates, donors are
usually selected to be CMV seronegative
and must be ABO compatible with the infant, in accordance with Standards, because there is a significant volume of red
cells in these products.32(p40) Many institutions also provide products that are
D-compatible (see Chapter 21).

Transfusion to Enhance Hemostasis
The elements of the hemostatic system of
the newborn are similar to those of older
children and adults, but the concentration
of many plasma proteins is decreased.
Coagulation factors do not cross the placenta, but are independently synthesized

Chapter 24: Neonatal and Pediatric Transfusion Practice

by the fetus. Plasma levels of coagulation
proteins increase progressively with gestational age. At birth, the infant’s prothrombin time and partial thromboplastin time are prolonged, compared to
older children and adults, primarily the
result of physiologically low levels of the
vitamin-K-dependent factors (II, VII, IX,
and X) and contact factors (XI, XII, prekallikrein, and high-molecular-weight
kininogen).73 Proteins C and S and antithrombin inhibitors of coagulation are
also at low levels. These two systems usually balance each other, so that spontaneous bleeding and thrombosis in the healthy
newborn are rare, but very little reserve
capacity exists for response to pathologic
insults. Therefore, serious bleeding may
occur in the first week of life in the sick
premature infant as a result of hemostatic
immaturity coupled with an acquired disorder of hemostasis.
In addition to having physiologically low
levels of the vitamin-K-dependent factors,
neonates may also become vitamin-K-deficient during the first 2 to 5 days of life, placing them at risk of bleeding. This “hemorrhagic disease of the newborn” is rare in
developed countries because intramuscular
vitamin K is routinely given at birth. If vitamin K therapy is omitted, especially if the
neonate is breast fed, life-threatening hemorrhage may occur. This should be treated
with FFP.2,74
Although hereditary deficiencies of coagulation factors may be apparent in the
newborn, significant bleeding is rare.
Coagulopathy more often results from an
acquired defect such as liver disease or
DIC.74 Although component therapy replacement may temporarily correct the
hemostatic problem, treatment of the underlying disease will ultimately reduce the
need to treat the acquired defect.
Newborns who are heterozygous for deficiencies of inhibitory proteins rarely expe-

571

rience complications in the absence of another pathologic insult. However, the homozygous form of protein C deficiency has
caused life-threatening thrombotic complications in the newborn period. In countries
where it is available, protein C concentrates
prepared from human plasma should be
used as the initial treatment for neonates
with homozygous protein C deficiency presenting with purpura fulminans. Protein C
concentrates may be available on a compassionate use basis in the United States.
Otherwise, plasma infusion is used as the
initial treatment during the acute event,
with subsequent anticoagulant therapy for
long-term management.74,75

Fresh Frozen Plasma
Fresh frozen plasma may be used to replace coagulation factors in newborns,
particularly if multiple factors are involved, such as in vitamin K deficiency.
The usual dose is 10 to 15 mL/kg, which
should increase factor activity by 15% to
20% unless there is marked consumptive
coagulopathy.74 As with red cell transfusions, there are several methods to provide small-volume FFP infusions while
limiting donor exposure and wastage of
components. Blood can be collected into
a system with multiple integrally attached
bags, creating aliquots that can be prepared for freezing.34 Once thawed, these
aliquots can be further divided and used
for several patients within a 24-hour period. If not used within 24 hours as aliquots,
the thawed plasma (stored at 1 to 6 C) can
still be used as a means to decrease donor
exposure. As with all patients, newborns
must receive FFP that is ABO compatible
and free of clinically significant unexpected antibodies. Group AB FFP is often
used because a single unit provides compatible small aliquots for several neonates
requiring FFP simultaneously.

572

AABB Technical Manual

Cryoprecipitate
Cryoprecipitate is rich in fibrinogen, coagulation Factors VIII and XIII, and von
Willebrand factor. This component is often used in conjunction with platelet
transfusions to treat DIC in the newborn.
In DIC, fibrinogen and platelets are the elements most often severely depleted. The
plasma in which the platelets are suspended is a source of stable coagulation
factors. Cryoprecipitate provides concentrated levels of additional fibrinogen and
storage-labile Factor VIII. For an infant, one
bag is sufficient to achieve hemostatic
levels. As with FFP and platelets, the
cryoprecipitate should be ABO compatible with the neonatal recipient. Directed
donor cryoprecipitate is not recommended
as first-line therapy in the newborn with
hemophilia A because safer alternatives are
available. Recombinant Factor VIII products or virus-inactivated, monoclonal-antibody-purified, plasma-derived products
are the standard treatment.76 Cryoprecipitate should be used to treat von Willebrand disease only as a last resort, when
safer products are not readily available.
More information on the use of cryoprecipitate can be found in Chapter 21.

Neonatal Polycythemia
A venous hematocrit greater than 65% or
hemoglobin in excess of 22 g/dL any time
in the first week of life defines polycythemia,
a condition that occurs in approximately
5% of all newborns. Small-for-gestationalage infants and infants of diabetic mothers are at increased risk for developing
polycythemia. As the hematocrit rises
above 65%, the viscosity of blood increases exponentially and oxygen transport decreases. For neonates, the exponential rise may occur at a hematocrit
closer to 40%. 77 Infants have a limited

ability to increase cardiac output to compensate for hyperviscosity and may
develop congestive heart failure. Impairment of blood flow can cause CNS abnormalities, pulmonary and renal failure, and
necrotizing enterocolitis. Phlebotomy can
be used to normalize the hematocrit to
55% to 60% and improve tissue perfusion,
while maintaining the blood volume.
To treat polycythemia, whole blood is removed and the volume replaced with
crystalloid (such as normal saline), the
choice being based on the quantity needed
and on the infant’s clinical condition.
Plasma is not recommended because the
volume administered will be insufficient to
correct any coagulopathy and because
necrotizing enterocolitis has been reported
when it is used in this procedure.78 A formula to approximate the volume of colloid
replacement required (and the volume of
blood to be drawn) for the exchange is47:
Volume of replacement fluid =
blood volume ×
(observed hematocrit – desired hematocrit)
observed hematocrit

Extracorporeal Membrane
Oxygenation
ECMO is a modified cardiopulmonary bypass technique that has been used for
short-term support for cardiac or respiratory failure. It is performed in specialized
centers and only for patients in whom
conventional medical therapy has failed
and anticipated survival with such therapy is limited (see Table 24-2). The use of
ECMO in patients other than neonates is
not widespread. The therapy is more successful in infants, whose small blood volume allows for total cardiorespiratory
support and whose primary respiratory
problem often resolves after 1 to 2 weeks

Chapter 24: Neonatal and Pediatric Transfusion Practice

Table 24-2. Disorders Treated by ECMO
■
■
■
■

Meconium aspiration
Diaphragmatic hernia
Persistent pulmonary hypertension of the
newborn
Severe group B streptococcal sepsis

of support. ECMO provides gas exchange
independent of the patient’s lungs, allowing them time to improve or heal without
exposure to aggressive ventilator support
and the secondary lung damage this may
79
cause.
Individual ECMO centers establish their
own specific criteria for transfusion and
blood component selection, and standard
transfusion practices are lacking. Because
of the combination of factors present (including systemic heparinization, platelet
dysfunction, thrombocytopenia, and other
coagulation defects) as well as the ECMO
circuitry itself, bleeding complications are
frequent. The ECMO team should be in
close communication with the blood bank
or transfusion service staff, and there
should be mutual agreement on protocols
to ensure consistency of care. Many infants
requiring ECMO have been transferred
from other hospitals, where they may already have received numerous transfusions. The amount of red cell, platelet, and
FFP support required to maintain hematologic and hemodynamic equilibrium will
vary depending on the clinical situation
and in accordance with the institutions’
practices.68 When platelet transfusion is required, some practitioners think it is important to transfuse through peripheral access to avoid platelet damage, whereas
others will transfuse directly into the ECMO
circuitry because all blood will eventually
flow through the equipment. It is also important to monitor ionized calcium levels

47,79

and supplement them as needed.
Tables 24-3 and 24-4.)

573

(See

Leukocyte Reduction
The benefit of leukocyte reduction of
components transfused to infants remains controversial. Difficulty in identifying transfusion reactions in this patient
population makes this question hard to
study. In addition, infants are rarely alloimmunized because of the immaturity of
their immune system during this period
of development. The reduction of risk of
CMV transmission to infants is the only
benefit of leukocyte reduction that has
been well documented, as discussed earlier.16,30,80
Of interest, a recent study in Canada
compared the clinical outcomes of premature infants weighing <1250 g before and
after implementation of universal leukocyte
reduction. Although neither mortality nor
bacteremia were reduced in the setting of
universal leukocyte reduction, other secondary clinical outcomes, such as retinopathy of prematurity and bronchopulmonary dysplasia, were improved. Length

Table 24-3. Risks of ECMO
■
■
■
■
■
■
■
■
■
■
■
■

Bleeding
Thrombosis
Thrombocytopenia
Neutropenia
Platelet dysfunction
Stroke
Seizure
Air embolism
Hemolysis
Systemic hypertension
Cannulization of carotid artery
Infectious complications of blood
transfusion

574

AABB Technical Manual

Table 24-4. Contraindications for ECMO
■
■
■
■
■

High risk for intraventricular hemorrhage
Irreversible lung disease
Systemic bleeding
Severe asphyxia
Presence of lethal malformations

of stay was decreased with universal leuko81
cyte reduction.

required, many of the methods described
to provide small-volume transfusions to neonates could be applied. All pediatric patients over 4 months of age, however, must
be tested for ABO and D type as well as for
the presence of clinically significant antibodies before red cell transfusions. Compatibility testing must be done in accordance with
Standards.32(pp37-41) Of note, a published abstract reported that leukocyte reduction
does decrease the occurrence of febrile nonhemolytic transfusion reactions in pediatric
hematology/oncology patients.82

Transfusion Practices in
Older Infants and Children

Red Cell Support for Children with
Hemoglobinopathies

The indications for transfusion of red cells
and other components in older infants
(>4 months) and children are similar to
those for adults but must take into account differences in blood volume, ability
to tolerate blood loss, and age-appropriate hemoglobin and hematocrit levels.
The most common indication for red cell
transfusion in children is to reverse or
prevent tissue hypoxia resulting from decreased red cell mass associated with surgical procedures or in response to anemia
of chronic diseases or hematologic malignancies. It is important to remember that
normal hemoglobin and hematocrit levels
are lower in children than adults. Pediatric patients may remain asymptomatic
despite extremely low levels of hemoglobin, particularly if the anemia has developed slowly.
The decision to transfuse should be
based not only on the hemoglobin level but
also on the presence or absence of symptoms, the functional capacity of the child,
the etiology of the anemia, the possibility of
using alternative therapies, and the presence or absence of additional clinical conditions that increase the risk for developing
hypoxia. If small-volume transfusions are

In certain childhood conditions, chronic
red cell transfusions are given not only to
treat tissue hypoxia but also to suppress
endogenous hemoglobin production. Approximately 6% to 10% of children with
sickle cell disease suffer a stroke, with
two-thirds experiencing a recurrence.83
The goal of transfusion for these patients
is to reduce the risk of stroke by decreasing the percentage of circulating red cells
capable of sickling, while simultaneously
avoiding an increase in blood viscosity. It
is important to remember that raising the
hematocrit, without significantly reducing the percent of sickle cells, could increase viscosity and negate any beneficial
effects of the transfusion.83,84 The rate of
recurrent stroke can be reduced to less
than 10% by maintaining a hemoglobin
level of 8 to 9 g/dL, with a hemoglobin S
level less than 30%, in children who have
had a cerebrovascular accident. This can
usually be achieved with a simple or partial exchange transfusion every 3 to 4 weeks.
Therapy is continued indefinitely, as cessation can lead to subsequent stroke.83,84
Because of concern about iron overload,
some workers follow several uneventful
years of transfusions to keep hemoglobin

Chapter 24: Neonatal and Pediatric Transfusion Practice

S below 30% with a less aggressive protocol that maintains hemoglobin S between
40% and 50%.84 Erythrocytapheresis has
been shown to improve iron balance in a
85
small cohort of patients. Adams et al
showed that transfusion to maintain a hemoglobin S level less than 30% in children
who have abnormal results on transcranial Doppler ultrasound reduced the
86
risk of a first stroke. The benefits of this
transfusion therapy, however, must be
weighed against the complications of
transfusion, such as iron overload and
alloimmunization, as well as the risks of
increased donor exposure during erythrocytapheresis. Although simple transfusion
increases blood viscosity, exchange transfusion does not. Blood for transfusion to a
patient with sickle cell disease should ideally be screened for hemoglobin S. In addition, most centers now provide leukocyte-reduced blood for patients with sickle
cell disease to prevent alloimmunization
to platelets, which could complicate trans87
plantation.
Red cell transfusions are also used to
treat acute complications associated with
sickle cell disease, such as splenic sequestration and aplastic crisis.88 Acute chest syndrome, a new pulmonary infiltrate in a patient with sickle cell disease, other than
atelectasis, with additional respiratory
symptoms and fever, carries a poor prognosis if untreated. Simple transfusion can be
used as a first-line therapy to improve oxygenation. For those patients who continue
to deteriorate or who do not improve, red
cell exchange transfusion should be performed. There are no randomized controlled trials comparing exchange and simple transfusion in this setting.89 A study
evaluating preoperative transfusion protocols found that a conservative protocol, in
which the hemoglobin was raised to 10 g/
dL, was as effective in preventing perioperative complications as an aggressive ap-

575

proach to decrease hemoglobin S levels to
below 30%.90
Patients with sickle cell disease might
also be at risk for severe delayed hemolytic
transfusion reactions that could be lifethreatening because of the coincident suppression of erythropoiesis. When a patient’s
hemoglobin level decreases after transfusion, this “hyperhemolytic” syndrome—
wherein it appears that autologous red cells
are destroyed through an innocent bystander mechanism—should be suspected.
In these circumstances, transfusion should
be stopped and corticosteroid therapy, or a
combination of corticosteroid therapy
and IGIV, considered, based on reports of
efficacy in case studies.91,92 Autoantibody
formation also occurs in these patients after transfusion.93 In the hope of decreasing
the need for transfusion, medical interventions are being explored. One therapy uses
hydroxyurea to increase the percentage of
hemoglobin F. By having a higher percentage of cells that are hemoglobin F, the formation of hemoglobin S polymers is reduced. In addition, the concomitant decrease
in neutrophil counts that accompanies
hydroxyurea administration was found to
be independently associated with a reduction in the rate of crisis.94 Marrow transplantation has also been used in some patients and may have a role in the future
treatment of patients with sickle cell disease.95,96
For children with thalassemia and severe
anemia, transfusion not only improves tissue oxygenation but also suppresses
erythropoiesis. By suppressing ineffective
erythropoiesis, many of the complications
associated with the disease are ameliorated. So-called hypertransfusion, in which
the pretransfusion hemoglobin is kept between 8 and 9 g/dL, allows normal growth
and development, as well as normal levels
of activity for the child’s age. Supertransfusion programs aim to maintain a pre-

576

AABB Technical Manual

transfusion hemoglobin concentration between 11 and 12 g/dL, in order to decrease
iron absorption from the gastrointestinal
tract. The results of maintaining near-normal hemoglobin levels are still controversial. Iron overload is a complication of
treatment, requiring chelation therapy be97
ginning early in childhood.

Antibody Production in Sickle Cell and
Thalassemia Patients
The frequency of red cell alloimmunization in chronically transfused children
varies with the disease, the age of first
transfusion, the number of transfusions
given, and the ethnic background of donors and recipients, although patients
with sickle cell disease have the highest
rates of alloimmunization of any patient
group.98-100 Antibodies to the common antigens of the Rh, Kell, Duffy, and Kidd
systems are often identified. It may be
prudent, therefore, to phenotype the patient’s red cell antigens as completely as
possible before beginning transfusion
therapy and to maintain a permanent record of the results. This can be helpful in
selecting compatible blood if alloimmunization occurs. There is evidence that
transfusing K, C, and E antigen-negative
red cells can significantly reduce the rate
of alloimmunization. 1 0 1 However, the
practice of transfusing only phenotypically matched units is controversial, especially in patients who have not yet developed the corresponding antibody, because
many of these units are difficult to ob83,102
A recent survey of 50 academic
tain.
medical centers in the United States and
Canada found that the most common
practice is to perform pretransfusion
phenotypic matching for C, E, and K.103 In
patients who have already become immunized and who are at high risk of developing additional antibodies, use of pheno-

typically matched units may be a reasonable approach to prevent further alloimmunization.102 Method 2.16 can be used
to perform red cell phenotyping on autologous red cells of recently transfused patients.
African Americans with sickle cell disease frequently become immunized because the majority of red cell transfusions
are obtained from Caucasian donors, with
major differences in antigen exposure. In
some areas, blood collectors have developed programs to specifically recruit African American donors to supply blood for
patients with sickle cell disease, in order to
101
reduce rates of alloimmunization. Leukocyte-reduced blood components may be of
particular value for these chronically transfused patients. One benefit would be to diminish the development of alloimmunization to HLA antigens, in light of the prospect
of future marrow transplantation.87 There
are conflicting data whether leukocyte reduction can prevent alloimmunization to
red cell transfusion.104,105 Preventing febrile
transfusion reactions is also important for
patients who may be receiving a unit of
phenotypically matched blood. Discarding
such a unit would be both wasteful and
may have an impact on the ability to adequately transfuse the patient.106

Platelets and Plasma
The indications for FFP and platelet
transfusions in older infants and children
parallel those for adults. Platelet transfusions are most often given as prophylaxis
to children receiving chemotherapy. Prophylactic platelet transfusions are seldom
given when platelet counts are above
10,000 to 20,000/µL, but, as with red cell
transfusion and hemoglobin, the indication for platelet transfusion should not be
based solely on the platelet count. When
additive risk factors such as fever, sepsis,

Chapter 24: Neonatal and Pediatric Transfusion Practice

DIC, or clotting abnormalities are present,
the platelet count may need to be higher
to prevent spontaneous hemorrhage. In the
absence of such factors, a much lower level
may be safe.56 In recent studies, the use of
ABO-compatible platelets has been associated with better clinical outcomes.107-109

References
1. Hume H, Bard H. Small volume red blood cell
transfusions for neonatal patients. Transfus
Med Rev 1995;9:187-99.
2. Roseff SD, ed. Pediatric transfusion: A physician’s handbook. 1st ed. Bethesda, MD:
AABB, 2003.
3. Brugnara C, Platt OS. The neonatal erythrocyte and its disorders. In: Nathan DG, Orkin
SH, eds. Nathan and Oski’s hematology of infancy and childhood. 5th ed. Philadelphia:
WB Saunders, 1998:19-52.
4. Blanchette V, Doyle J, Schmidt B, Zipursky A.
Hematology. In: Avery GB, Fletcher MA, MacDonald MG, eds. Neonatology: Pathophysiology and management of the newborn.
4th ed. Philadelphia: JB Lippincott, 1994:
952-99.
5. Wallgren G, Hanson JS, Lind J. Quantitative
studies of the human neonatal circulation.
Acta Paediatr Scand 1967;179(Suppl):43-54.
6. Ohls RK. Evaluation and treatment of anemia in the neonate. In: Christensen RD, ed.
Hematologic problems of the neonate. Philadelphia: WB Saunders, 2000:137-69.
7. Ohls RK. Erythropoietin to prevent and treat
the anemia of prematurity. Curr Opin Pediatr
1999;11:108-14.
8. Maier RE, Obladen M, Müller-Hansen I, et al.
Early treatment with erythropoietin β ameliorates anemia and reduces transfusion requirements in infants with birth weights below 1000 g. J Pediatr 2002;141:8-15.
9. Widness JA, Seward VJ, Kromer IJ, et al.
Changing patterns of red cell transfusion in
very low birth weight infants. J Pediatr 1996;
129:680-7.
10. Vamvakas EC, Strauss RG. Meta-analysis of
controlled clinical trials studying the efficacy of rHuEPO in reducing blood transfusions in the anemia of prematurity. Transfusion 2001;41:406-15.
11. Luban NLC, Mikesell G, Sacher RA. Techniques for warming red blood cells packaged
in different containers for neonatal use. Clin
Pediatr 1985;24:642-5.

577

12. Simister NE. Placental transport of immunoglobulin G. Vaccine 2003;21:3365-9.
13. DePalma L. Red cell alloantibody formation
in the neonate and infant: Considerations
for current immunohematologic practice.
Immunohematology 1992;8:33-7.
14. Sanders MR, Graeber JE. Posttransfusion graftversus-host disease in infancy. J Pediatr 1990;
117:159-63.
15. Ohto H, Anderson KC. Posttransfusion graftversus-host disease in Japanese newborns.
Transfusion 1996;36:117-23.
16. Stauss RG. Data-driven blood banking practices for neonatal RBC transfusions. Transfusion 2000;40:1528-40.
17. Strauss RG. Transfusion therapy in neonates.
Am J Dis Child 1991;145:904-11.
18. Strauss RG. Neonatal transfusion. In: Anderson KC, Ness PM, eds. Scientific basis of
transfusion medicine. Implications for clinic a l p ra c t i c e. 2nd ed . Ph i l a d e l p h i a : WB
Saunders, 2000:321-6.
19. Strauss RG. Routinely washing irradiated red
cells before transfusion seems unwarranted.
Transfusion 1990;30:675-7.
20. McDonald TB, Berkowitz RA. Massive transfusion in children. In: Jeffries LC, Brecher
ME, eds. Massive transfusion. Bethesda, MD:
AABB, 1994:97-119.
21. Harris B, Lumadue J, Luban NLC, Pollack M.
Transfusion-related hyperkalemic arrest
from irradiated packed red blood cells (abstract). Transfusion 1998;38(Suppl):69S.
22. Hall TL, Barnes A, Miller JR, et al. Neonatal
mortality following transfusion of red cells
with high plasma potassium levels. Transfusion 1993;33:606-9.
23. Pisciotto PT. Cytomegalovirus safety issues
in neonatal transfusion. In Hermann JH,
Manno CS, eds. Pediatric transfusion therapy. Bethesda, MD: AABB Press, 2002:171-92.
24. Delage G. Transfusion-transmitted infections in the newborn. Transfus Med Rev
1995;9:271-6.
25. Hamprecht K, Maschmann J, Vochem M, et
al. Epidemiology of transmission of cytomegalovirus from mother to preterm infant by
breastfeeding. Lancet 2001;357:513-8.
26. Adler SP, Chandrika T, Lawrence L, Baggett J.
Cytomegalovirus infections in neonates acquired by blood transfusions. Pediatr Inf Dis
1983;2:114-8.
27. Brady MT, Milam JD, Anderson DC. Use of
deglycerolized red blood cells to prevent posttransfusion infection with cytomegalovirus
in neonates. J Infect Dis 1984;150:334-9.
28. Strauss RG. Selection of white cell-reduced
blood components for transfusions during
infancy. Transfusion 1993;33:352-7.

578

AABB Technical Manual

29. Bowden RA, Slichter SJ, Sayers M, et al. A
comparison of filtered leukocyte-reduced
and cytomegalovirus (CMV ) seronegative
blood products for the prevention of transfusion-associated CMV infection after marrow
transplant. Blood 1995;86:3598-603.
30. Gilbert GL, Hayes K, Hudson H, et al. Prevention of transfusion-associated cytomegalovirus infection in infants by blood filtration
to remove leukocytes. Lancet 1989;i:1228-31.
31. Ludvigsen C, Swanson JL, Thompson TR,
McCullough J. The failure of neonates to
form red cell alloantibodies in response to
multiple transfusions. Am J Clin Pathol 1987;
87:250-1.
32. Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.
33. Pridjian G. Fetomaternal interactions: Placental physiology and its role as a go-between. In: Avery GB, Fletcher MA, MacDonald
MG, eds. Neonatology: Pathophysiology and
management of the newborn. 5th ed. Philadelphia: JB Lippincott, 1999:125-41.
34. Roseff SD. Pediatric blood collection and
transfusion technology. In: Her man J K,
Manno CS, eds. Pediatric transfusion therapy.
Bethesda, MD: AABB Press, 2002:217-47.
35. Levy GJ, Strauss RG, Hume H, et al. National
survey of neonatal transfusion practices, I:
Red blood cell therapy. Pediatrics 1993;91:
523-9.
36. Cook S, Gunter J, Wissel M. Effective use of a
strategy using assigned red cell units to limit
donor exposure for neonatal patients. Transfusion 1993;33:379-83.
37. Wang-Rodriguez J, Mannino FL, Liu D, Lane
TA. A novel strategy to limit blood donor exposure and blood waste in multiply transfused
premature infants. Transfusion 1996;36:6470.
38. Strauss RG, Burmeister LF, Johnson K, et al.
Feasibility and safety of AS-3 red blood cells
for neonatal transfusions. J Pediatr 2000;136:
215-9.
39. Goodstein MH, Herman JH, Smith JF, et al.
Metabolic consequences in very low birth
weight infants transfused with older AS-1
preserved erythrocytes. Pediatr Pathol Lab
Med 1999;18:173-85.
40. Rock G, Poon A, Haddad R, et al. Nutricel as
an additive solution for neonatal transfusion.
Transfus Sci 1999;20:29-36.
41. Luban NLC, Strauss RG, Hume HA. Commentary on the safety of red cells preserved in extended-storage media for neonatal transfusions. Transfusion 1991;31:229-35.
42. Tuchschmid P, Mieth D, Burger R, et al. Potential hazard of hypoalbuminemia in new-

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.
57.

58.

born babies after exchange transfusions with
Adsol red blood cell concentrates (letter). Pediatrics 1990;85:234-5.
Brecher ME, ed. Collected questions and answers. 6th ed. Bethesda, MD: AABB, 2000:
73-5.
Burch KJ, Phelps SJ, Constance TD. Effect of
an infusion device on the integrity of whole
blood and packed red blood cells. Am J Hosp
Pharm 1991;48:92-7.
Criss VR, DePalma L, Luban NLC. Analysis of
a linear peristaltic infusion device for the
transfusion of red cells to pediatric patients.
Transfusion 1993;33:842-4.
Longhurst DM, Gooch W, Castillo RA. In vitro
evaluation of a pediatric microaggregate
blood filter. Transfusion 1983;23:170-2.
Behrman RE, Kleigman RM, Jenson HB, eds.
Nelson’s textbook of pediatrics. 16th ed. Philadelphia: WB Saunders, 2000.
Koenig JM. Evaluation and treatment of
erythroblastosis fetalis in the neonate. In:
Christensen RD, ed. Hematologic problems
of the neonate. Philadelphia: WB Saunders,
2000:185-207.
American Academy of Pediatrics Subcommittee on Hyperbilirubinemia. Management of
hyperbilirubinemia in the newborn infant 35
or more weeks of gestation. Clinical Practice
Guideline. Pediatrics 2004;114:297-316.
Ballard RA, Vincour B, Reynolds JW, et al.
Transient hyperammonemia of the preterm
infant. N Engl J Med 1978;299:920-5.
Leonard JV. The early detection and management of inborn errors presenting acutely in
the neonatal period. Eur J Pediatr 1985;143:
253-7.
Chan G, Schoff D. Variance in albumin loading in exchange transfusions. J Pediatr 1976;
88:609-13.
Strauss RG, Levy GJ, Sotelo-Avila C, et al. National survey of neonatal transfusion practices: II. Blood component therapy. Pediatrics 1993;91:530-6.
Maier RF, Sonntag J, Walka MW, et al. Changing practices of red blood cell transfusions in
infants with birth weights less than 1000 g. J
Pediatr 2000;136:220-4.
Blanchette VS, Kuhne T, Hume H, Hellman J.
Platelet transfusion therapy in newborn infants. Transfus Med Rev 1995;9:215-30.
Beutler E. Platelet transfusions: The 20,000/
µL trigger. Blood 1993;81:1411-13.
Andrew M, Vegh P, Caco C, et al. A randomized, controlled trial of platelet transfusions
in thrombocytopenic premature infants. J
Pediatr 1993;123:285-91.
Pisciotto P, Snyder EL, Snyder JA, et al. In vitro characteristics of leukocyte-reduced sin-

Chapter 24: Neonatal and Pediatric Transfusion Practice

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

gle unit platelet concentrates stored in syringes. Transfusion 1994;34:407-11.
Sweetman RW, Cairo MS. Blood component
and immunotherapy in neonatal sepsis.
Transfus Med Rev 1995;9:251-8.
Vamvakas EC, Pineda AA. Meta-analysis of
clinical studies of the efficacy of granulocyte
transfusions in the treatment of bacterial
sepsis. J Clin Apheresis 1996;11:1-9.
Rosenthal J, Cairo MS. Neonatal myelopoiesis and immunomodulation of host defenses. In: Petz LD, Swisher SN, Kleinman S,
et al, eds. Clinical practice of transfusion
m e d i c i n e. 3 rd e d . Ne w Yo rk : C h u rc h i l l
Livingstone, 1996:685-703.
Jenson HB, Pollock BH. The role of intravenous immunoglobulins for the prevention
and treatment of neonatal sepsis. Semin
Perinatol 1998;22:50-63.
Sandberg K, Fasth A, Berger A, et al. Preterm
infants with low immunoglobulin G levels
have increased risk of neonatal sepsis but do
not benefit from prophylactic immunoglobulin G. J Pediatr 2000;137:623-8.
Hill HR. Additional confirmation of the lack
of effect of intravenous immunoglobulin in
the prevention of neonatal infection (editorial). J Pediatr 2000;137:595-7.
Schibler KR, Osborne KA, Leung LY, et al. A
randomized, placebo-controlled trial of
granulocyte colony-stimulating factor administration to newborn infants with
neutropenia and clinical signs of early-onset
sepsis. Pediatrics 1998;102:6-13.
Calhoun DA, Lunoe M, Du Y, et al. Granulocyte colony-stimulating factor serum and
urine concentrations in neutropenic neonates before and after intravenous administration of recombinant granulocyte colonystimulating factor. Pediatrics 2000;105:392-7.
Price TH. The current prospects for neutrophil transfusion for the treatment of granulocytopenic infected patients. Transfus Med
Rev 2000;14:2-11.
Roseff SD, Luban NLC, Manno CS. Guidelines for assessing appropriateness of pediatric transfusion. Transfusion 2002;42:1398413.
Christensen RD, Bradley PP, Rothstein G. The
leukocyte left shift in clinical and experimental neonatal sepsis. J Pediatr 1981;98:
101-5.
Cairo MS, Rucker R, Bennetts GA, et al. Improved survival of newborns receiving leukocyte transfusions for sepsis. Pediatrics 1984;
74:887-92.
Cairo MS, Worcester CC, Rucker RW, et al.
Randomized trial of granulocyte transfusions versus intravenous immune globulin

72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

579

therapy for neutropenia and sepsis. J Pediatr
1992;120:281-5.
Hübel K, Dale DC, Liles WC. Granulocyte
transfusion therapy: Update on potential
clinical applications. Curr Opin Hematol
2001;8:161-4.
Andrew M, Paes B, Johnston M. Development of the hemostatic system in the neonate and young infant. Am J Pediatr Hematol
Oncol 1990;12:95-104.
Andrew M. Transfusion in the newborn:
Plasma products. In: Kennedy M, Wilson S,
Kelton J, eds. Perinatal transfusion medicine.
Arlington, VA: AABB, 1990:145-77.
Monagle P, Michelson AD, Bovill E, Andrew
M. Antithrombotic therapy in children.
Chest 2001;119(Suppl):344-70S.
Pressey JG, Manno CS. Therapy for hemop h i l i a a n d vo n Wi l l e b ra n d d i s e a s e. In:
Herman JK, Manno CS, eds. Pediatric transfusion therapy. Bethesda, MD: AABB Press,
2002:355-82.
Lindemann R, Haga P. Evaluation and treatment of polycythemia in the neonate. In:
Christensen RD, ed. Hematologic problems
of the neonate. Philadephia: WB Saunders,
2000:171-83.
Black VD, Ru mack CM, Lubchenco LD,
Koops BL. Gastrointestinal injury in polycythemic term infants. Pediatrics 1985;76:
225-31.
Kevy SV. Extracorporeal therapy for infants
and children. In: Petz L D, Swisher SN,
Kleinman S, et al, eds. Clinical practice of
transfusion medicine. 3rd ed. New York:
Churchill Livingstone, 1996:733-55.
Strauss RG. Selection of white cell-reduced
blood components for transfusions during
early infancy. Transfusion 1993;33:352-7.
Fergusson D, Hébert PC, Lee SK, et al. Clinical outcomes following institution of universal leukoreduction of blood transfusions in
premature infants. JAMA 2003;289:1950-6.
Young G, Jubran RF, Luban NLC. Febrile
transfusion reactions in pediatric hematology/oncology patients: The effect of leukodepletion (abstract). Blood 1999;94(Suppl 1):
3371a.
Sharon BI, Honig GR. Management of congenital hemolytic anemias. In: Simon TL,
Dzik WH, Snyder ES, et al, eds. Rossi’s principles of transfusion medicine. 3rd ed. Philadelphia: Lippincott Williams and Wilkins,
2002:463-82.
Cohen AR, Norr is CF, Smith-Whitley K.
Transfusion therapy for sickle cell disease.
In: Capon SM, Chambers LA, eds. New directions in pediatric hematology. Bethesda,
MD: AABB, 1996:39-88.

580

AABB Technical Manual

85. Adams DM, Schultz WH, Ware RF, Kinney TR.
Erythrocytapheresis can reduce iron overload and prevent the need for chelation therapy in chronically transfused pediatric patients. J Pediatr Hematol Oncol 1996;18:3-7.
86. Adams RJ, McKie VC, Hsu L, et al. Prevention
of a first stroke by transfusions in children
with sickle cell anemia and abnormal results
on transcranial Doppler ultrasonography. N
Engl J Med 1998;339:5-11.
87. Friedman DF, Lukas MB, Jawad A, et al. Alloimmunization to platelets in heavily transfused patients with sickle cell disease. Blood
1996;88:3216-22.
88. National Heart Lung and Blood Institute. The
management of sickle cell disease. 4th ed.
No. 02-2117. Bethesda, MD: National Institutes of Health, 2002.
89. Platt OS. The acute chest syndrome of sickle
cell disease. N Engl J Med 2000;342:1904-7.
90. Vichinsky EP, Heberkern CM, Neumayr L, et
al. A comparison of conservative and aggressive transfusion regimens in the perioperative management of sickle cell disease. N
Engl J Med 1995;333:206-13.
91. Petz LD, Calhoun L, Shulman IA, et al. The
sickle cell hemolytic transfusion reaction
syndrome. Transfusion 1997;37:382-92.
92. Win N, Doughty H, Telfer P, et al. Hyperhemolytic transfusion reaction in sickle cell
disease. Transfusion 2001;41:323-8.
93. Garratty G. Autoantibodies induced by blood
transfusion (editorial). Transfusion 2004;44:
5-9.
94. Charash S, Barton FB, Moore RD, et al. Hydroxyurea and sickle cell anemia. Clinical utility of
a myelosuppressive “switching” agent. The
Multicenter Study of Hydroxyurea in Sickle
Cell Anemia. Medicine (Baltimore) 1996;75:
300-26.
95. Steinberg MH. Management of sickle cell disease. N Engl J Med 1999;340:1021-30.
96. Woodard P, Jeng M, Handgretinger R, et al.
Summary of symposium: The future of stem
cell transplantation for sickle cell disease. J
Pediatr Hematol Oncol 2002;24:512-4.

97. Hoffbrand AV, Al-Refaie F, Davis B, et al.
Long-term trial of deferiprone in 51 transfusion-dependent iron overload patients. Blood
1998;91:295-300.
98. Rosse WF, Gallagher D, Kinney TR, et al.
Transfusion and alloimmunization in sickle
cell disease. Blood 1990;76:1431-7.
99. Spanos T, Karageorge M, Ladis V, et al. Red
cell alloantibodies in patients with thalassemia. Vox Sang 1990;58:50-5.
100. Rosse WF, Telen M, Ware RE. Transfusion
support for patients with sickle cell disease.
Bethesda, MD: AABB Press, 1998.
101. Smith-Whitley K. Alloimmunization in patients with sickle cell disease. In: Herman JK,
Manno CS, eds. Pediatric transfusion therapy.
Bethesda, MD: AABB Press, 2002:249-82.
102. Tahhan HR, Holbrook CT, Braddy LR, et al.
Antigen-matched donor blood in the transfusion management of patients with sickle cell
disease. Transfusion 1994;34:562-9.
103. Afenyi-Annan A, Brecher ME. Pre-transfusion
phenotype matching for sickle cell disease
patients (letter). Transfusion 2004;44:619-20.
104. Blumberg N, Heal JM, Gettings KF. Leukoreduction of red cell transfusions is associated with a decreased incidence of red cell
alloimmunization. Transfusion 2003;43:94552.
105. Van de Watering L, Jermans J, Witvliet M, et
al. HLA and RBC immunization after filtered
and buffy coat-depleted blood transfusion in
cardiac surgery: A randomized controlled
trial. Transfusion 2003;43:765-71.
106. Lane TA, Anderson KC, Goodnough LT, et al.
Leukocyte reduction in blood component
therapy. Ann Intern Med 1992;117:151-62.
107. Larsson LG, Welsh VJ, Ladd DJ. Acute intravascular hemolysis to out-of-group platelet
transfusion. Transfusion 2000;40:902-6.
108. Heal JM, Blumberg N. The second century of
ABO: And now for something completely different. Transfusion 1999;39:1155-9.
109. Blumberg N, Heal JM, Hicks GL, Risher WH.
Association of ABO-mismatched platelet
transfusions with morbidity and mortality in
cardiac surgery. Transfusion 2001;41:790-3.

Chapter 25: Cell Therapy and Cellular Product Transplantation

Chapter 25

Cell Therapy and Cellular
Product Transplantation

HERAPEUTIC CELLS INCLUDE cell
populations collected and processed to provide a special therapeutic effect. The classic example of cell
therapy is the use of pluripotent stem
cells, which are capable of self-renewal
and differentiation into all blood-cell lineages. These stem cells—transplanted in
preparations of marrow, stimulated peripheral blood mononuclear preparations,
or cord blood—also give rise to other regenerative tissues such as hepatocytes,
endothelial cells, and other tissue under
the proper microenvironmental circum1
stances. Other cell populations may serve
therapeutic purposes such as immune
modulation in posttransplant donor lymphocyte infusion (DLI). The hematopoietic progenitor cells (HPCs), which are
committed to a blood-cell lineage, were
first studied in the laboratory for their
power to give rise to complete, sustained
hematopoietic engraftment when given in

sufficient numbers. This chapter primarily
discusses the hematopoietic repopulation
cells. Cell preparations for HPC transplantation are thought to contain both hematopoietic stem cells (HSCs) capable of
self-renewal and HPCs committed to a
blood-cell lineage. However, committed
progenitor cells lack capacity for sustained self-renewal or the ability to differentiate into other blood-cell lineages. The
committed cells are important for the
speed of engraftment.2 Current measurement methods cannot easily separate the
earlier and more committed cell populations. Both cell populations are referred to
as HPCs in this chapter. HPCs collected
from peripheral blood via apheresis are
referred to as HPC-A. Those collected by
harvesting marrow are termed HPC-M and
those from cord blood are called HPC-C.
HPC transplantation has advanced from
a research procedure performed in a few
centers to a common medical procedure
581

582

AABB Technical Manual

performed in many tertiary care centers.
HPC transplantation can be classified according to the source of the HPCs used for
engraftment as autologous, allogeneic,
syngeneic, and xenogeneic.
Autologous HPC transplantation is technically not a transplant but, rather, the “rescue” of a patient with the patient’s own
HPCs, which are removed and stored frozen
or in nonfrozen conditions for later infusion
as protection from the lethal effects of therapeutic or ablative irradiation or chemotherapy. The autologous graft may be manipulated to retain HPCs and leave behind
or exclude tumor cells or damaging immune cells. Autologous HPCs are reinfused
to repopulate the patient’s marrow after an
otherwise lethal or near-lethal dose of radiation or chemotherapy given to treat malignancies of the marrow and metastatic or recurrent solid tumors. In the special case of
an identical twin donor and recipient, such
transplants are referred to as syngeneic.
Allogeneic HPC transplantation involves
the infusion of HPCs from another human
(an HLA-matched related or unrelated
donor) in order to establish donor cell
chimerism to rescue the patient from doseintense therapy and/or as an active immunotherapy against a disease after a potentially
lethal dose of radiation or chemotherapy.
Such transplants are preferred in patients
who have acute myelogenous leukemia
(AML), acute lymphocytic leukemia (ALL),
severe immunodeficiency, aplastic anemia,
marrow involvement with their malignancy,
or those who are incapable of supplying
their own autologous “normal” HPCs, as
with hemoglobinopathies (thalassemias or
sickle cell disease). As new indications for
transplantation are developed, some others
are drastically changed by new discoveries.
For instance, patients with chronic myelogenous leukemia (CML) used to receive the
largest number of adult hematopoietic
transplants; however, imatinib mesylate

has been such a successful treatment for
this disease that the number of CML transplants has dropped sharply since licensure
of this drug.3
Another advance in marrow transplantation has been the recognition and exploitation of the effects of transplanted allogeneic
immune cells on the malignancy of the patient. Because the transplanted cells are the
treatment modality for the patient, the
preparation for transplantation needs only
to modify the patient’s immune system to
allow the new cells to engraft. This nonmyeloablative transplantation expands the
number of patients eligible for transplant
by accepting patients of more advanced age
and with more health problems. These patients would not be candidates to enter
more toxic myeloablative regimens because
the risk of the treatment would be so great.
In this setting, chemotherapy and/or lowdose total body irradiation targets the recipient’s T lymphocytes to allow tolerance
for the healthy, allogeneic graft.
In successful marrow transplants, the
cells gradually produce full chimerism in
the patient’s marrow and attack and eliminate the tumor cells immunologically.4-6 In
patients where healthy cell replacement is
the intended result of treatment, such as in
children with immune deficiencies, this
method allows replacement cells to engraft
without putting the child through the danger of full myeloablation. The patient does
not have to undergo extensive periods of
cytopenia with exposure to infection and
bleeding risk. The engrafted cells become
the treatment agent, allowing the chemotherapy and/or radiation to be low-dose
and relatively low toxicity. This approach
uses the immune reconstitution as the tool
to control disease, but it is not without risk
because the immune cells can also attack
the healthy tissues of the patient, causing
graft-vs-host disease (GVHD).7 The number
of nonmyeloablative transplants has in-

Chapter 25: Cell Therapy and Cellular Product Transplantation

creased from 20 in 1997 to 870 in 2001, according to the Center for International
Blood and Marrow Transplant Research
(CIBMTR) reporting system.3
Xenogeneic HPC transplantation would
involve HPC transplants derived from a
nonhuman species. However, because of
currently insurmountable immunologic
barriers and disease concerns, these transplants are not now clinically feasible.
Sources of progenitor cells include marrow, peripheral blood, umbilical cord
blood, and fetal liver (although this source
is experimental and not in routine clinical
use).1 Once collected, the HPCs may be
subjected to ex-vivo processing, eg, removal of incompatible red cells or plasma
and/or cell selection (purging) before the
transplantation procedure. In some cases,
certain cell populations are “positively” selected (selectively isolated) for their special
therapeutic effects. T lymphocytes may be
isolated and later infused in measured
doses for antitumor effects, thus minimizing GVHD. Separation of cell populations
can be accomplished using a cell separation device approved by the Food and Drug
Administration (FDA), such as the Isolex
system (Baxter Healthcare, Deerfield, IL).
Alternatively, some cell populations may be
“negatively selected” (culled out or destroyed), as by antibody-mediated lysis of
malignant cells or by flow cytometry separation of cell populations in clinical trial situations.

583

lignant diseases have also been treated with
life-saving transplantation, as a group,
they represent less than 15% of all trans3,9
plants. The success rate of HPC transplantation depends on the condition and age
of the patient, type and stage of the disease being treated, the cell dose, and degree of HLA matching between the donor
and the patient. Overall, long-term survival rates are generally 30% to 60% for
otherwise fatal diseases. Table 25-1 describes general outcomes.10

Sources of Hematopoietic
Progenitor Cells
Historically, marrow was the primary source
of hematopoietic cells for transplantation.
However, HPC-A transplants constituted
approximately 90% of adult autologous
and about half of allogeneic transplantation procedures in the 1998-2000 International Bone Marrow Transplant Registry
report. 3 Data on umbilical cord blood
transplants have shown promising results
in pediatric patients for whom a matched
unrelated allogeneic HPC-M or HPC-A donor is unavailable. Clinical studies are in
progress to determine 1) the safety and efficacy of cord blood transplantation, 2)
whether adults can be successfully transplanted, and 3) the extent of HLA mismatch that can safely and effectively provide durable engraftment.

HPCs from Marrow (Autologous)

Diseases Treated with
Hematopoietic Cell
Transplantation
Many diseases have been treated with
8
HPC transplantation. Malignant diseases
are the most frequent indications for HPC
transplantation. Although many nonma-

Currently, only about 5% of autologous
transplants are performed with HPC-M.
This source has been largely replaced by
mobilized autologous HPC-A.

HPCs from Marrow (Allogeneic)
Allogeneic transplants address the problem of the inability to obtain a tumor-free

584

AABB Technical Manual

Table 25-1. Examples of Diseases Responsive to Hematopoietic Stem Cell
10
Transplantation
Disease

Type of Transplant

Timing

Clinical Results

AML

Allogeneic

First CR

OS 40%-50%

ALL (children)

Allogeneic

Second CR

OS 40%-65%

ALL (high risk)

Allogeneic

First CR

OS 50%

Chronic phase CML

Allogeneic

Chronic phase (CP)

OS 50%-80%

Accelerated phase CML

Allogeneic

Individualized

OS 30%-40%

Blast phase CML

Allogeneic

Second CP

OS 15%-25%

Myelodysplastic
syndrome

Allogeneic

Age <60

OS 40%

Aplastic anemia

Allogeneic

Individualized

OS 70%-90%

CLL

Allogeneic or
autologous

Participation in
clinical trial

Small series of patients
with durable CR;
nonablative transplants
under investigation

Intermediate-grade NHL

Autologous

Chemosensitive
relapse

OS 40%-50%

High-risk NHL

Autologous

First CR

OS 50%-60%

Low-grade NHL

Allogeneic or autologous

Chemosensitive
relapse

DFS 25%-50% at 5 years

Mantle cell lymphoma

Allogeneic or autologous
clinical trial

First CR

Small series with durable
CR rates of 25%-50%

Lymphoblastic lymphoma

Allogeneic or autologous
clinical trial

Chemosensitive
Small series with durable
relapse or first CR
CR

NHL or Hodgkin's disease

Allogeneic clinical trial

Advanced refractory
disease

DFS 15%-25%

Multiple myeloma

Autologous

Chemosensitive relapse or first CR

OS 50% at 5 years; DFS
20%

High-risk breast,
testicular, or ovarian
cancer

Autologous clinical trial

Chemosensitive
disease

Improved survival over
historical controls not
confirmed in
randomized trials

Renal cell carcinoma

Nonablative allogeneic

Clinical trial

Small series with durable
CR

Thalassemia

Allogeneic

Clinical trial

OS 75% for patients
without cirrhosis

Sickle cell anemia

Allogeneic

Clinical trial

OS 75%

Autoimmune disorders

Allogeneic

Clinical trial

Small series of remissions

CR = complete response; DFS = disease-free survival; OS = overall survival; AML = acute myelogenous leukemia; CML =
chronic myelogenous leukemia; ALL = acute lymphocytic leukemia; CP = chronic phase; NHL = non-Hodgkin's lymphoma.
Netter examples used with permission from Icon Learning Systems, a division of MediMedia USA, Inc. All rights reserved.

Chapter 25: Cell Therapy and Cellular Product Transplantation

graft with an autologous transplant for a
patient with malignant disease and provide possible immune help after transplantation from graft-vs-tumor response.
For other patients, such as those with
marrow failure, immunodeficiency, inborn errors of metabolism, or hemoglobinopathy, an allogeneic transplant is the
only appropriate type of graft. Sources of
allogeneic HPC-M may be from matched
or partially matched related or unrelated
donors.

Matched, Unrelated Donor Transplantation
Matched, unrelated donor searches can
be initiated for the 60% to 70% of candidates without an HLA-identical related
(usually sibling) donor. Several marrow
donor databases are available worldwide.
The largest is the National Marrow Donor
Program (NMDP) database. Since its
founding in 1986, the NMDP has facilitated approximately 12,000 transplant
procedures with a total of over 2000 transplants per year. A directory of transplant
centers, outcome results, and charges is
available from the NMDP. (See Appendix
11 at the end of the book).
Upon initial search, 80% of transplantation candidates usually find an HLA phenotypic match. However, patients from racial
or ethnic minorities have a lower chance of
success in such a donor search (Caucasian,
81%; Hispanic, 64%; Asian/Pacific Islander,
55%; African American, 47%; and American
Indian/Alaska Native, 50%; according to the
NMDP). The median time from initiating a
search to receiving a transplant has been
120 days, but a new expedited search and
donor preparation program is being implemented to shorten this time.
The NMDP is conducting a clinical trial
of HPC-A collections from unrelated, HLAmatched volunteers stimulated with granulocyte colony-stimulating factor (G-CSF or

585

filgrastim). The donors’ physical symptoms as well as their attitudes and feelings
about the process are being monitored for
the trial. The use of HPC-A offers the potential advantage of improved engraftment kinetics and enhanced graft-vs-leukemia
(GVL) effect. In allogeneic, related transplant settings clinical trials comparing
HPC-M to HPC-A, the latter showed improved survival. Although chronic GVHD
was a problem in the HPC-A study arm, it
was manageable in a sufficient number of
cases to give those patients a survival advantage.12
Initially, the most common diagnosis in
patients undergoing matched, unrelated
donor transplantation was CML. The discovery of imatinib mesylate has revolutionized the treatment of CML such that the
most common indication for allogeneic
transplantation is acute leukemia followed
by non-Hodgkin’s lymphomas, multiple
myeloma, and other diseases of marrow
failure such as aplastic anemia.13
Traditionally, HLA typing for Class I
(HLA-A and HLA-B) has been dependent
on serologic techniques. However, it is
likely that the posttransplant complications
of GVHD and failure to engraft result from
use of phenotypically matched, unrelated
donors with significant disparities in alloantigens that were not identified through
serologic matching techniques.14 HLA-A
and HLA-B molecular Class I typing, intermediate resolution, and high-resolution
typing of Class II alleles are the current
standard of care, particularly in unrelated
transplants.
The risk of GVHD is greater with HLA
Class II disparity than with Class I dispar15
ity. HLA typing for HLA-DR and HLA-DQ
is routinely performed by DNA-based techniques. Molecular technology provides
greater resolution, including subtypes of alleles identified as cross-reactive groups using conventional serologic techniques. Mis-

586

AABB Technical Manual

matching for a single Class I or Class II
antigen has no effect on survival, but mortality increases with more than one Class I
mismatch or simultaneous mismatches in
Class I and Class II antigens.15 Recent studies demonstrated the importance of recipient HLA-DRB1 and HLA-DQB1 allele dis16,17
parity in the development of GVHD.
With further experience in molecular typing and transplant outcomes, the extent to
which successfully transplanted cells can
tolerate disparities in specific alleles will be
elucidated. Although the importance of
HLA-A, -B, and -DR disparities is well
known, the significance of HLA-C disparity
is being investigated. HLA-C typing has been
hampered by poor serologic identification,
and its significance has been thought, until
recently, to play a minor role in the T-cell
immune response because of its reduced
polymorphism and low level of cell surface
expression. Early studies showed that
HLA-C antigens can be recognized by
alloreactive cytotoxic T lymphocytes and
natural killer cells, which may be associated
with an increased risk of graft failure.18

Graft-vs-Host Disease
The negative outcomes of HLA mismatching are graft rejection, host-vs-graft reactions, and graft-vs-host reactions. In acute
GVHD, the transplanted cells may attack
the tissues of the recipient early in the
engraftment—within 100 days after the
initial engraftment-associated events. The
skin, the gastrointestinal tract, and the
liver are most commonly involved, although usually not concurrently. The site
and severity determine the clinical grade
of acute GVHD. The risk of GVHD is
greater with HLA-mismatched, unrelated
and related transplants than with HLAidentical transplants.19
Chronic GVHD characteristically occurs
spontaneously months after transplanta-

tion or after acute GVHD (generally after
posttransplant day 50) and may severely affect the patient’s quality of life. In addition
to the symptoms found in the acute form,
chronic autoimmune-type disorders such
as biliary cirrhosis, Sjogren’s syndrome, and
systemic sclerosis may develop as the
transplanted immune cells attack the secretory epithelial cells in the saliva glands, the
biliary tree, or the patient’s connective tissue. Chronic GVHD was reported in 55% to
65% of allogeneic transplant patients who
survived beyond day 100 in a large study.2
Both forms of GVHD impair the patient’s
immune response and predispose the patient to infections. To decrease or eliminate
GVHD in these transplants, HPCs can
undergo procedures for T-cell reduction
(depletion) and the patient can be treated
prophylactically with a variety of immunosuppressive drug therapies.
GVHD has been associated with both a
decreased disease relapse and an improved
overall survival in leukemia patients if the
GVHD is relatively mild. Such a GVL effect
is believed to be secondary to the graft attacking residual malignant cells. A major
clinical challenge is to maximize the GVL
effect while minimizing the adverse sequelae of GVHD. The mechanism of the
GVL effect is incompletely understood, but
donor-derived cytotoxic T lymphocytes
specific for the patient’s minor histocompatibility antigens may contribute to the effect. Donor lymphocyte infusion of therapeutic T cells in patients with leukemic
relapse after allogeneic transplantation has
been attempted to induce a GVL effect.20
Conflicting data exist about whether a GVL
effect can occur independently of GVHD. A
strategy to maximize the GVL effect and
minimize GVHD has been to titrate the
number of donor lymphocytes until GVHD
Grade II-III occurs in order to create a GVL
effect without severe GVHD. However, the
optimal number of donor lymphocytes ca-

Chapter 25: Cell Therapy and Cellular Product Transplantation

pable of inducing a GVL effect without significant GVHD is not known. Immunosuppression, number of T cells transfused,
T-cell phenotype, myelosuppression, and
the timing of the DLI of therapeutic T cells
all play a role in the balance between the
GVL effect and GVHD. The GVL effect is best
documented in CML and less well shown in
AML, ALL, and other lymphoid malignancies.
However, because it is difficult to identify
a good HLA match, allogeneic transplantation is associated with a major risk that
immunocompetent donor T cells reacting
against recipient tissues will cause GVHD.
Even in HLA-identical (six-antigen match)
donor/recipient pairs, up to 6% of the grafts
will fail and GVHD will occur in 20% to 60%
of cases as a result of nucleated cells exhibiting minor histocompatibility antigens that
are not linked to the major histocompatibility complex antigens.21 Improved HLA
typing techniques now employ molecular
techniques that give a fuller picture of the
antigen mapping and match/mismatch
picture, but serologic terms of estimation of
number of matches are still frequently used
in clinical descriptions for reference. This
occurs despite immunosuppressive therapy
administered for several months after the
procedure.22 New and emerging immune
cell manipulations are being explored to
exploit the cancer-controlling effects of
these cells while seeking to avoid the unwanted consequences of GVHD. Extracorporeal photopheresis is being used to treat
patients with GVHD and to reduce the dose
of immunosuppressive medications re23
quired.

HPCs by Apheresis (Autologous)
Autologous HPC-A collection involves
mobilizing the hematopoietic cells from
the patient’s marrow compartment into
the peripheral blood with hematopoietic
growth factors, most commonly filgrastim,

587

with or without treatment with chemotherapy before collection. Once in the
circulation, the HPCs are collected by
leukapheresis. HPC-A collection carries
no anesthesia risk, is less invasive, and
contains fewer tumor cells than marrow
harvests.

HPCs by Apheresis (Allogeneic)

Related Transplantation
For adult allogeneic transplantation, the
best clinical results are obtained with a
completely HLA-matched, related donor.
The best chance of finding a six-antigen
HLA match is among the patient’s genetic
sisters and brothers. Parents and children
will be at least a haplotype match.
Genetically, there is a 25% chance of a
sibling being a complete match, a 50%
chance of a haplotype match, and a 25%
chance of a complete mismatch. Pediatric
patients are more tolerant of partially mismatched grafts and, therefore, have a larger
available donor pool.24 In the rare instance a
recipient has an identical twin, a syngeneic
transplant may be optimal because the donor and recipient cells are genotypically
identical and the risk of GVHD is reduced.
However, syngeneic grafts do not provide
the graft-vs-tumor effect found in allogeneic transplants.
HPC-A has largely replaced HPC-M for
related transplantation.25 The use of G-CSF
in healthy donors has been shown to mobilize sufficient HSCs for allogeneic transplantation,26 thus avoiding the need for anesthesia and a marrow harvest. Clinical
data comparing allogeneic HPC-A vs allogeneic HPC-M from HLA-identical siblings
have shown that HPC-A recipients have
faster engraftment, improved immune reconstitution, lower transplant-related morbidity, and a similar incidence of acute
2,27
GVHD. Retrospective analyses reported a
higher incidence of chronic GVHD in re-

588

AABB Technical Manual

lated allogeneic HPC-A recipients. However, a recent prospective randomized
study found no difference in the risk of
chronic GVHD.12

HPCs from Cord Blood
Despite the fact that over 3 million individuals are registered with the NMDP, patients in need of an allogeneic HPC transplant have ≤85% chance of finding a
matched donor. Finding and qualifying a
willing donor for HPC-A or HPC-M collection typically takes weeks. Because of the
time and availability constraints, attention has turned to the third available
source—HPC-C. Umbilical cord blood,
which in clinical practice is routinely discarded, is being banked as an alternative
source of HPCs, especially for children,
for whom smaller collection numbers of
HPCs are adequate for successful engraftment. The advantages of cord blood as a
source of HPCs for transplantation include: no risk to the donor, potential
availability of cord blood from donor populations underrepresented in the NMDP,
more rapid availability, and possibly
lower risk of viral infection and GVHD. Areas of concern regarding HPC-C include
ethical and informed consent issues, speed
of engraftment, higher mortality in the
posttransplant period, and ability to
achieve engraftment in adults as a result
of the limited number of nucleated cells
in cord blood.
Cord blood obtained from a delivered
placenta is known to be rich in early and
committed progenitor cells.29 Since the first
cord blood transplant was reported in 1989
for Fanconi anemia,30 more than 2500 patients have received cord blood for a variety
of hematologic malignant and nonmalignant conditions.31
Cord blood is collected from the placenta
at the time of delivery using a variety of

techniques, but the preferred system uses a
small collection bag (150 to 250 mL) with
appropriate anticoagulant. In-vitro data
have suggested that placental blood has an
increased capacity for proliferation.32 The
volume of units retained for processing is
typically 80 to 100 mL (range, 40-240 mL),
with a median nucleated cell count of 1.2 ×
109 in the units chosen for transplantation
and a median CD34+ cell count of 2.7 ×
105/kg.33 Clinical studies have reported successful engraftment in children.34,35 The median time to neutrophil engraftment (500/
µL) is 30 days; median time to platelet
engraftment (50,000/µL) is 56 days.36 Compared with engraftment observed after allogeneic marrow transplantation, neutrophil
and platelet engraftment appear to be delayed. Clinical studies have also suggested
that unrelated HPC-C transplants are associated with a lower risk of GVHD compared
with unrelated HPC-M transplants in children, even considering the lower risk sus31,34,36,37
ceptibility of pediatric patients to GVHD.
Two studies have compared engraftment
and outcomes of transplantation of adults
with one- or two-antigen mismatched
HPC-C, matched HPC-M, and one-antigen
mismatched HPC-M. The findings in the
two studies were very similar: HPC-M
transplant recipients recovered neutrophils
on day 18 to 19, and HPC-C recipients recovered neutrophils on day 26 to 27.38,39
Platelet engraftment in these studies repeated the finding that HPC-M transplantation provided more rapid recovery at 29
days compared with 60 days for HPC-C.
Both of these studies found that the HPC-C
transplantation compares favorably with
one-antigen mismatched HPC-M transplantation, with equal advantage for survival for each group. A matched HPC-M
transplant was superior to HPC-C or mismatched HPC-M, but the advantage was
small. This is encouraging news for adults
who need an HPC-M transplant but have

Chapter 25: Cell Therapy and Cellular Product Transplantation

difficulty finding a suitably matched donor.
Current clinical trials are testing whether
multiple HPC-C units received by adults
will facilitate faster engraftment. A study of
23 such recipients with advanced-stage disease showed a mean neutrophil engraftment of 23 days and a 57% chance of disease-free survival after 1 year. In these
two-unit transplants, the cells of one of the
units prevailed and provided the lasting
40
engraftment population.

589

ucts in 1992. The program has stored in
excess of 14,000 cord blood units and has
provided cells for more than 1500 transplant procedures. The outcomes of the
first 562 procedures have been reported
in detail.34 The NMDP has developed a
registry of participating cord blood banks.
The National Cord Blood Bank of the New
York Blood Center, NetCord, Bone Marrow
Donors Worldwide, and the Caitlin Raymond Registry are additional search sites
available today.

Related Cord Blood Donors
Sibling-derived cord blood has been used
as a source of hematopoietic engraftment
in more than 250 allogeneic transplants in
Europe and North America, representing
15% of the allogeneic cord blood transplants.31,35 The kinetics of hematopoietic
recovery are similar to those observed with
unrelated cord blood recipients.31 Times to
both platelet and neutrophil engraftment
are slower than those observed in HLAmatched sibling marrow transplants. However, recipients of cord blood from HLAidentical siblings have a lower incidence
of acute and chronic GVHD compared with
marrow from HLA-identical siblings.31

Autologous Cord Blood
Companies are marketing the freezing and
long-term storage of an infant’s cord blood
cells to parents, in case the child may need
them. 41,42 The chance of an individual
needing a cord blood transplant by age 18
is estimated to be 1 in 200,000.42 The first
successful autologous cord blood transplantation procedure was reported in a 14month-old patient with neuroblastoma.43

Cord Blood Banks
The first large-scale community cord
blood program was the Placental Blood
Program at the New York Blood Center,
which began collecting placental prod-

Donor Eligibility
Donor Evaluation: Autologous Setting
In the autologous setting, the major concern regarding eligibility for transplantation arises from the condition of the patient. For HPC-M, the patient’s marrow
first should be assessed for residual malignancy and marrow cellularity. Patients
scheduled for an autologous transplant
should undergo an extensive history and
physical examination to identify any risks
from the marrow harvest and/or apheresis procedures. For HPC-A, the patient/
donor is evaluated for the likelihood that
he or she can undergo successful mobilization and collection.

Donor Evaluation: Allogeneic Setting
Allogeneic donors must be selected according to their degree of HLA match;
qualifications according to the FDA regulations; standards of the AABB, NMDP,
and Foundation for the Accreditation of
Cellular Therapy (FACT); and physical
ability and willingness of the donor to undergo the collection procedure. Ideally, a
full six-antigen match should be found;
however, transplant procedures have been
performed successfully using one-antigen
mismatched and haploidentical donors.

590

AABB Technical Manual

Cytomegalovirus (CMV) status of the donor is also a deciding factor in the selection process in the case of CMV-negative
recipients. The use of parous females or
gender-mismatched individuals as donors
is associated with an increased risk of
GVHD, as is a history of transfusion in the
donor.44-46 Therefore, the preferred HLAidentical donor would be CMV negative
(in the case of CMV-negative recipients);
of the same gender as the recipient; if female, nonparous; and untransfused.

Infectious Disease Testing
Autologous and tested allogeneic donors
must be screened and tested for certain
infectious diseases.47 In the Code of Federal Regulations (CFR) Title 21, sections
of the regulations relevant to cell therapy
donors are Donor Screening (1271.75),
Donor Testing: General (1271.80), and Donor Testing: Specific Requirements
(1271.85). The rules are described by the
FDA as “comprehensive, but flexible.” The
donor screening rules are consistent with
the 1994 Centers for Disease Control and
Prevention (CDC) guidelines to prevent
transmission of human immunodeficiency
virus (HIV), and 1993 FDA guidelines for
prevention of transmission of hepatitis B
virus (HBV) and hepatitis C virus (HCV).
Testing for syphilis is required, and tissue
rich in leukocytes must be tested for human T-cell lymphotropic virus (HTLV )
and CMV. They also include requirements
for questioning donors about risk factors
for variant Creutzfeldt-Jakob disease and
new or emerging pathogens such as West
Nile virus and severe acute respiratory
syndrome, and requirements for tests for
carriers of these illnesses as soon as they
are practically available. The regulations
and guidance are extensive and fit the
FDA’s stated “layered approach” that intensifies the amount of screening and test-

ing of the donor to the level of risk to
which the recipient may be subjected.
Thus, unrelated allogeneic donors represent the highest level of risk, whereas
family members or regular sexual partners
are considered a lower risk source of new
exposure to patients.
The donor must be screened in order to
minimize the risk of disease transmission
to an already immunocompromised recipient. FDA regulations specify that the HPC
donor sample should normally be tested up
to 7 days before collection, or at the time of
collection but before release of the product.
HPC-A donor samples may be obtained up
to 30 days before collection in order to have
infectious disease testing completed before
the patient begins myeloablative chemotherapy [21 CFR 1271.80(b)]. For purposes
of optimal donor selection, it may be advisable to test the donor earlier in the transplantation workup period as well.48,49
The use of approved nucleic acid tests
for HIV and HCV has considerably shortened the possible “window period” in donors who may have contracted either of
these infections but who do not yet test
positive on antibody tests. Donors being
stimulated with filgrastim develop higher
sample-to-cutoff ratios. This makes falsepositive reactions more likely in the
immunoassays currently used for infectious
disease. False-positive test results for hepatitis B surface antigen and HCV antibody
have been associated with G-CSF administration in normal donors.50,51
Donors who are confirmed positive for
HIV should not be used as a source for the
transplant. Other positive disease markers
do not necessarily prohibit use of collections from a particular donor (eg, anti-HBc
positive), and special evaluation of donors
with this marker is under way by the NMDP
at this time. The allograft material from
such donors may be used when the patient’s transplantation physician informs

Chapter 25: Cell Therapy and Cellular Product Transplantation

him or her of the status of the donor and
documents consent of the patient. Segregated methods of storage must be used for
the biohazardous material collected until it
is infused so that cross-contamination of
other stored products is prevented.48(p78),49,52
In cases of allogeneic HPC-C transplants,
a sample for infectious disease studies
should be obtained from the donor’s
mother within 48 hours of collection of the
cord blood and tested. Any positive results
should be reported to the mother and the
mother’s physician. The HPC-C donation
involving a mother or a child who has a
positive infectious disease history or results
should either be discarded or the patient
must go through a special notification and
consent process.52 In cases of allogeneic
transplantation, CMV status of the donor
and the recipient should be carefully considered.
A recipient may develop primary CMV
infection if he or she is CMV negative and
receives a CMV-positive graft but may have
some protection from the immunity in the
graft. CMV-positive individuals receiving a
CMV-negative graft may have a severe primary infection of the graft from virus resident in the recipient’s tissue.46

Collection of Products
HPC-M Collection
A marrow harvest is the same for an allogeneic donor as for an autologous patient.
The procedure is performed under sterile
conditions in the operating room. The
posterior iliac crest provides the richest
site of marrow. In the autologous patient,
prior radiation therapy to an aspiration
site may result in hypocellular yields. In
general, these sites are unsuitable for harvest and should be avoided. Once aspirated, the marrow should be mixed and

591

diluted with an anticoagulant (usually
preservative-free heparin and/or ACD).
The marrow aliquots are pooled into a
sterile vessel or a harvest collection bag
equipped with filters of graduated pore
size. The marrow is then transferred to a
sterile blood bag and transported to the
processing laboratory, where samples are
removed for graft evaluation, quality assurance testing, possible manipulation of
the product, final labeling, and/or cryopreservation.

Collection Targets
The recipient’s body weight and type of
manipulation of the collection, if any, will
determine the volume of marrow to be
collected. A frequently used minimum
target (after processing), for both autologous
and allogeneic transplants, is 2.0 × 108 nucleated cells per kilogram of recipient
body weight. Marrow harvests in autologous patients who have received alkylating agents as therapy may yield fewer progenitor cells relative to the total nucleated
cells collected. In these cases, extra marrow should be obtained if possible.53 If the
harvested product is to be processed (ie,
T-cell depletion, ex-vivo tumor purging),
additional marrow (up to double the reinfusion target) should be collected. The
NMDP limits the volume harvested from
its donors to a maximum of 1500 mL. Many
centers use the cell number requested for
transplant and the cell count on the product during collection to determine the final collection volume.

Clinical Considerations
The age of the donor and, in the autologous setting, the underlying disease and
previous treatment regimen influence the
HPC yield (Table 25-2).54 Autologous or allogeneic donors may require RBC transfusions to replace blood taken with marrow

592

AABB Technical Manual

collection. Frequently, allogeneic marrow
donors will have an autologous RBC unit
collected 2 to 3 weeks before the harvest.
Most RBC transfusions occur after the harvest to avoid marrow dilution. Allogeneic
blood components must be irradiated if
given before or during the procedure, to
incapacitate donor lymphocytes from replication and attack of the transplant recipient, possibly causing GVHD. In transplants that require marrow manipulation,
such as red cell depletion, the recovered
red cells may be returned to the donor.

well as the pluripotent stem cells. CD34+
cells purified from marrow are capable of
55
trilineage reconstitution in humans. One
to three percent of normal marrow cells
express this antigen. In normal peripheral
blood, CD34+ cells circulate in low numbers (0.01-0.1%).56 Monoclonal antibodies
(MoAbs) to CD34 have been developed
and are used in flow cytometric assays to
measure the stem/progenitor content of
HPC collections and CD34+ cell concentration in donor peripheral blood.57

HPC-A Collection

HPC-A Collection (Autologous)

HPCs can be mobilized into the peripheral blood with the use of recombinant
colony-stimulating factors and collected
in sufficient numbers to achieve longterm hematopoietic engraftment in a transplant recipient.

In the autologous setting, HPC-A donors
are routinely mobilized with hematopoietic growth factors (with or without chemotherapy), which include G-CSF and
granulocyte-macrophage colony-stimulating factor (GM-CSF) or a combination
of the two. Mobilization with chemotherapy or growth factors alone results in a
10- to 30-fold increase in the concentration of HPCs over the steady state.8 Combined mobilization with growth factors
and chemotherapy enriches HPC concentrations 50- to 200-fold.58 Transplantation
of autologous HPC-A results in a reduced
time to hematopoietic recovery compared
with autologous HPC-M.59 Platelet reconstitution is most striking and patient hos-

CD34
CD34 is the cluster designation given to a
transmembrane glycoprotein present on
immature hematopoietic cells, some mature endothelial cells, and stromal cells.55
The antigen has an approximate molecular weight of 110 kD and carries a negative
charge. Cells expressing the CD34 antigen
encompass the lineage-committed cells as

Table 25-2. Factors Reported to Affect the Mobilization of Hematopoietic Cells
1.

2.
3.
4.
5.

Mobilization technique
a. Chemotherapy—degree of transient myelosuppression
b. Growth factors—type, schedule, dose
c. Use of combined chemotherapy and growth factors
Extent of prior chemotherapy/radiation
Type of underlying disease
Age of patient
Presence of marrow metastases

Adapted with permission from Lane. 54

Chapter 25: Cell Therapy and Cellular Product Transplantation

pital stays are reduced by 7 to 10 days.
60
Schwella and colleagues demonstrated
that the median time to an unsupported
platelet count of ≥20,000/µL was 10 days
in the group receiving chemotherapy plus
G-CSF-mobilized HPCs vs 17 days in the
group receiving autologous HPC-M. Effective mobilization in the autologous patient is influenced by previous chemotherapy or radiation.
There is a linear relation between the
precollection peripheral blood CD34 count
and the volume of blood that needs to be
processed to achieve a target dose. Graphs
and predictive models would be expected
to vary based on the efficiency of CD34+
collection related to the specific equipment
and software employed. It would also be
expected to be least predictive at low peripheral blood CD34+ concentrations, where
the enumeration is least accurate. White
cell counts, the number of mononuclear
cells, and the number of circulating CD34+
cells have all been used as surrogate markers for the timing of the HPC-A collection.59,61-63 It has been suggested that the optimal time to begin HPC collection in patients who have been primed with chemotherapy is when the white cell count first
exceeds 5 × 109/L.59 However, the white cell
concentration does not correlate with the
number of HPCs in the peripheral blood.
Phenotypic analysis of CD34+ cells by flow
cytometry provides a more real-time measurement of CD34+ cell content in an HPC
collection or hematopoietic graft. The technique can be used to indicate the timing of
the first collection and the total number of
CD34+ cells/kg finally collected (as a func56,57,64-66
tion of the volume of blood processed).
The length of time for engraftment correlates with the number of CD34+ cells in the
collection.65,67-69 In general, a peripheral blood
CD34+ cell concentration of 10/µL can be
expected to result in a yield of at least
1.0 × 106 CD34+ cells/kg.60,61,64

593

In most autologous patients, venous access is obtained through a dual- or triplelumen central venous apheresis catheter.
Operators of blood cell separators generally
process two to three blood volumes per
procedure. In the pediatric setting, it may
be necessary to prime the cell separator
with compatible, irradiated red cells. The
donor will undergo daily procedures of approximately 2 to 5 hours each. Large-volume leukapheresis procedures (processing
at least three blood volumes or 15-20 liters)
are performed at many centers to reduce
the overall number of collections. Investigational studies have suggested that there is
equilibration of noncirculating HPCs into the
peripheral circulation with large-volume
collections.70-72 Hillyer73 reported a 2.5-fold
increase in colony-forming units–granulocyte-macrophage (CFU-GM) per mL processed in collection when the collection
volume was 15 liters compared to the yield
in the first blood volume of the collection.
Collection Targets. The adequacy of
autologous HPC collection is gauged by the
CD34+ dose (the number of CD34+ cells per
kilogram of recipient body weight). The reported minimum threshold of CD34+ cells
necessary for neutrophil and platelet engraftment in the autologous patient has ranged
from 0.75 to 1.0 × 106/kg.8,64 This minimum
target is a broad guideline and higher doses
have been associated with accelerated
platelet engraftment,74 reduced febrile complications, and use of antibiotic therapy af75
ter transplantation. Recent data support
an economic benefit associated with greater
CD34+ cell collections (greater or equal to
5.0 × 106/kg) compared to the minimum acceptable collections required for engraft6
76
ment (1.0 × 10 /kg). The type of processing
will also influence the volume necessary for
collection.
Poor Autologous HPC Mobilization. Patients who have been heavily pretreated
with chemotherapy and/or radiation ther-

594

AABB Technical Manual

apy may fail to mobilize enough HPCs after
stimulation with growth factors/chemotherapy. The best way to obtain an adequate collection from poorly mobilized patients is unknown. Collection of HPC-M in
combination with, or in place of, HPC-A is
frequently ineffective in improving engraftment.77 Second attempts at mobilization
with G-CSF alone have been successful in
obtaining adequate CD34+ cell counts and
can be as effective as G-CSF and chemotherapy.78 Increasing the dose of G-CSF or
combining with GM-CSF has also successfully mobilized some autologous donors after prior failed attempts.79
Mobilization can be very difficult in some
patients, particularly if they have been
heavily pretreated or are older. Another approach to increase production of stem/progenitor cells by growth factors is manipulation of the chemokines that attach the cells
to their microenvironment. The interaction
of stromal-derived growth factor-1 (SDF-1)
and CXCR4, its ligand, controls mobilization. The molecule AMD 3100 was developed originally as a potent and selective
inhibitor of CXCR4 in order to control replication of HIV-containing cells. An observed
side effect of the drug was a rapid increase
in white cells. Broxmeyer and colleagues
measured a 40-fold increase in HPCs after
injection of AMD 3100 into human volunteers.80 Phase I and II clinical trials are under way in conjunction with G-CSF to enhance mobilization and HPC-A yields in
81
difficult-to-mobilize patients.
Clinical Considerations for Autologous
HPC-A Collection. The frequency and length
of HPC-A collections may result in donor
discomfort and side effects. Complete blood
counts are performed before and after each
apheresis procedure to monitor the hematocrit and platelet values. Red cell and/or
platelet transfusions may be required. Red
cell loss should be minimal in HPC-A collection, but platelet counts typically de-

crease from 25% to 40% because the HPCs
lie close to the platelet layer in the buffy
coat. Collection can be performed via peripheral or central venous access. Patients
who have received prior chemotherapy are
more likely to need a central venous catheter because of poor peripheral access. Catheter-associated thrombosis (either in the
catheter or in the vein surrounding it) is the
most common complication associated
with HPC-A collection.82

HPC-A Collection (Allogeneic)
Collection of allogeneic HPCs from HLAmatched relatives is primarily performed
using G-CSF mobilization. Typically, donors are given 10 to 16 µg/kg as a subcutaneous injection once or twice daily. The
concentration of CD34+ cells in the peripheral blood begins to rise after 3 days
of G-CSF administration and peaks after 5
to 6 days. Standard volume leukapheresis
(processing 8-9 L) results in a component
with the following median values: white
cells, 32.4 × 109; mononuclear cell count,
9
6
31.4 × 10 ; CD34+ cells, 330 × 10 ; plate9
83
lets, 470 × 10 ; and RBCs, 7.6 mL. Clinical
trials have suggested a minimum dose of
2.0 × 106/kg of CD34+ cells to be adequate
84
for allogeneic HPC-A transplants.
Allogeneic donors with poor venous access may require central venous catheters.
In the NMDP report on normal donor collections of HPCs, 5% of men and 20% of
women require central venous access for
85
collection. There is a 1% risk of complications associated with these catheters including infection, hemorrhage, and pneumothorax. Thrombocytopenia has been reported
as a complication of G-CSF administration
86,87
and HPC-A collection in healthy donors.
There is also a risk in exposing normal
donors to G-CSF. Ninety percent of donors
experience side effects.88 The most common complaint is bone pain followed by

Chapter 25: Cell Therapy and Cellular Product Transplantation

headaches, body aches, fatigue, nausea,
and/or vomiting. Bone pain, headaches,
and body aches can be successfully managed with nonsteroidal analgesics such as
acetaminophen. As mentioned earlier,
false-positive infectious disease serologies
have been reported.51 Serious adverse effects are rare but include reports of splenic
rupture,89,90 neutrophilic infiltration of the
91
skin (Sweet’s syndrome), arterial thrombo92
93
sis, and an anaphylactoid reaction. Donors with complex hemoglobinopathies
(eg, sickle trait and β+ thalassemia) have
been observed to have complications with
G-CSF stimulation for the purpose of be94,95
coming an HPC donor.

HPC-C Collection
Umbilical cord blood can be collected from
either a delivered or an undelivered placenta.96 Cord blood collection should not
interfere with obstetric care of the mother
or the infant. If the birth provider intends
to collect the cord blood, plans should be
made in advance to abandon the collection if care of the mother or the infant requires the care provider’s attention instead. Cord blood collected after delivery
of the placenta is preferably initiated
within 15 minutes of parturition, if adequate volumes are to be obtained.97 To
minimize the risk of bacterial contamination, the surface of the cord should be
cleaned with alcohol, then disinfected, in
a similar manner to the preparation of skin
for a blood donation. A large-bore needle
connected to a blood collection bag containing CPDA, CPD, or ACD anticoagulant
is inserted into the umbilical vein so that
the placental blood drains by gravity into
the bag. Alternatively, a delivered placenta can be suspended above the collection bag. CPDA or CPD are the preferred
anticoagulants because they are isotonic

595

and have a neutral pH. However, other
closed system arrangements using ACD or
heparin are in use in some centers. Informed consent from the biologic mother
or legal representative of the child must
be obtained, preferably before delivery.
A personal and family medical history of
the biologic mother (and, if available, of the
infant donor) must be obtained and documented before, or within 48 hours of, the
collection. If available, his medical history
should be obtained from the biologic father. HPC-C collections are not acceptable
for allogeneic use if there is a family history
(biologic mother, father, or sibling) of genetic disorders that may affect the graft’s effectiveness in the recipient or otherwise expose the recipient to a genetic disorder
through the transplant.
Red cells from the HPC-C collection or
from the infant donor must be typed for
ABO/Rh and a screen for unexpected red
cell antibodies must be performed using either the mother’s serum or plasma or a
sample from either the infant donor or the
52,56
cord blood collection. White cells from the
cord blood must be typed for HLA (HLA-A
and -B antigen testing, and DNA-based
Class II typing) by a laboratory accredited
by the American Society of Histocompatibility and Immunogenetics (ASHI) or an
equivalent accrediting organization.49,52 In
order for HPC-C to be a feasible alternative
to allogeneic HPC-M or HPC-A transplants,
it is essential to have a frozen inventory of
ready-to-use HLA-typed products.
Samples, preferably from an attached
segment, should be frozen for pretransplant confirmation of HLA type by the
transplant facility. Many centers freeze a
sample of the mother’s serum to allow new
infectious disease tests to be done if they
become available after the HPC-C has been
collected and are thought to be important
at the time the transplant is planned. The
emergence of West Nile virus as a threat to

596

AABB Technical Manual

mothers, infants, and transplant recipients
is an example. A sample of the mother’s
blood containing cells or DNA (a dried filter
paper sample is adequate) may be useful if
questions concerning the child’s HLA type
arise. These samples from the mother were
more useful when HLA typing was done by
serologic methods to investigate ambiguous types in the infant. CD34+ cell enumeration at the time of thawing may also be
performed to estimate the stem cell content of the HPC-C graft but is useful only if
a viability marker is employed so that only
viable cells are counted.
The desire to increase the dose of stem
cells in HPC-C has fueled interest in ex-vivo
expansion of the cells in a laboratory setting.98 Early clinical trials with expanded
cord blood progenitors are promising, having shown successful engraftment; however, no improvement in patient survival
has been reported.99,100 Ex-vivo expansion of
cord blood is an area of active experimental
investigation.

Processing of Hematopoietic
Progenitor Cells
Techniques for Cell Selection and/or
Purging of Hematopoietic Progenitor Cells

Selection of the CD34+ Cells
As an HPC undergoes differentiation and
maturation, CD34 antigen levels decrease.
This property, coupled with the use of MoAbs
specific for the different epitopes of the
CD34 molecule, permits physical separation procedures. There are several methods of immunoselection available, such as
fluorescence-activated cell sorting (FACS)
101
and immunomagnetic beads. Selection
of CD34+ HPCs may be associated with a
reduction of tumor cells (autologous grafts)
or T cells (allogeneic grafts). The clinical
utility of CD34 and AC133 selection for tu-

mor or T-cell reduction is under investigation.102
Immunomagnetic Separation. Various
immunomagnetic separation techniques,
direct or indirect, are available. Typically, a
CD34 antibody is coupled to a magnetic
bead. This complex is incubated with mononuclear cells, and the cells expressing the
CD34 antigen bind to the antibody-coated
beads, forming rosettes. A magnet is applied to separate the rosetting CD34+ cells
from the nonrosetting cells. Bead detachment, which varies among methods, may
be accomplished through anti-Fab fragments or enzymatic treatment (eg, chymo103
papain). The Isolex 300 system (Baxter
Healthcare Immunotherapy Division, Irvine,
CA), a magnetic cell separator, is a semiautomated instrument for clinical-scale
CD34+ selection applications. This method
uses antibody-coated Dynal paramagnetic
beads to rosette the CD34+ cells. A fully automated device (Isolex 300i, Baxter) and a
peptide release agent are available for clinical use.104 Other techniques of magnetic cell
separation employ superparamagnetic microbeads that remain attached to the HPC surface.105 One such method, magnetic cell
sorting (MACS, Miltenyi Biotec, Bergisch
Gladbach, Germany), was first introduced
on a small scale by Miltenyi. 1 0 6 The
CliniMACS (Miltenyi Biotec) is a clinicalscale version of the MACS system and is
available in the United States for investigational use only. This system employs antibody-conjugated iron-dextran microbeads.
The magnetically stained cells are separated over a high gradient magnetic column
and a microprocessor controls the elution
of the CD34+ cells.
Counterflow Centrifugal Elutriation.
Counterflow centrifugal elutriation (CCE) is
a method of separating cells based on their
size and density. A continuous-flow centrifuge (Beckman Instruments, Palo Alto, CA,
and Gambro, Golden, CO) and unique

Chapter 25: Cell Therapy and Cellular Product Transplantation

chamber design allow for the basic separation principle: two opposing forces (centrifugal force and counter media flow) acting
upon cells at the same time. As cells are
pumped into the chamber (centripetal direction), they align according to their sedimentation properties. With adjustment of
the counterflow rate, the centrifugal and
counterflow forces are balanced, and a gradient of flow rates exists across the chamber. By gradually increasing the flow rate of
the medium or decreasing the speed of the
rotor, cells can be eluted out of the chamber and collected. Smaller, slower sedimenting cells elute first. Although this technique is used primarily for T-cell depletion,
CCE has been applied to CD34+ cell selection. CD34+ cells are heterogeneous and
will elute in subset fractions that are useful
for repopulation experiments and ex-vivo
107
expansion trials.

Autologous Tumor Purging
Purging or negative selection refers to the
removal of tumor cells that may contaminate the autologous graft. In patients with
hematopoietic disease or malignancies
that frequently involve the marrow (eg,
lymphomas), minimal residual disease
contributes to relapse.108 Although autologous HPC-A collections have a lower probability than HPC-M of tumor contamination and fewer tumor cells/mL, they still
may contain large numbers of viable tumor cells. Studies have demonstrated that
tumor cells may be mobilized from the
marrow into the peripheral circulation. 1 0 9 , 1 1 0 Whether tumor purging decreases the likelihood of relapse is controversial. Because some purging methods
may damage HPCs, the probability of residual disease or relapse should be carefully balanced against the higher graft
failure rate or increased mortality from
prolonged aplasia that may be associated

111

597

with tumor purging. Numerous techn i q u e s f o r a u t o l o g o u s p u r g i n g a re
available. The goal of all purging methods— whether physical, immunologic, or
pharmacologic—is the destruction or removal of the malignant clone while maintaining the efficacy of the HPCs necessary
for engraftment.112
Pharmacologic Techniques. In-vivo
antineoplastic therapy produces greater tumor cell kill because of the differential sensitivity of malignant cells over normal cells.
In-vitro pharmacologic purging is an effort
to expand this therapeutic ratio. In-vitro
purging allows for dose and exposure intensification without concern for organ toxicity because higher drug concentrations
can be used on isolated hematopoietic
grafts ex vivo, which then can be administered in vivo. However, the drug concentration must be at nontoxic levels before reinfusion. Activated oxazaphosphorines
(4-hydroperoxycyclophosphamide, mafosfamide) were the most frequently used
compounds but are generally no longer in
use in the United States. Other chemotherapeutic agents have been investigated
in preclinical models but are not in clinical
use. Results from purged autologous transplants for AML with either of these drugs
showed prolonged aplasia with less than
1% CFU-GM survival.112
Physical Techniques. Separation methods based on cell size and density through
gradient-generating reagents or CCE do not
achieve adequate tumor cell depletion.111
However, the combined use of physical and
immunologic techniques requires further
study.
Immunologic Techniques. The development of MoAbs coupled with the discovery
of tumor-associated antigens opened the
field for immunologic purging.113 MoAbs
may be directed at tumor-specific antigens
or cell-differentiation antigens.111 The immunologic techniques differ primarily by

598

AABB Technical Manual

the method of target cell removal. MoAbs
are used in conjunction with complement,
bound to toxins, or coupled to magnetic
beads. The choice of MoAb and the heterogeneity of antigen expression on the target
cell affect the success of the purge or the
level of depletion. Many investigators employ a cocktail of MoAbs in an effort to enhance the purging efficiency.114,115 Complement-mediated cytotoxicity is frequently used
if the antibody is of IgM isotype or if the expected level of antigen density is high.111
Immunomagnetic cell separation, either
by direct or indirect method, employs an
antibody-coated magnetic bead to target
the antigen or antigens of interest. A recent
study described a 5-log tumor cell depletion with two cycles using the indirect
method for B-cell lymphoma. Colony assays
showed only a 20% reduction in CFU-GM
and multipotential CFU (CFU-GEMM).114

T-Cell Depletion
GVHD is a significant complication in
allogeneic HPC transplantation. Because
the disease process is mediated by donor
T cells that are reactive against the host,
depletion strategies are used to target and
remove these cells in an effort to decrease
the incidence or at least lessen the severity of GVHD. Many of the techniques outlined for positive selection and tumor purging are applicable to T-cell depletion (Table
25-3).17
In T-cell-depleted grafts, two major areas
of concern are graft failure and recurrent
leukemia. Most instances of graft failure
(initial or late) are caused by immunologic
rejection. Early transplants involving depleted grafts in patients with CML showed
a 50% or greater relapse rate. In contrast,
patients who developed GVHD had a lower
relapse rate. Subsequent clinical and investigational data provided evidence of an immune-mediated GVL effect.116

Table 25-3. Methods of T-Cell Depletion
Nonimmunologic
Counterflow centrifugal elutriation
Pharmacologic/cytotoxic drugs
Immunologic
Monoclonal antibodies with or without
complement
Immunotoxins
Immunomagnetic beads

Research and developmental efforts have
focused on determining the optimal level of
T-cell depletion. Dreger et al115 reported a
comparison of T-cell depletion methods
(CAMPATH-1 plus complement, immunomagnetic CD34+ selection, and biotinavidin-mediated CD34+ selection). The
immunomagnetic method provided a 4-log
reduction in T cells vs a 3.1-log reduction
with the biotin-avidin method. MoAb treatment with autologous complement yielded
a 2.1-log reduction. The challenge of minimizing the severity of GVHD and maintaining the GVL effect is ongoing. Additional
studies are examining the role of subpopulations of T cells and/or cytokines in
potentiating the GVL effect.117

ABO Incompatibility
Although HLA compatibility is crucial in
the successful engraftment of myelosuppressed or ablated patients, ABO compatibility is not. Pluripotent and very early
committed HPCs do not possess ABH antigens, allowing engraftment to occur successfully regardless of the ABO compatibility between the recipient and donor.
ABO incompatibility does not affect neutrophil or platelet engraftment, graft failure,
or rejection. However, delayed red cell
engraftment may occur after a major
ABO-incompatible transplant and delayed
hemolysis may occur after a minor ABOincompatible transplant where the donor

Chapter 25: Cell Therapy and Cellular Product Transplantation

has antibodies to the recipient’s blood
cells (Table 25-4).118

Major ABO Incompatibility
Major incompatibility occurs when the
recipient has ABO antibodies or other red
cell antibodies, such as anti-D or antiKell, against the donor red cell antigens.
The HPC preparation can be processed to
remove mature red cells. The group O recipient who receives a group A graft may
continue to produce anti-A and anti-B for
3 to 4 months or longer in rare instances,
and the presence of anti-A will delay
erythropoiesis by the group A graft; group
A red cells appear in the circulation when
the recipient’s anti-A disappears. Granulo-

599

cyte and platelet production are not
affected.119 Red cell engraftment may be
delayed to ≥40 days after the transplantation.120 Red cells used for transfusion of the
recipient must be compatible with both the
donor and the recipient. In some centers,
group O red cells are given to all major
ABO-incompatible transplant recipients
in order to avoid confusion. Recommendations for optimal blood component selection are shown in Table 25-5.

Minor ABO Incompatibility
Minor ABO incompatibility occurs when
the donor has antibodies against the recipient red cell antigens (such as a group
O donor to a non-group O recipient). Be-

Table 25-4. Potential Problems with ABO- and Rh-Incompatible HPC
Transplantation
Example
Incompatibility

Donor

Patient

Potential Problems

ABO (major)

Group A

Group O

ABO (minor)

Group O

Group A

Negative

Positive

Negative
(with
anti-D)
Positive

Hemolysis of infused donor RBCs, delay
of RBC engraftment, hemolysis at the
time of donor RBC engraftment
Hemolysis of patient RBCs from infused
donor plasma; in pediatric cases,
hemolysis of patient RBCs 7-10 days
after transplant caused by passenger
lymphocyte-derived
isohemagglutinins
Hemolysis of patient RBCs by donor
anti-D produced after engraftment
Hemolysis of donor RBCs from newly
engrafted HPCs (rare), or delayed
RBC recovery
Hemolysis of patient RBCs by donor antibodies in plasma produced after
engraftment
Hemolysis of donor RBCs from newly
engrafted HPCs (rare), or delayed
RBC recovery

Other RBC
antigens

Negative

Positive

Negative
(with
antibody)

600

Table 25-5. Transfusion Support for Patients Undergoing ABO-Mismatched Allogeneic HPC Transplantation

Recipient

Donor

Mismatch
Type

A
B
AB
AB
AB
O
O
O
A
B
A
B

O
O
O
A
B
A
B
AB
AB
AB
B
A

Minor
Minor
Minor
Minor
Minor
Major
Major
Major
Major
Major
Minor & major
Minor & major

All
Components

Recipient
Recipient
Recipient
Recipient
Recipient
Recipient
Recipient
Recipient
Recipient
Recipient
Recipient
Recipient

Phase II
First
Choice
Platelets

RBCs

O
O
O
A, O
B, O
O
O
O
A, O
B, O
O
O

A, AB
B, AB
AB
AB
AB
A
B
AB
AB
AB
AB
AB
121

Next Choice
Platelets*

AB; B; O
AB; A; O
A; B; O
A; B; O
B; A; O
AB; B; O
AB; A; O
A; B; O
A; B; O
B; A; O
A; B; O
B; A; O

*Platelet concentrates should be selected in the order presented. Modified from Friedberg et al.
Phase I = the time when the patient/recipient is prepared for HPC transplantation.
Phase II = from the initiation of myeloablative therapy until:
For RBC—DAT is negative and antidonor isohemagglutinins are no longer detectable (ie, the reverse typing is donor type).
For FFPs—recipient’s erythrocytes are no longer detectable (ie, the forward typing is consistent with donor’s ABO group).
Phase III = after the forward and reverse type of the patient are consistent with donor’s ABO group.
Beginning from Phase I, all cellular components should be irradiated and leukocyte reduced.

Phase III

FFP

A, AB
B, AB
AB
AB
AB
A, AB
B, AB
AB
AB
AB
AB
AB

All
Components

Donor
Donor
Donor
Donor
Donor
Donor
Donor
Donor
Donor
Donor
Donor
Donor

AABB Technical Manual

Phase I

Chapter 25: Cell Therapy and Cellular Product Transplantation

fore infusion of a graft from a plasma-incompatible donor, the plasma may be removed to avoid infusion of preformed
anti-A and/or anti-B. Minor ABO-incompatible transplants can be characterized by
rather abrupt onset of immune hemolysis,
which begins about 7 to 10 days after transplantation and may last for 2 weeks. The
direct antiglobulin test (DAT) is positive;
anti-A and/or anti-B can be recovered in
the eluate and hemoglobinemia and/or
hemoglobinuria may occur. An additional
30% of such transplant recipients develop
a positive DAT without experiencing gross
hemolysis. This phenomenon is the result
of red cell antibodies produced by passenger B lymphocytes in the stem cell
121
graft. Non-ABO antibodies from passenger lymphocytes in HPC transplants have
122
also been reported. Although transient,
the hemolysis may persist for up to 2 weeks
and may require transfusion with group O
red cells. Minor ABO-mismatched HPC
transplants may be associated with a
higher risk of hemolysis, possibly caused
123
by greater B-cell content. In all cases,
plasma used for transfusion should be
compatible with both the donor and the
recipient. In some centers, group AB plasma
products are given to all minor ABO-incompatible transplant recipients in order
to avoid confusion. RBC transfusions, beginning with the conditioning regimen,
should be of donor type. Recommendations for optimal blood component selection are shown in Table 25-5.

Chimerism
In spite of intensive pretransplant chemotherapy and irradiation, some of the host’s
hematopoietic cells may survive and subsequently coexist with cells produced by
the transplanted HPCs. This dual cell population, called partial hematopoietic
chimerism, may have an effect on immu-

601

124

nologic tolerance. This tolerance develops in successful transplants and allows
normal immune reconstitution. Full chimerism (replacement of host marrow cells
entirely by donor cells) usually occurs in
successful HPC-M transplantation.

Processing in the Presence of ABO
Incompatibilities
Major ABO Incompatibility. Two approaches have been employed with major
ABO incompatibilities: 1) removal of or
decrease in the isoagglutinin level in the
recipient or 2) removal of the red cells in
the HPC-M collection. Processing is not
generally required with HPC-A because
such a small volume of red cells is usually
included.
Attempts to remove or decrease the isoagglutinin titer in recipients involve the use
of one or more large-volume plasma exchanges with or without the subsequent infusion of donor-type red cells as a secondary
effort to absorb any additional isoagglutinins.125,126
Other approaches to ABO-incompatible
products include red cell depletion. The
most prevalent method in use is red cell
sedimentation.
Some institutions accomplish red cell
depletion using density gradient separation
and cell washers (Gambro BCT, Lakewood,
CO); others use continuous-flow apheresis
machines such as the Spectra (Gambro) and
the Fenwal CS-3000 (Baxter Healthcare),
with mononuclear cell recoveries of 94%
and 87% and red cell depletion of 99% and
98%, respectively.
Minor ABO Incompatibility. Theoretically, the level of hemolysis is dependent on
the volume of HPC-M infused relative to
the recipient’s plasma volume and the IgM
titer of the donor. Incompatible plasma can
be easily removed by centrifugation.
Plasma removal using this method removes

602

AABB Technical Manual

approximately 75% of the plasma volume
while recovering >70% of the initial nucleated cell count. Plasma removal is not generally required in HPC-A because the volume of incompatible plasma is usually small.

Umbilical Cord Blood Processing
Advances in the processing of umbilical
cord blood cells have solved the early
problems of unacceptably high progenitor cell losses with manipulation.127 Unlike
cryopreservation laboratories that store
relatively few autologous or allogeneic
HPC-M or HPC-A products (typically, less
than a few hundred products at any one
time), a successful cord bank would be
expected to store thousands of products
for prolonged periods. Therefore, attempts
to reduce umbilical cord blood bulk (and,
therefore, storage space, liquid nitrogen
requirements, and cost) have been actively pursued. Currently, many centers
are following the approach used by the
New York Blood Center, which involves
sedimentation and volume reduction before cryopreservation.128

Cultures for Microbial Contamination
Sterility is essential in blood products for
infusion to immunosuppressed patients.

This is particularly important with HPC-M,
which is frequently collected in an open
system, and with products that require
129-134
multiple manipulations (Table 25-6).
Culture growth can result from contamination during collection or product processing, or as a result of an infected catheter or the patient’s sepsis. Skin commensals
are the predominant isolates from these
cultures. In all cases it is important to
identify the source, the degree of contamination, and the causative organism,
given the fact that this product is intended for transplantation in an immunocompromised recipient. Each product
must be tested for microbial contamination at least once during the course of
processing, usually just before freezing or
infusion with allogeneic transplants.48 A
positive culture does not necessitate immediate discard of the product because
these are frequently irreplaceable cells.135
Thus, positive results need to be reviewed
by the appropriate physician on a caseby-case basis and sensitivity tests ordered
to better define the organism and to allow
for appropriate clinical management at
the time of transplant. This should also
serve as the impetus to review all procedures and techniques to ensure that they

Table 25-6. Percent Microbial Contamination of Cell Therapy Products
Source of HPCs
Marrow

No. of Products Tested
291
227
317
194

% Contaminated
0
1.3
6.0
<0.1

Reference
129
130
131
132

Peripheral blood

1380
560
576
1040

0.65
0.7
0.5
0.2

129
130
131
133

Cord blood

1000

<1.0*

134

*Initial rates were as high as 28%; however, with improved donor selection and technique, the rate fell to <1%.

Chapter 25: Cell Therapy and Cellular Product Transplantation

provide safeguards to prevent contamination.

Stem Cell Enumeration: CD34 Analysis,
Aldehyde Dehydrogenase Activity, and Others
As stem cells differentiate into hematopoietic cells, CD34 surface markers appear. As the cells continue to differentiate
and acquire other surface markers, CD34
expression diminishes. Stages of differentiation may be studied by the expression
or co-expression of other antigens or
properties. There are currently two assays
for CD34 that are FDA-cleared for in-vitro
diagnostic use. These CD34+ measurement methods are the most widely used
for the evaluation of grafts, have the longest history of use, and show the most
clinical correlations in the marrow transplantation literature.
The principle of gain and later loss of
CD34 expression has been applied to evaluate the quality of HPC grafts. The current
approach is to use a method based on
multiparameter flow cytometry to determine the number of CD34+ cells and to calculate a subsequent CD34+ dose based on
the recipient’s weight. Numerous flow
cytometric analysis methods for CD34 enumeration exist.56,136,137 Because these methods differ, correlation of CD34 dose values
from site to site is often unreliable. Multiple
studies have documented the variability of
CD34 analysis methods and results and
have examined possible causes of the variability.56,57,138,139 Because of the “rare event”
nature of CD34 enumeration, several procedural components play a critical role in
the assay. Selection of the CD34 antibody
clone, fluorescent conjugate, lysing solution, lyse-wash format, gating strategy, and
the number of events analyzed are some of
the factors that influence the end result.140
Growing awareness of, and concern
about, the need for a standardized ap-

603

proach to CD34 analysis have prompted
several collaborative groups such as the International Society for Cellular Therapy
(ISCT) to propose guidelines for CD34+ cell
determination by flow cytometry.56,141 Alternate approaches are available and will continue to be developed and investigated as
more is discovered about the CD34 antigen
and other markers for the pluripotent stem
142
cell.
A second method for enumeration of
early cells has gained FDA clearance. The
cytosolic expression of aldehyde dehydrogenase (ALDH) in stem cells was found to
be the protective property of HSCs in cyclophosphamide treatment of patients and in
preservation of stem cells in graft purging
with 4HC. Flow cytometric analysis of
ALDH-positive cells with the enzymatic excitation and trapping inside the stem cells
of a fluorescent substrate (because the
fluorophore becomes charged and does not
diffuse freely) allows measurement of a viable population enriched in colony-forming
progenitor cells. Both long-term repopulating and committed progenitor cells are
enriched and the ALDHbr contains the population responsible for hematopoietic engraftment of NOD-SCID mice.143,144 This method
br
lo
to enumerate ALDH SSc cells identifies
only cells with intact membranes and is
well correlated with engraftment in postthaw HPC-A grafts.145 Another measurement technique involving the use of Flk-2
and Thy 1.1lo showed that Flk-2–, Thy 1.1lo
cells represented long-term repopulating
lo
cells, whereas Flk-2+, Thy 1.1 cells were
short-term engraftment-enhancing cells.
These tools may be developed for use in future graft engineering approaches to finely
146
separate early cell populations.

Colony-Forming Cell Assays
Culture systems are available that can
demonstrate in-vitro proliferative capacity

604

AABB Technical Manual

of a hematopoietic sample. It is thought
that short-term (generally within 2 weeks)
repopulating potential is produced from
the committed HPCs. Long-term repopulating ability is thought to be a result of
the pluripotential HPCs that are principally necessary for a complete and sustained engraftment. Cultures may be useful to assess engraftment potential; however,
because media, culture techniques, and
colony identification are quite variable,
interinstitutional comparisons are difficult. Reported CFU-GM doses below
which engraftment may be delayed range
from 1.5 × 105 to 5.0 × 105/kg.147
Long-term cultures are not routinely used
clinically because of the 5- to 8-week incubation requirements. However, they may be
useful in evaluating developmental procedures involving product manipulation.

Freezing and Storage
Although easily performed by many transplant centers, HPC cryopreservation and
reinfusion are not without risk of loss of
HPCs. The infusion of cryopreserved cells
also results in risks to the recipient, many
of which are life-threatening, and some of
which are not well understood. The loss of
HPCs from cryopreservation and the effect of this loss on engraftment speed have
not been quantified. There are multiple
aspects to successful cryopreservation of
HPCs. Each of these variables affects the
recovery of HPCs during the cryopreservation steps and, as with any manufacturing
process, must be rigidly controlled for reproducible results. The question that still
faces cryopreservation facilities is whether
other solutions or processing techniques
will provide better cryosurvival of cells, at
less cost, with greater simplicity, or with
less toxicity to either the recipient or to
the cell type being frozen.

HPC-A, HPC-M, and HPC-C are frozen
and stored using the same techniques. The
general parameters include cryopreservation
in dimethylsulfoxide (DMSO) and a source
of plasma protein with or without hydroxyethyl starch (HES), cooling at 1 to 3 C/
minute, and storage at –80 C or colder. Variations on this technique include the concentration at which the cells are frozen, the
amount and source of the plasma protein,
148
and the cooling techniques used. Most of
these variations probably have little effect
on the survival of the HPCs as shown by the
consistent engraftment of cryopreserved
components. However, cryopreservation
results in the loss of an undefined but potentially substantial proportion of HPCs, and
delay in engraftment can occur if the component being frozen has borderline quantities of HPCs. There is also considerable but
generally minor toxicity associated with the
infusion of cryopreserved cells that must be
considered when developing cryopreservation techniques.

Allogeneic Products
The collection of allogeneic marrow is
generally timed to coincide with the completion of the recipient’s preparative regimen. Because of the brief storage period
required, the product is maintained in the
liquid state. Unseparated HPC-M can be
stored in the liquid state for up to 3 days
at either 4 C or 22 C without any significant loss in viability of either uncommitted or committed progenitors. The ability
to store unmanipulated marrow under
these conditions is vital in the context of
the NMDP or other transplant registries
where unrelated HPC-M units are collected and then transported great distances for transplantation. Similarly,
HPC-A units may be stored in the liquid
state under the same conditions as HPC-M
149,150
The cell confor up to 3 days at 4 C.

Chapter 25: Cell Therapy and Cellular Product Transplantation

centration is important in determining
the storage time and temperature that the
cells will tolerate without damage. High
concentrations of white cells in liquid
storage at room temperature quickly expend the supplies of oxygen and glucose
and cause a decrease in pH in the storage
medium that is toxic to the cells. A lower
concentration of cells (higher volume of
suspending medium, often autologous
plasma) and refrigerated storage are protective.

Autologous Products
Because of the length of time required for
treatment and/or HPC product collection,
autologous products are usually cryopreserved and stored until the time of infusion. Cryopreservation also allows HPCs to
be collected while the patient is in remission and stored for use in case a relapse
occurs (prophylactic storage). At present,
no expiration date has been defined for
these products; however, HPC-M stored
for 11 years has been used for transplantation, with sustained engraftment.151

Computer-Controlled Cryopreservation
The purpose of cryopreservation is to freeze
cells in such a way as to allow for their
long-term storage with a minimal loss of
cell viability or reconstitutive ability upon
thaw. The main obstacle to maintaining
viability during cryopreservation and
storage is the formation of intracellular
ice crystals along with an increase in external osmolarity, causing exit of water from
the cell, both resulting in cell lysis. Thermal shock, the time required for phase
change (liquid state to solid state), and
the posttransition freezing rate also present specific conditions to be managed for
cryopreservation.152
The adverse effects caused by the formation of intracellular ice crystals or by cell

605

dehydration can be minimized by a slow
cooling rate and the addition of a cryoprotective agent such as DMSO. Cryoprotectants such as DMSO prevent the formation of large ice crystals within the cells
by penetrating the cell and providing some
balance of the external hyperosmolar solute conditions in the freezing medium surrounding the cell. 153 A commonly used
cryoprotectant consists of 20% DMSO and
20% plasma or albumin prepared in a buffered electrolyte solution or tissue culture
media. The plasma or albumin provides a
protein source, which aids in preventing cell
damage during freezing and thawing. The
cryoprotectant is combined with an equal
volume of product immediately before
freezing, resulting in a final cell suspension
containing 10% DMSO and 10% plasma.
Phase transition time is a physical phenomenon of freezing water where forming
ice crystals release heat energy into the cell
solution that needs to be offset by continued cooling to prevent crystallization of ice,
thawing, and recrystallization during this
energy exchange, with damage to the cell
membrane.152 This method of freezing requires physical conditions that support this
process. This may be accomplished by a
computerized programmable freezing
chamber that adds liquid nitrogen in sufficient amounts to push the freezing process
through the phase change smoothly. The
freezer is programmed to cool cells at an
optimal rate of 1 to 3 C/minute until a temperature of –90 to –100 C is reached.154-156 With
the combination of a cryoprotectant and a
programmed rate of freezing, cryopreservation and long-term storage of HPCs are
possible, with minimal damage to the cells.

Passive Controlled-Rate Freezing
Mechanical, methanol bath immersion,
or “passive controlled-rate” freezing is advocated by some investigators as a simple,

606

AABB Technical Manual

reliable, cost-effective, and validated
method of managing the physical conditions of cooling as an alternative to a controlled-rate device.157-159 The principle is
that products can be cooled without the
aid of a programmable freezer or liquid
nitrogen if the conditions of freezing are
predetermined, measured, and performed
according to standard operating procedures. The products are stored in a –80 C
mechanical freezer or colder after the initial freeze. HPCs stored in this fashion
have successfully engrafted after as long
as 2 years of storage.159 In some cases, the
combination of the metal canisters the
bags are placed in, the bag itself, and the
volume of product frozen produces a
freezing rate of approximately 3 C/minute, which falls within the optimal range
previously discussed.160 The major deterrent to use of mechanical freezers is fear
of mechanical failure and the lack of data
on long-term storage and engraftment.

Frozen Storage
Although products frozen in a mechanical
freezer are stored at –80 to –150 C, products cryopreserved using a programmable
freezer generally are stored in a liquid nitrogen freezer. The storage temperature
achieved with vapor phase, although not
as cold as liquid phase, averages –140 C, a
temperature that has been shown to allow
for viable long-term marrow storage.151
The major drawbacks to vapor phase storage include the potential for large fluctuation in temperature when the freezer is
entered as well as the variation in storage
temperature throughout the freezer itself,
dependent on the design of the storage
chamber and the procedure for entering
and retrieving units. The time to rising
temperatures of the stored products in
cases of electrical or liquid nitrogen supply emergencies is significantly shorter

than with liquid phase. The advantage of
vapor phase storage is the possibly decreased risk of cross-contamination that
has become an issue with liquid phase
storage.161

Storage of Untested or Infectious Products
Regulations [21 CFR 1271.60(a) and
1271.65(a)] and standards48(p35),49 require alternative storage for products that are untested or have a positive disease marker
test result. Some institutions comply by
storing all products in vapor phase and
physical separation by category of product. Other institutions use an overwrap,
placing the component in an outer plastic
bag that is sealed before storage. A third
alternative is a separate storage compartment for units that are untested or positive for infectious disease markers.

Final Suitability Criteria
Before each autologous or allogeneic unit
is released, it must undergo review and
meet the predetermined release criteria
described in the facility’s quality plan.
The pertinent facts describing donor selection, product collection, processing,
and storage must be reviewed. The container label and associated information
must accurately reflect the product classification, storage/preservative medium in
the final container, the product content
(usually cell content), and results of release testing including infectious disease
tests. If exceptions to standard practice
were made, they must be explained either
on the label or in the accompanying release material.

Transportation and Shipping
In some cases, a hematopoietic component
must be transported from one center to
another. The product must be positively

Chapter 25: Cell Therapy and Cellular Product Transplantation

identified upon its removal from inventory in preparation for shipping. In all
cases, precautions should be taken to protect the component from rough handling,
out-of-range temperatures, X-ray examination, breakage, and spillage. The shipping container must undergo quality control to ensure that it is capable of holding
the expected temperature during shipping. In the case of cryopreserved components, the use of a liquid nitrogen “dry
shipper” is desirable. Such “dry shippers”
have liquid nitrogen absorbent material
between the walls of the container that allows the inside of the container to maintain temperatures in the range of –180 C
for up to 10 to 14 days if they are properly
filled with liquid nitrogen and shipped in
the upright position. Tipping or inversion
of the container during shipping permits
the liquid nitrogen to drain out, allowing
the container to warm toward ambient
temperature.

Thawing and Infusion
For all components, final identification is
done by the nurse or physician performing the infusion. Flow through the central
venous catheter is confirmed and the cells
are infused by gravity drip, calibrated
pump, or manual push with or without an
in-line filter (a standard 170-micron red
cell infusion filter is acceptable). Although
DMSO was thought to be toxic to HPCs, it
is now known to be nontoxic after shortterm exposure (up to 1 hour) at the concentration used for cryopreservation of
HPCs.162 However, prolonged exposure to
DMSO ex vivo at 22 to 37 C may be harmful to HPCs. To minimize the exposure of
thawed cells to DMSO, many centers rapidly thaw one bag at a time near the bedside. Some centers place product bags in
secondary containment bags before thaw-

607

ing; others immerse the bag (all but the
access ports) directly into sterile water or
saline at 37 to 40 C.152 The bag is kneaded
gently until all solid clumps have thawed.
The cells are then infused (usually 10-15
mL per minute). Products may also be
washed and resuspended in the laboratory
before infusion to prevent cell aggregation.152
Side effects associated with infusions include nausea, diarrhea, flushing, bradycardia, hypertension, and abdominal pain.
In general, such side effects may justify
slowing, but not halting, the infusion until
the symptoms pass. A limit of 1 gram of
DMSO per kilogram of body weight of the
patient at one infusion is recommended to
allow the patient to tolerate both the DMSO
and the volume infusion effects of the administration. Sudden and severe hypotension can occur in the absence of adequate
antihistamine premedication. The patient
should receive fluids and treatment to ensure that the urine is “alkalinized.” This facilitates the clearance of hemoglobin caused
by red cell lysis, which occurs during freezing and reduces the risk of renal complications. If the total infusion volume exceeds
10 mL/kg of recipient body weight, many
centers divide the volume over a morning
and an afternoon infusion or over 2 consecutive days.
It is best to collect postthaw laboratory
samples directly from the patient’s infusion
bags instead of freezing separate individual
specimens because these samples are identical to the infused product.163

Evaluation and Quality Control
of Hematopoietic Products
Cell Counts
Each hematopoietic product is analyzed
to determine the total cell concentration
and the mononuclear cell concentration,

608

AABB Technical Manual

which are used to calculate the number of
cells per kilogram (of the recipient) or cell
dose for each product.48(p55),49 These doses,
in combination with other assays, determine the number of collections necessary
to achieve engraftment. 135 In addition,
they are used to calculate the percent recovery, providing a quality control measure for processing procedures and
equipment.164
In general, automated cell counts provide
the most rapid and accurate value. However, platelet/cellular aggregates in HPC-A
or fat globules in HPC-M specimens can
falsely decrease or increase cell counts.135,165
In such cases, manual cell counts may be
preferable. It is important that cell counts
are not overestimated because this may result in inaccurate estimation of time to
engraftment or graft failure.

Engraftment Data
Ultimately, engraftment of neutrophils,
platelets, and red cells is the primary determinant of graft quality. Monitoring and
documenting days to engraftment for
neutrophil and platelet lineage are required by FACT49 and AABB48(p41) for accreditation.

Tumor Cell Detection
Tumor cell detection techniques have been
developed to screen products suspected
of tumor cell contamination and to evaluate purged products. The majority of these
assays use MoAbs that specifically bind
tumor antigens. Detection and quantitation can be done by flow cytometry, immunofluorescence, or immunohistochemical
staining. Sensitivity varies with technique
from 0.1% to 0.0004% of cells examined.166
Preliminary studies indicate that the pres-

ence of tumor cells may be associated
167
with a reduced disease-free survival.

Regulations
In 1997, the FDA announced a new comprehensive approach to the regulation of
cellular and tissue-based products. 1 6 8
HPCs from placental/umbilical cord
blood and peripheral blood were covered
by this proposal. Many of the policies,
proposed regulations, and guidance documents needed to implement this ap47,169-173
proach have been published.
These regulations require establishment
registration and listing of facilities collecting, processing, or distributing tissue or cell
therapy products. Although the regulations
are similar to those for blood donor qualification, there are differences appropriate to
the specific tissue source under consideration. The cGTP regulations (found in Title
21 CFR 1271.145 to 320) are specific instructions intended to ensure that facilities
establish and maintain a quality program
that documents that personnel, procedures, facilities, equipment, supplies, and
reagents are set up and maintained in an
acceptable and a standard manner. Process
control is required: there must be written
procedures and documented validation.
Products are required to be collected,
tested, labeled, and stored in ways that preserve their identity and prevent contamination and cross-contamination. It may be
necessary to demonstrate regulatory compliance during FDA inspection visits. Reporting of adverse events (when applicable)
is also required within 15 days of receipt of
the information if the event is serious as defined in 21 CFR 1271.350. This body of regulations represents a major effort on the part
of the FDA to ensure that tissue and cell
therapy products are safe for the recipient.

Chapter 25: Cell Therapy and Cellular Product Transplantation

Standards
The AABB and FACT have published separate, but substantially similar, standards
for HPCs.48,49 The AABB Standards for Cellular Therapy Product Services addresses
the collection, processing, storage, and
distribution of HPCs. Other organizations
such as the NMDP also publish voluntary
standards. FACT standards address clinical issues provided by HPC clinical transplant programs as well as laboratory services performing the collection, processing,
storage, and distribution of HPCs. Both
AABB and FACT provide an inspection
and certification program for requesting
members. These program reviews are an
important, external, and impartial look at
program organization and how the program’s organization affects patient outcome. As an outgrowth of the standards
writing process, representatives from the
constituent organizations of AABB and
FACT cooperated to produce a uniformly
endorsed Circular of Information for the
Use of Cellular Therapy Products.174 In this
circular, the organizations agreed upon a
number of principles, including a common set of names for cell therapy products. This agreement on product names
allowed the International Committee on
Commonality of Blood Bank Automation’s
(ICCBBA) North American Task Force to
approve progenitor cell label designs, including ICCBBA’s quality design requirements.

References

10.

11.

12.

13.

1. Korbling M, Estrov Z. Adult stem cells for tissue repair—a new therapeutic concept? N
Engl J Med 2003;349:570-82.
2. Champlin RE, Schmitz N, Horowitz MM, et
al. Blood stem cells compared with bone
marrow as a source of hematopoietic cells
for allogeneic transplantation. IBMTR Histocompatibility and Stem Cell Sources Work-

14.

609

ing Committee and the European Group for
Blood and Marrow Transplantation (EBMT).
Blood 2000;95:3702-9.
Report on state of the art in blood and marrow transplantation—the IBMTR/ABMTR
summary slides with guide (2002). IBMTR/
ABMTR Newsletter 2002;9(1):4-11. [Available
at http://www.ibmtr.org/newsletter/pdf/
2002Feb.pdf.]
Champlin R, Khouri I, Anderlini P, et al. Nonmyeloablative preparative regimens for allogeneic hematopoietic transplantation. Biology
and current indications. Oncology (Hunting), 2003;17:94-100.
Sandmaier BM, McSweeney P, Yu C, Storb R.
Nonmyeloablative transplants: Preclinical
and clinical results. Semin Oncol 2000;27:
78-81.
Storb RF, Champlin R, Riddell SR, et al. Nonmyeloablative transplants for malignant disease. Hematology (Am Soc Hematol Educ
Program) 2001;375-91.
Giralt S, Anagnostopoulos A, Shahjahan M,
et al. Nonablative stem cell transplantation
for older patients with acute leukemias and
myelodysplastic syndromes. Semin Hematol
2002;39:57-62.
To LB, Haylock DN, Simmons PJ, Juttner CA.
The biology and clinical uses of blood stem
cells. Blood 1997;89:2233-58.
National Marrow Donor Program Overview
(2001 slide presentation). [Available at http://
www.nmdp.org/nmdp/slide_presentation.
html.]
Powderly J, Shea T. Hematopoietic cell transplantation. In: Runge MS, Greganti MA, eds.
Netter’s internal medicine. Teterboro, NJ:
Icon Learning Systems, 2003:401-05.
Stroncek DF, Confer DL, Leitman SF. Peripheral blood progenitor cells for HPC transplants involving unrelated donors. Transfusion 2000;40:731-41.
Bensinger WI, Martin PJ, Storer B, et al.
Transplantation of bone marrow as compared with peripheral-blood stem cells from
HLA-identical relatives in patients with
hematologic malignancy. N Engl J Med 2001;
344:175-81.
Beatty PG, Kollman C, Howe CW. Unrelateddonor marrow transplants: The experience
of the National Marrow Donor Program. Clin
Transpl 1995:271-7.
Scott I, O’Shea J, Bruce M, et al. Molecular
typing shows a high level of HLA class I incompatibility in serologically well matched
donor/patient pairs: Implications for unrelated bone marrow donor selection. Blood
1998;92:4864-71.

610

AABB Technical Manual

15. Petersdorf EW, Gooley TA, Anasetti C, et al.
Optimizing outcome after unrelated marrow
transplantation by comprehensive matching
of HLA class I and II alleles in the donor and
recipient. Blood 1998;92:3515-20.
16. Petersdorf EW, Longton GM, Anasetti C, et al.
Definition of HLA-DQ as a transplantation
antigen. Proc Natl Acad Sci U S A 1996;93:
15358-63.
17. Gajewski J, Gjertson D, Cecka M, et al. The
impact of T-cell depletion on the effects of
HLA DR beta 1 and DQ beta allele matching
in HLA serologically identical unrelated bone
marrow transplantation. Biol Blood Marrow
Transplant 1997;3:76-82.
18. Petersdorf EW, Longton GM, Anasetti C, et al.
Association of HLA-C disparity with graft failure after marrow transplantation from unrelated donors. Blood 1997;89:1818-23.
19. Kanfer E. Graft-versus-host disease. In:
Treleaven J, Wiernik P, eds. Color atlas and
text of bone marrow transplantation. London: Mosby-Wolfe, 1995:185-8.
20. Kolb HJ, Schattenberg A, Goldman JM, et al.
Graft-versus-leukemia effect of donor lymphocyte transfusion in marrow grafted patients. Blood 1995;86:2041-50.
21. Champlin R. Bone marrow transplantation
from HLA-matched unrelated donors as treatment for leukemia. J Hematother 1993;2:
323-7.
22. Champlin R, Kyoung L. T-cell depletion to
prevent graft-versus-host disease following
allogeneic bone marrow transplantation. In:
Areman EM, Deeg HJ, Sacher RA, eds. Bone
marrow and stem cell processing: A manual
of current techniques. Philadelphia: FA Davis, 1992:163-217.
23. Apisarnthanarax N, Donato M, Korbling M, et
al. Extracorporeal photopheresis therapy in
the management of steroid-refractory or steroid-dependent cutaneous chronic graft-versus-host disease after allogeneic stem cell
transplantation: Feasibility and results. Bone
Marrow Transplant 2003;31:459-65.
24. Chan KW, Wadsworth LD. Pediatric bone
marrow transplantation and processing. In:
Areman EM, Deeg HJ, Sacher RA, eds. Bone
marrow and stem cell processing: A manual
of current techniques. Philadelphia: FA Davis, 1992:363-85.
25. Stroncek D, Anderlini P. Mobilized PBPC concentrates: A maturing blood component (editorial). Transfusion 2001;41:168-71.
26. Bensinger WI, Clift R, Martin P, et al. Allogeneic peripheral blood stem cell transplantation in patients with advanced hematologic
malignancies: A retrospective comparison

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

with marrow transplantation. Blood 1996;88:
2794-800.
Ottinger HD, Beelen DW, Scheulen B, et al.
Improved immune reconstitution after allotransplantation of peripheral blood stem
cells instead of bone marrow. Blood 1996;88:
2775-9.
Storek J, Gooley T, Siadak M, et al. Allogeneic
peripheral blood stem cell transplantation
may be associated with a high risk of chronic
graft-versus-host disease. Blood 1997;90:
4705-9.
Broxmeyer HE, Gluckman E, Auerbach AD, et
al. Human umbilical cord blood: A clinically
useful source of transplantable hematopoietic stem/progenitor cells. Int J Cell Cloning
1990;8:76.
Gluckman E, Broxmeyer HE, Auerbach AD, et
al. Hematopoietic reconstitution in a patient
with Fanconi anemia by means of umbilical-cord blood from an HLA-identical sibling. N Engl J Med 1989;321:1174-8.
Rocha V, Wagner JE, Sobocinski KA, et al.
Graft-versus-host disease in children who
have received a cord-blood or bone marrow
transplant from an HLA-identical sibling. N
Engl J Med 2000;342:1846-54.
Laughlin MJ, Barker J, Bambach B, et al.
Hematopoietic engraftment and survival in
adult recipients of umbilical-cord blood from
u n r e l a t e d d o n o r s. N E n g l J Me d 2 0 0 1 ;
344:1815-22.
Wagner J, Barker J, DeFor T, et al. Transplantation of unrelated donor umbilical cord
blood in 102 patients with malignant and
non-malignant diseases: Influence of CD34
cell dose and HLA disparity on treatment-related mortality and survival. Blood 2002;
100:1611-18.
Rubinstein R, Carrier C, Scaradavou A, et al.
Outcomes among 562 recipients of placental-blood transplants from unrelated donors.
N Engl J Med 1998;339:1565-77.
Wagner JE, Kernan NA, Steinbuch M, et al.
Allogeneic sibling umbilical-cord-blood
transplantation in children with malignant
and non-malignant disease. Lancet 1995;
346:214-9.
Locatelli F, Rocha V, Chastang C, et al. Factors
associated with outcome after cord blood
transplantation in children with acute leukemia. Eurocord-Cord Blood Transplant Group.
Blood 1999;93:3662-71.
Rocha V, Cornish J, Sievers EL, et al. Comparison of outcomes of unrelated bone marrow
and umbilical cord blood transplants in children with acute leukemia. Blood 2001;97:
2962-71.

Chapter 25: Cell Therapy and Cellular Product Transplantation

38. Laughlin MJ, Eapen M, Rubinstein P, et al.
Outcomes after transplantation of cord blood
or bone marrow from unrelated donors in
adults with leukemia. N Engl J Med 2004;351:
2265-75.
39. Rocha V, Labopin M, Sanz G, et al. Transplants
of umbilical-cord blood or bone marrow
from unrelated donors in adults with acute
leukemia. N Engl J Med 2004;351:2276-85.
40. Barker JN, Weisdorf DJ, DeFor TE, et al. Transplantation of two partially HLA-matched
umbilical cord blood units to enhance engraftment in adults with hematologic malignancy.
Blood 2005;105:1343-7.
41. Mehta SN. Umbilical-blood storage: Uncertain benefits, high price. The Wall Street
Journal 1995 Nov 14;Sect B:1-2.
42. Rubin R. A hard sell to bank your baby’s
blood. US News & World Report 1996 July
29:60-1.
43. Ferreira E, Pasternak J, Bacal N, et al. Autologous cord blood transplantation (letter). Bone
Marrow Transplant 1999;24:1041.
44. Flowers MED, Pepe MS, Longton G, et al.
Previous donor pregnancy as a risk factor for
acute graft-versus-host disease in patients
with aplastic anemia treated by allogeneic
marrow transplantation. Br J Haematol 1990;
74:492-6.
45. Gale RP, Bortin MM, Van Bekkum DW, et al.
Risk factors for acute graft-versus-host disease. Br J Haematol 1987;67:397-406.
46. Bensinger WI, Deeg JH. Transfusion support
and donor considerations in marrow transplantation. In: Sacher RA, AuBuchon JP, eds.
Marrow transplantation: Practical and technical aspects of stem cell reconstitution.
Bethesda, MD: AABB, 1992:157-84.
47. Food and Drug Administration. Eligibility
determination for donors of human cells, tissues, and cellular and tissue-based products;
final rule (May 25, 2004). Fed Regist 2004;69:
29786-834.
48. Szczepiorkowski ZM, ed. Standards for cellular therapy product services. Bethesda, MD:
AABB, 2004.
49. Warkentin PI, ed. Standards for hematopoietic progenitor cell collection, processing
and transplantation. 2nd ed. Omaha, NE:
Foundation for the Accreditation of Cellular
Therapy, 2002.
50. Mair DC, Brecher ME, Hom E, et al. Falsepositive hepatitis B surface antigen screening test results in patients receiving granulocyte-colony-stimulating factor. Transfusion
1996;36:948-51.
51. Kaufman RM, Goodnough LT, Lublin DM, et
al. False-positive hepatitis C screening tests
results in healthy allogeneic peripheral blood

52.

53.

54.

55.

56.

57.

58.

59.
60.

61.

62.

63.

64.

611

stem cell donors (abstract). Transfusion 2000;
40(Suppl):S34.
International standards for cord blood collection, processing, testing, banking, selection and release. 2nd ed. Omaha, NE: Foundation for the Accreditation of Cellular
Therapy, 2001.
Treleaven JG. Bone marrow harvesting and
reinfusion. In: Gee AP, ed. Bone marrow processing and purging. Boca Raton, FL: CRC
Press, 1991:32-4.
Lane TA. Mobilization of hematopoietic progenitor cells. In: Brecher ME, Lasky LC,
Sacher RA, Issitt LA, eds. Hematopoietic progenitor cells: Processing, standards and
practice. Bethesda, MD: AABB, 1995:59-108.
Krause DS, Fackler MJ, Civin CI, et al. CD34:
Structure, biology, and clinical utility. Blood
1996;87:1-13.
Sutherland DR, Anderson L, Keeney M, et al.
The ISHAGE guidelines for CD34+ cell determination by flow cytometry. J Hematother
1996;5:213-26.
Brecher ME, Sims L, Schmitz J, et al. North
American multicenter study on flow cytometr ic enumeration of CD34+ hematopoietic stem cells. J Hematother 1996;5:22736.
Lie AK, Rawlings TP, Bayly JL, To LB. Progenitor cell yield in sequential blood stem cell
mobilization in the same patients: Insights
into chemotherapy dose escalation and
combination of haemopoietic growth factor
and chemotherapy. Br J Haematol 1996;95:
39-44.
Leitman SF, Read SJ. Hematopoietic progenitor cells. Semin Hematol 1996;33:341-58.
Schwella N, Beyer J, Schwaner I, et al. Impact
of preleukapheresis cell counts on collection
results and correlation of progenitor-cell
dose with engraftment after high-dose chemotherapy in patients with germ cell cancer.
Blood 1994;83:3787-94.
Elliot C, Samson DM, Armitage S, et al. When
to harvest peripheral blood stem cells after
mobilization therapy: Prediction of CD34
positive cell yield by preceding day CD34
positive concentration cell in peripheral
blood. J Clin Oncol 1996;14:970-3.
Owen HG, Brecher ME. Paired randomized
comparative study of PBSC collection (abstract). J Clin Apheresis 1997;12:29.
Bandarenko N, Sims LC, Brecher ME. Circulating CD34+ counts are predictive of PBSC
CD34+ yields (letter). Transfusion 1997;37:
1218.
Perez-Simon JA, Caballero MD, Corral M, et
al. Minimum number of circulating CD34+
cells to ensure successful leukapheresis and

612

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

AABB Technical Manual

engraftment in autologous peripheral blood
progenitor cell transplantation. Transfusion
1998;38:385-91.
Green RHA, Morrison AE, Watson D, et al.
The use of peripheral blood CD34 measurements in determining the optimal timing of
peripheral blood progenitor cell (PBPC) collections (abstract). J Clin Apheresis 1997;12:
47.
Yu J, Leisenring W, Bensinger WI, et al. The
predictive value of white cell or CD34+ cell
count in the peripheral blood for timing
apheresis and maximizing yield. Transfusion
1999;39:442-50.
Shpall EJ, Champlin R, Glaspy JA. Effect of
CD34+ peripheral blood progenitor cell dose
on hematopoietic recovery. Biol Blood Marrow Transplant 1998;4:84-92.
Schulman KA, Birch R, Zhen B, et al. Effect of
CD34+ cell dose on resource utilization in patients after high-dose chemotherapy with peripheral-blood stem-cell support. J Clin
Oncol 1999;17:1227.
Bensinger W, Appelbaum F, Rowley S, et al.
Factors that influence collection and engraftment of autologous peripheral-blood stem
cells. J Clin Oncol 1995;13:2547-55.
Malachawski ME, Comenzo RL, Hillyer CD, et
al. Large-volume leukapheresis for peripheral blood stem cell collection in patients
with hematologic malignancies. Transfusion
1992;32:732-5.
Passos-Coelho JL, Braine HG, Davis JM, et al.
Predictive factors for peripheral-blood progenitor-cell collections using a single largevolume leukapheresis after cyclophosphamide and granulocyte-macrophage colony-stimulating factor mobilization. J Clin
Oncol 1995;13:705-14.
Hillyer CD, Lackey DA, Hart KK, et al. CD34+
progenitors and colony-forming units-granulocyte macrophage are recruited during
large-volume leukapheresis and concentrated by counterflow centrifugal elutriation.
Transfusion 1993;33:316-21.
Hillyer CD. Large volume leukapheresis to
maximize peripheral blood stem cell collection. J Hematother 1993;2:529-32.
Glaspy JA, Shpall EJ, LeMaistre CF, et al. Peripheral blood progenitor cell mobilization
using stem cell factor in combination with
filgrastim in breast cancer patients. Blood
1997;90:2939-51.
Scheid C, Draube A, Reiser M, et al. Using at
least 5 × 106/kg CD34+ cells for autologous
stem cell transplantation significantly reduces febrile complications and use of antibiotics after transplantation. Bone Marrow
Transplant 1999;23:1177-81.

76. Glaspy JA. Economic considerations in the
use of peripheral blood progenitor cells to
support high-dose chemotherapy. Bone Marrow Transplant 1999;23(Suppl 2):S21-7.
77. Watts MJ, Sullivan AM, Leverett D, et al.
Back-up bone marrow is frequently ineffective in patients with poor peripheral stem cell
mobilization. J Clin Oncol 1998;16:1554-60.
78. Gazitt Y, Shaughnessy P, Liu Q. Differential
mobilization of CD34+ cells and lymphoma
cells in non-Hodgkin’s lymphoma patients
mobilized with different growth factors. J
Hematother Stem Cell Res 2001;10:167-76.
79. Jennis A, Pecora A, Preti R, et al. High dose
G-CSF stem cell mobilization after initial
stem cell mobilization failure (abstract).
Blood 1998;92(Suppl 1):271a.
80. Broxmeyer HE, Hangoc G, Cooper S, Bridger
G. Interference of the SDF-1/CXCR4 axis in
mice with AMD 3100 induces rapid high level
mobilization of hematopoietic progenitor
cells and AMD 3100 acts synergistically with
G-CSF and MIP-1 alpha to mobilize progenitors (abstract). Blood 2001;96(Suppl 1):3371a.
81. Devine S, Flomenberg N, Vesole D, et al.
Safety and effect on WBC count and CD34
cell mobilization following administration of
AMD 3100 to patients with multiple myeloma
or non-Hodgkin’s lymphoma. Presented at
the Eighth Congress of the European Hematology Association, Rotterdam, The Netherlands, July 5-9, 2003.
82. Goldberg SL, Mangan KF, Klumpp TR, et al.
Complications of peripheral blood stem cell
harvesting: Review of 554 PBSC leukaphereses. J Hematother 1995;4:85-90.
83. Stroncek DF, Clay ME, Smith J, et al. Composition of peripheral blood progenitor cell
components collected from healthy donors.
Transfusion 1997;37:411-7.
84. Ilhan O, Arslan O, Arat M, et al. The impact of
the CD34+ cell dose on engraftment in allogeneic peripheral stem cell transplantation.
Transfus Sci 1999;20:69-71.
85. Anderlini P, Rizzo JD, Nugent ML, et al. Peripheral blood stem cell donation: An analysis from the International Bone Marrow
Transplant Registry (IBMTR) and European
Group for Blood and Marrow Transplant
(EBMT) databases. Bone Marrow Transplant
2001;27:689-92.
86. Stroncek DF, Clay ME, Smith J, et al. Changes
in blood counts after the administration of
granulocyte-colony-stimulating factor and
the collection of peripheral blood stem cells
from healthy donors. Transfusion 1996;36:
596-600.
87. Bandarenko N, Brecher ME, Owen H, et al.
Thrombocytopenia in allogeneic peripheral

Chapter 25: Cell Therapy and Cellular Product Transplantation

88.

89.

90.

91.

92.

93.

94.
95.

96.

97.

98.

99.

blood stem cell collections (letter). Transfusion 1996;36:668.
Stroncek DF, Clay ME, Petzoldt ML, et al.
Treatment of normal individuals with granulocyte colony-stimulating factor: Donor experiences and the effects on peripheral blood
CD34+ cell counts and on the collection of
peripheral blood stem cells. Transfusion
1996;36:601-10.
Becker PS, Wagle M, Matous S, et al. Spontaneous splenic rupture following administration of granulocyte colony-stimulating factor (G-CSF): Occurrence in an allogeneic
donor of peripheral blood stem cells. Biol
Blood Marrow Transplant 1997;3:45-9.
Falzetti F, Aversa F, Minelli O, Tabilil A. Spontaneous rupture of spleen during peripheral
blood stem cell mobilization in a healthy donor (letter). Lancet 1999;353:555.
Arbetter KR, Hubbard KW, Markovic SN, et
al. Case of granulocyte colony-stimulating
factor-induced Sweet’s syndrome. Am J
Hematol 1999;61:126-9.
Kawachi Y, Watanabe A, Uchida T, et al.
Acute arterial thrombosis due to platelet aggregation in a patient receiving granulocyte
colony-stimulating factor. Br J Haematol
1996;94:413-6.
Adkins DR. Anaphylactoid reaction in a normal donor given granulocyte colony-stimulating factor (letter). J Clin Oncol 1998;16:
812-3.
Blau CA. Adverse effects of G-CSF in sickle
cell syndromes (letter). Blood 2001;97:3682.
Wei A, Grigg A. Granulocyte colony-stimulating factor-induced sickle cell crisis and
multiorgan dysfunction in a patient with
compound heterozygous sickle cell/β + thalassemia. Blood 2001;97:3998-9.
Rubinstein P, Carrier C, Taylor P, Stevens CE.
Placental and umbilical cord blood banking
for unrelated marrow reconstitution. In:
Brecher ME, Lasky LC, Sacher RA, Issitt LA,
eds. Hematopoietic progenitor cells: Processing, standards and practice. Bethesda,
MD: AABB, 1995:1-17.
Yao AC, Minian M, Lind J. Distribution of
blood between infant and placenta after
birth. Lancet 1969;ii:871-3.
Di Gusto DL, Lee R, Moon J, et al. Hematopoietic potential of cryopreserved and ex
vivo manipulated umbilical cord blood progenitor cells evaluated in vitro and in vivo.
Blood 1996;87:1261-71.
Pecora AL, Stiff P, Jennis A, et al. Prompt and
durable engraftment in two older adult patients with high risk chronic myelogenous
leukemia (CML) using ex vivo expanded and
unmanipulated unrelated umbilical cord

100.

101.

102.

103.

104.

105.

106.

107.

108.

109.

613

blood. Bone Marrow Transplant 2000;25:
797-9.
Kogler G, Nurnberger W, Fuscher J, et al. Simultaneous cord blood transplantation of ex
vivo expanded together with non-expanded
cells for high risk leukemia. Bone Marrow
Transplant 1999;24:397-403.
Silvestri F. CD34+ cell purification by immunomagnetic beads/chymopapain system
from normal and myeloid leukemias bone
marrow and peripheral blood for research
use. In: Wunder E, Sovalat H, Henon P, Serke
S, eds. Hematopoietic stem cells: The Mulhouse manual. Dayton, OH: AlphaMed Press,
1995:161-9.
Gallacher L, Murdoch B, Wu DM, et al. Isolation and characterization of human
CD34 – Lin – and CD34 + Lin – hematopoietic
stem cells using cell surface markers AC133
and CD7. Blood 2000;95:2813-20.
Wu nd er E , d eWynt er E . Pu r i fi c a t i o n o f
CD34+ cells. In: Wunder E, Sovalat H, Henon
P, Serke S, eds. Hematopoietic stem cells:
T h e Mu l h o u s e m a n u a l . D a y t o n , O H :
AlphaMed Press, 1995:125-82.
Marolleau JP, Brice P, Cortivo LD, et al.
CD34+ selection by immunomagnetic selection (Isolex 300) for patients with malignancies (abstract). Blood 1996;88(Suppl 1):110a.
Devernardi N, Camilla C, Traore Y, et al. Antibody-coated magnetic particles for CD34+
cell separation. In: Wunder E, Sovalat H,
Henon P, Serke S, eds. Hematopoietic stem
cells: The Mulhouse manual. Dayton, OH:
AlphaMed Press, 1995:183-200.
Miltenyi S, Guth S, Radbruch A, et al. Isolation of CD34+ hematopoietic progenitor
cells by high-gradient magnetic cell sorting
(MACS). In: Wunder E, Sovalat H, Henon P,
Serke S, eds. Hematopoietic stem cells: The
Mulhouse manual. Dayton, OH: AlphaMed
Press, 1995:201-13.
Wunder E, Herbein G, Sovalat H, et al. Separation of hematopoietic cells of different maturity in mononuclear cell subsets generated
by elutriation. In: Wunder E, Sovalat H,
Henon P, Serke S, eds. Hematopoietic stem
cells: The Mulhouse manual. Dayton, OH:
AlphaMed Press, 1995:271-9.
Brenner MK, Rill DR, Moen RC, et al. Genemarking to trace origin of relapse after autologous bone-marrow transplantation. Lancet 1993;341:85-6.
Ross AA, Cooper BW, Lazarus HM, et al. Detection and viability of tumor cells in peripheral blood stem cell collections from breast
cancer patients using immunocytochemical
and clonogenic assay techniques. Blood
1993;82:2605-10.

614

AABB Technical Manual

110. Brugger W, Bross KJ, Glatt M, et al. Mobilization of tumor cells and hematopoietic progenitor cells into peripheral blood of patients
with solid tumors. Blood 1994;83:636-40.
111. Rowley SD, Davis JM. Purging techniques in
autologous transplantation. In: Areman EM,
Deeg HJ, Sacher RA, eds. Bone marrow and
stem cell processing: A manual of current
techniques. Philadelphia: FA Davis, 1992:21835.
112. Rowley SD, Davis JM. The use of 4-HC in
autologous purging. In: Gee AP, ed. Bone
marrow processing and purging. Boca Raton,
FL: CRC Press, 1991:248-62.
113. Ball ED. Monoclonal antibodies and complement for autologous marrow purging. In: Gee
AP, ed. Bone marrow processing and purging.
Boca Raton, FL: CRC Press, 1991:281-8.
114. Kvalheim G, Wang MY, Pharo A, et al. Purging
of tumor cells from leukapheresis products:
Experimental and clinical aspects. J Hematother 1996;5:427-36.
115. Dreger P, Viehmann K, Steinmann J, et al.
G-CSF mobilized peripheral blood progenitor cells for allogeneic transplantation: Comparison of T cell depletion strategies using
different CD34+ selection systems or
CAMPATH-1. Exp Hematol 1995;23:147-54.
116. van der Straaten HM, Fijnheer R, Dekker AW,
et al. Relationship between graft-versus-host
disease and graft-versus-leukaemia in partial
T cell-depleted bone marrow transplantation. Br J Haematol 2001;114:31-5.
117. Krenger W, Ferrara J. Dysregulation of cytokines during graft-versus-host disease. J
Hematother 1996;5:3-14.
118. Lopez-Plaza I, Triulzi DJ. Transfusion support in HSCT. In: Ball ED, Lister J, Law P, eds.
Hematopoietic stem cell therapy. Philadelphia: Churchill Livingstone, 2000:589-97.
119. Long GD, Blume KG. Allogeneic and autologous bone marrow transplantation. In: Beutler
E, Lichtman MA, Coller BS, Kipps TL, eds.
Williams’ hematology. 5th ed. New York:
McGraw-Hill, 1995:172-94.
120. Worel N, Greinix HT, Schneider B, et al. Regeneration of erythropoiesis after related and
unrelated donor BMT or peripheral blood
HPC transplantation: A major ABO mismatch
means problems. Transfusion 2000;40:54360.
121. Friedberg RC, Andrzejewski C. Transfusion
therapy in hematopoietic stem cell transplantation. In: Mintz PD, ed. Transfusion
therapy: Clinical principles and practice. 2nd
ed. Bethesda, MD: AABB Press, 2005:279-96.
122. Leo A, Mytilineos J, Voso MT, et al. Passenger
lymphocyte syndrome with severe hemolytic
anemia due to an anti-Jk a after allogeneic

123.

124.
125.

126.

127.

128.

129.

130.

131.

132.

133.

134.

135.

PBPC transplantation. Transfusion 2000;40:
632-6.
Salmon JP, Michaux S, Hermanne JP, et al. Delayed massive immune hemolysis mediated
by minor ABO incompatibility after allogeneic peripheral blood progenitor cell transplantation. Transfusion 1999;39:824-7.
Good R. Mixed chimerism and immunological tolerance. N Engl J Med 1993;328:801-2.
Buckner CD, Clift RA, Sanders JE, et al. ABO
incompatible marrow transplants. Transplantation 1978;26:233-8.
Hershko C, Gale RP, Ho W, Fitchen J. ABH antigens and bone marrow transplantation. Br J
Haematol 1980;44:65-73.
Broxmeyer HE, Douglas GW, Hangoc G, et al.
Human umbilical cord blood as a potential
source of transplantable hematopoietic stem/
progenitor cells. Proc Natl Acad Sci U S A
1989;86:3828-32.
Rubinstein P, Dobrila L, Rosenfield RE, et al.
Processing and cryopreservation of placental/umbilical cord blood for unrelated bone
marrow reconstitution. Proc Natl Acad Sci
U S A 1995;92:10119-22.
Webb IJ, Coral FS, Andersen JW, et al. Sources
and sequelae of bacterial contamination of
hematopoietic stem cell components: Implications for the safety of hematotherapy and
gra ft engi neer i ng. Tra nsfu si o n 1996;36:
782-8.
Cohen A, Tepperberg M, Waters-Pick B, et al.
The significance of microbial cultures of the
hematopoietic support for patients receiving
high-dose chemotherapy. J Hematother
1996;5:289-94.
Padley D, Koontz F, Trigg ME, et al. Bacterial
contamination rates following processing of
bone marrow and peripheral blood progenitor cell preparations. Transfusion 1996;36:
53-6.
Lazarus HM, Mogalhaes-Silverman M, Fox
RM, et al. Contamination during in vitro processing of bone marrow for transplantation:
Clinical significance. Bone Marrow Transplant 1991;7:241-6.
Espinosa MT, Fox R, Creger RJ, Lazarus HM.
Microbiologic contamination of peripheral
blood progenitor cells collected for hematopoietic cell transplantation. Transfusion
1996;36:789-93.
Armitage D, Warwick R, Fehily D, et al. Cord
blood banking in London: The first 1000 collections. Bone Marrow Transplant 1999;24:
130-45.
Davis JM, Schepers KG. Quality control of
hematopoietic progenitor cell products. In:
Brecher ME, Lasky LC, Sacher RA, Issitt LA,
eds. Hematopoietic progenitor cells: Process-

Chapter 25: Cell Therapy and Cellular Product Transplantation

136.

137.

138.

139.

140.

141.

142.

143.

144.

145.

146.

147.

ing, standards and practice. Bethesda, MD:
AABB, 1995:159-81.
Siena S, Bregni M, Brando B, et al. Flow
cytometry for clinical estimation of circulating hematopoietic progenitors for autologous transplantation in cancer patients.
Blood 1991;77:400-9.
Roscoe RA, Rybka WB, Winkelstein A, et al.
Enumeration of CD34+ hematopoietic stem
cells for reconstitution following myeloablative therapy. Cytometry 1994;16:74-9.
Johnsen HE. Report from a Nordic Workshop
on CD34+ cell analysis: Technical recommendations for progenitor cell enumeration
in leukapheresis from multiple myeloma patients. J Hematother 1995;4:21-8.
Chang A, Ma DDF. The influence of flow
cytometric gating strategy on the standardization of CD34+ cell quantitation: An Australian multicenter study. J Hematother 1996;
5:605-16.
Säberlich S, Kirsch A, Serke S. Determination
of CD34+ hematopoietic cells by multiparameter flow cytometry: Technical remarks. In: Wunder E, Sovalat H, Hénon P,
Serke S, eds. Hematopoietic stem cells: The
Mulhouse manual. Dayton, OH: AlphaMed
Press, 1994:45-60.
Johnsen HE. Toward a worldwide standard for
CD34+ enumeration? (letter) J Hematother
1997;6:83-4.
Sims LC, Brecher ME, Gertis K, et al. Enumeration of CD34 positive stem cells: Evaluation and comparison of three methods. J
Hematother 1997;6:213-26.
Storms RW, Trujillo AP, Springer JB, et al. Isolation of primitive human hematopoietic
progenitors on the basis of aldehyde dehydrogenase activity. Proc Natl Acad Sci
U S A 1999;96:9118-23.
Hess DA, Meyerrose TE, Wirthlin L, et al.
Functional characterization of highly purified
human hematopoietic repopulating cells
isolated according to aldehyde dehydrogenase activity. Blood 2004;104:1648-55.
Fallon P, Gentry T, Balber AE, et al. Mobilized
peripheral blood SSClo ALDHbr cells have the
phenotypic and functional properties of
primitive hematopoietic progenitor cells and
their number correlates with engraftment
following autologous transplantation. Br J
Haematol 2003;122:99-108.
Christensen JL, Weissman IL. Flk-2 is a
marker in hematopoietic stem cell differentiation: A simple method to isolate long-term
stem cells. Proc Natl Acad Sci U S A 2001;98:
14541-6.
Wunder E, Sovalat H, Henon P, Serke S, eds.
Hematopoietic stem cells: The Mulhouse

148.

149.

150.

151.

152.

153.
154.

155.

156.

157.

158.

159.

160.

161.

615

Manual; Dayton, OH: AlphaMed Press, 1995:
126-82.
Moroff G, Seetharman S, Kurtz JW, et al. Retention of cellular properties of PBPCs following liquid storage and cryopreservation.
Transfusion 2004;44:245-52.
Hechler G, Weide R, Heymanns J, et al. Storage of noncryopreserved peripheral blood
stem cells for transplantation. Ann Hematol
1996;72:303-6.
Sugrue SR, Hubel AH, McCullough J, et al.
The effect of overnight storage of leukapheresis stem cell products on cell viability,
recovery and cost. J Hematother 1998;7:431-6.
Aird W, Laborpin M, Gorin NC, Anten JH.
Long term cryopreservation of human stem
cells. Bone Marrow Transplant 1992;9:48790.
Gorin NC. Cryopreservation and storage of
stem cells. In: Areman EM, Deeg JH, Sacher
RA, eds. Bone marrow and stem cell processing: A manual of current techniques. Philadelphia: FA Davis, 1992:138-41.
Meryman HT. Cryoprotective agents. Cryobiology 1971;8:173-83.
Leibo SP, Farrant J, Mazur P, et al. Effects of
freezing on marrow stem cell suspensions:
Interactions of cooling and warming rates in
the presence of PVP, sucrose or glycerol.
Cryobiology 1970;6:15-32.
Mazur P. Theoretical and experimental effects of cooling and warming velocity in the
survival of frozen and thawed cells. Cryobiology 1966;2:181-92.
Lewis JP, Passovoy M, Trobaugh FE. The effect of cooling regimens on the transplantat i o n p o t e n t i a l o f m a r r ow. Tr a n s f u s i o n
1967;7:17-32.
Hernandez-Navarro F, Ojeda E, Arrieta R, et
al. Hematopoietic cell transplantation using
plasma and DMSO without HES, with nonprogrammed freezing by immersion in a
methanol bath: Results in 213 cases. Bone
Marrow Transplant 1998;21:511-7.
Galmes A, Besalduch J, Bargay J, et al. Cryopreservation of hematopoietic progenitor
cells with 5-percent dimethyl sulfoxide at
–80 degrees C without rate-controlled freezing. Transfusion 1996;36:794-7.
Stiff PJ, Murgo AJ, Zaroules CG, et al. A simplified bone marrow cryopreservation method.
Blood 1988;71:1102-3.
Stiff PJ, Murgo AJ, Zaroules CG, et al. Unfractionated marrow cell cryopreservation
using dimethylsulfoxide and hydroxyethyl
starch. Cryobiology 1983;21:17-21.
Tedder RS, Zuckerman MA, Goldstone AH, et
al. Hepatitis B transmission from contami-

616

162.

163.

164.

165.

166.

167.

168.

169.

AABB Technical Manual

nated cryopreservation tank. Lancet 1995;
346:137-40.
Rowley SD, Anderson GL. Effect of DMSO exposure without cryopreservation on hematopoietic progenitor cells. Bone Marrow Transplant 1993;11:389-93.
Gee AP. Quality control in bone marrow processing. In: Gee AP, ed. Bone marrow processing and purging: A practical guide. Boca
Raton, FL: CRC Press, 1991:19-27.
Lasky LC, Johnson NL. Quality assurance in
marrow processing. In: Areman EM, Deeg HJ,
Sacher RA, eds. Bone marrow and stem cell
processing: A manual of current techniques.
Philadelphia: FA Davis, 1992:386-443.
Bentley SA, Taylor MA, Killian DE, et al. Correction of bone marrow nucleated cell counts
for the presence of fat particles. Am J Clin
Pathol 1995;140:60-4.
Moss TJ. Detection of metastatic tumor cells
in bone marrow. In: Gee AP, ed. Bone marrow
processing and purging: A practical guide.
Boca Raton, FL: CRC Press, 1991:121-35.
Pecora AL, Lazarus EM, Cooper B, et al.
Breast cancer contamination in peripheral
blood cell (PBPC) collections association
with bone marrow disease and type of mobilization (abstract). Blood 1997;89(Suppl):99a.
Food and Drug Administration. A proposed
approach to the regulation of cellular and tissue-based products (March 4, 1997). Fed
Regist 1997;62:9721-2.
Food and Drug Administration. Human tissue
intended for transplantation; final rule, 21

170.

171.

172.

173.

174.

CFR 16 and 1270 ( July 29, 1997). Fed Regist
1997;62:40429-47.
Food and Drug Administration. Guidance for
industry: Screening and testing of donors of
human tissue intended for transplantation.
( July 29, 1997) Rockville, MD: CBER Office of
Communication, Training, and Manufacturers Assistance, 1997.
Food and Drug Administration. Draft guidance for industry: Preventive measures to reduce possible risk of transmission of Creutzf e l d t - Ja k o b d i s e a s e ( C J D ) a n d v a r i a n t
Creutzfeldt-Jakob disease (vCJD) by human
cells, tissues, and cellular and tissue-based
products (HCT/Ps) ( June 25, 2002). Fed
Regist 2002;67:42789-90.
Food and Drug Administration. Request for
proposed standards for unrelated allogeneic
peripheral and placental/umbilical cord
blood hematopoietic stem/progenitor cell
products; request for comments ( January 20,
1998). Fed Regist 1998;63:2985-8.
Food and Drug Administration. Current good
tissue practice for human cell, tissue, and
cellular and tissue-based product establishments; inspection and enforcement (November 24, 2004). Fed Regist 2004;69:68612-88.
AABB, American Red Cross, America’s Blood
Centers, American Society for Blood and
Marrow Transplantation, Foundation for the
Accreditation of Cellular Therapy, International Society for Cellular Therapy, National
Marrow Donor Program. Circular of information for the use of cellular therapy products.
Bethesda, MD: AABB, 2003.

Chapter 26: Tissue and Organ Transplantation

Chapter 26

Tissue and Organ
Transplantation

N RECENT YEARS, the numbers of
cornea, bone, skin, heart valve, and other
1-3
tissue donations and transplants have
4
exceeded those of solid organs, such as
kidneys, livers, hearts, and lungs. The
hospital blood bank, transfusion service,
community blood center, and regional
blood center are uniquely qualified to
provide essential support for organ and
tissue transplantation and to serve as tis5
sue banks (Table 26-1). It is common for
hospital blood banks to provide transfusion support for organ and tissue recipients and, in some cases, to store, keep records
of, and dispense tissue for allografts. AABB
Standards for Blood Banks and Transfu6(pp8,14,15,60,79)
addresses the resion Services
ceipt, storage, transportation, and records
of tissue allografts. Additional guidance for
the collection and preparation of tissue
and organ allografts is available from federal and state regulations, Public Health

Service Guidelines, and the standards,
guidelines, and technical manuals of other
7-14
national or local organizations.

Transplant-Transmitted
Diseases and Preventive
Measures
The widened availability of tissue and organ grafts has encouraged new clinical
uses and highlighted not only their effectiveness and advantages but also their
drawbacks, side effects, and complications.
Organs and tissues can transmit bacterial,
fungal, viral, and prion diseases from the
donor to the recipient3,15-26 (Table 26-2),
but careful donor screening and testing,
along with disinfection and sterilization
steps for specific tissues, can markedly reduce the risk.
617

618

AABB Technical Manual

Table 26-1. Skills and Experience Appropriate for Institutions Undertaking
Tissue Banking
■

Community support

■

Public accountability

■

Public education with a broad-based public information system

■

Donor recruitment

■

Counseling

■

Medical overview

■

Donor selection

■

Donor testing including automated virology testing to avoid transcription errors

■

Cellular cryopreservation

■

Temperature-controlled and monitored storage

■

Regulatory compliance

■

Transportation infrastructure

■

Financial relations with hospitals

■

Computerized inventory control

■

Record-keeping

■

Logistics management

■

Investigation of adverse reactions

■

Peer review of medical, scientific, and operational practice

■

Recipient matching

■

Concern over the balance of the adequacy and safety of supply

■

Reputation for dependable service

■

Commitment to research and development

■

24-hours-per-day, 7-days-per-week operation

Reproduced with permission from Warwick et al.5

Risk Reduction for Tissues
Crucial to the safety of transplanted tissue
is an evaluation of the potential donor’s
eligibility. Listed below are the questions,
examinations, and tests undertaken to ensure that material from the potential donor
poses a low threat of disease transmission.
Additional measures apply to reproductive
10,11
tissue donors.
1.
Review of health history, through interviews with next of kin and/or significant other, and possibly health-

care provider, and review of medical
records. Review includes an evaluation (although not necessarily rejection for asterisked items*) of the potential donor for:
a.
History of infection,* malignant
disease,* or neurodegenerative
disease
b.
History of autoimmune processes*
c.
History of exposure to hormone
derived from human pituitary
gland or dura mater transplant

Chapter 26: Tissue and Organ Transplantation

619

Table 26-2. Infectious Diseases Reported to Have Been Transmitted by Organ and
15-26
Tissue Allografts
Allograft

Infectious Disease/Disease Agent

Bone

HIV-1
Hepatitis C
Hepatitis, unspecified type
Bacteria
Tuberculosis

Cornea

Hepatitis B
Creutzfeldt-Jakob disease
Rabies
Cytomegalovirus (?)
Bacteria
Fungus

Dura mater

Creutzfeldt-Jakob disease

Heart valve

Hepatitis B
Tuberculosis

Skin

Bacteria
Cytomegalovirus (?)
HIV-1 (?)

Pericardium

Creutzfeldt-Jakob disease
Bacteria

Solid organ (eg, kidney, liver,
heart)

HIV-1
Hepatitis B
Hepatitis C
Cytomegalovirus
Epstein-Barr virus
Parvovirus
Toxoplasmosis
Chagas’ disease
Malaria
Bacteria
Tuberculosis
HHV-8
Strongyloidiasis
Sarcoidosis
West Nile virus
Rabies
Lymphocytic choriomeningitis
(cont’d)

620

AABB Technical Manual

Table 26-2. Infectious Diseases Reported to Have Been Transmitted by Organ and
15-26
(cont'd)
Tissue Allografts
Allograft

Infectious Disease/Disease Agent

Pancreatic islet

Bacteria

Semen

Hepatitis B
Hepatitis C (?)
Gonorrhea
Syphilis (?)
HIV-1
HTLV-I (?)
Human papilloma virus (?)
Trichomonas vaginalis
Chlamydia trachomatis
Cytomegalovirus (?)
Ureaplasma urealyticum
HSV-2
Mycoplasma hominis
Group B streptococcus

Review for evidence of high-risk behavior (exclusion for any of the donor-deferral criteria included in items
9c, 9e, and 10 of Standard 5.4.1A of
Standards for Blood Banks and Trans6(pp62-65)
fusion Services.
Serologic testing on suitable blood
10
specimens (see below for more detail).
a.
Hepatitis B surface antigen
(HBsAg)
b.
Antibodies to human immunodeficiency viruses 1 and 2 (antiHIV-1 and -2)
c.
Antibody to hepatitis C virus
(anti-HCV)
d.
Antibody to human T-cell lymphotropic virus types I and II
(anti-HTLV-I and -II)
e.
Serologic test for syphilis

Physical examination to detect:
a.
Evidence of intravenous drug use
b.
Jaundice
c.
External signs of infection
d.
Signs of AIDS
Review of results of autopsy examination, if performed.

Consent and Donor Eligibility
Written consent for clinical use of any tissue or organ must be obtained from a living donor or from the next of kin of a deceased donor (formerly referred to as a
cadaveric donor), except when corneas are
procured under statutory consent. State
and federal referral statutes and regulations exist, mandating that hospitals 1)
maintain policies for notifying organ procurement organizations and appropriate

Chapter 26: Tissue and Organ Transplantation

tissue banks, including eye banks, when
death of a patient has occurred or is imminent and 2) designate requestors to approach families about organ and tissue
donation. All applicable federal, state, and
local laws concerning the consent of next
of kin must be obeyed. When the next of
kin signs consent for tissue donation, he
or she should specify which tissues may
be donated and whether the permission
includes tissue to be used for research or
other specific uses.
Living donors and the families of deceased donors are not responsible for expenses involved in recovering and processing donated tissues and organs. Donors do
not receive compensation for the donation,
although donors of reproductive tissue are
often compensated for their time, risk, and
inconvenience, and donor families may receive incentives, such as a contribution toward funeral expenses, for donation from
deceased donors. Tissues can be recovered
up to 24 hours after death if the body is refrigerated within 12 hours of death. Tissues
can be recovered up to 15 hours after death
if not refrigerated. Organs must be recovered from a donor whose neurologic death
has been declared and whose circulation
has been maintained (see Table 26-3). Eyes
as a source of corneal allografts should be
removed as soon as possible after death to
ensure viability of endothelial cells.
Each prospective deceased donor must
be evaluated against eligibility criteria for
the specific tissue(s) and organ(s) to be collected; eg, deceased newborns are not suitable for bone donation because of their
cartilaginous skeletal structure but may be
candidates for heart valve donation. Although exceptions may be made in specific
cases, the medical eligibility of a donor is
determined on the basis of absence of infection and malignancy as revealed by the
medical history, physical examination, laboratory tests, and autopsy, if performed.

621

Donation of organs and tissues does not ordinarily cause delays in funerals or prevent
family viewing of the body.

Serologic Testing
Federal regulations require that donors of
tissues be tested for HBsAg, anti-HIV-1/2,
and anti-HCV, and given a syphilis screening test, with tests licensed by the Food
and Drug Administration (FDA). Testing
must be performed by a laboratory that is
certified under the provisions of the Clinical Laboratory Improvement Amendments
of 19887,8 or that has met equivalent requirements as determined by the Centers
for Medicare and Medicaid Services. A
screening test approved for cadaveric
27
specimens must be used when available.
National standard-setting organizations,
such as the American Association of Tissue Banks (AATB),10 Eye Bank Association
12
of America (EBAA), and the United Net13
work for Organ Sharing (UNOS), may require additional tests for infectious disease markers. The American Society for
Reproductive Medicine also has issued
recommendations for gamete and embryo
donation. 11 For anonymous semen donors, tests for anti-HIV-1/2 and anti-HCV
must be repeated on a sample obtained 6
months after donation and the results
found negative before semen can be released for use. 9,10 Testing for anti-HBc
must also be performed on this sample to
rule out hepatitis B infection at the time
of collection. ABO typing is required for
organ grafts. HLA typing is essential for
organ grafts and hematopoietic progeni13
28
tor cells but not for tissues. Other tests,
such as antibody to cytomegalovirus (antiCMV), are also generally performed on organ donors. For deceased donors, tests are
optimally performed on a blood sample
obtained before administration of transfusions or fluids; these patients may have re-

622

AABB Technical Manual

Table 26-3. Kinds of Donors Providing Organs and Tissue for Transplantation

Living Donor

Deceased Donor
(Neurologic Death)

Deceased Donor
(Cardiorespiratory and
Neurologic Death)

Amniotic membrane

Heart

Bone

Bone
Fetal tissues (mother is the
donor)
Foreskin

Kidney
Liver

Cartilage
Cornea

Lung

Dura mater

Pancreas (with or without
small intestine)

Fascia lata
Heart valve

Kidney
Liver
Lung
Marrow

Marrow
Pericardium
Skin
Tendon

Milk
Pancreas
Parathyroid (frequently
autologous)

Vascular tissue

Peripheral blood progenitor
cells
Reproductive tissue
Umbilical cord blood
Umbilical vein

ceived large volumes of replacement
fluids shortly before death, and the consequent plasma dilution may cause falsenegative results.19,20
If donor serum collected before blood
transfusion or intravenous fluid administration is not available from other sources, a
pretransfusion sample is often available
from the blood bank because blood banks
hold specimens collected for compatibility
for at least 7 days. For samples obtained after transfusion or infusion of intravenous
fluids, algorithms for determining the suitability of a donor sample are available.7,8 For
example, the sample is not suitable for infectious disease marker testing if either one
of the following situations exists:

The total volume of colloid (plasma,
dextran, platelets, or hetastarch) transfused in 48 hours plus the total volume of crystalloid infused in the
hour before the sample is obtained
exceed the patient’s plasma volume.
2.
The sum of the volume of blood
transfused (RBCs, whole blood) and
colloid transfused in 48 hours plus
the total volume of crystalloid infused
in the hour before the sample is obtained exceed the patient’s blood
volume.
Testing of cadaveric blood specimens can
be complicated by postmortem hemolysis,
which can cause misleading test results (eg,
15,29
Many tests lifalse-positive HBsAg).

Chapter 26: Tissue and Organ Transplantation

censed for use on blood donor specimens
are not licensed for use with postmortem
specimens, but licensed assays for postmortem specimens are available for antiHIV-1/2, HBsAg, and HIV-1/HCV nucleic
acid.27

Bone Banking
Except for blood cells, bone is the most
1
commonly transplanted tissue or organ.
When bone grafting is needed, fresh autologous bone, usually removed from the
iliac crest during surgery, is generally considered the most effective graft material.
As with blood, the use of autologous bone
for graft material is not risk free and there
may be morbidity and infectious complications. The quantity of bone graft needed
for some surgical procedures may make
the use of autologous bone impractical.
Allografts are used for these patients and
for patients in whom the prolongation of
surgery, extra bleeding, and potential
complications of autograft collection are
considered undesirable. Bone allografts
have achieved widespread clinical application for acetabular and proximal femur
support in revisions of failed hip prostheses; packing of benign bone cysts; spinal
fusion to treat disc disease or scoliosis; reconstruction of maxillo-facial defects; and
correction of healed fractures. Demineralized bone powder is commonly used by
periodontal surgeons to restore alveolar
bone in periodontal pockets.
The surgeon today has access to a wide
choice of processed bone allografts: freezedried or frozen, cancellous or cortical, with
or without treatment with sterilants. Common preparations include frozen or freezedried cancellous cubes or chips, cortical
struts, and cortical-cancellous blocks and
dowels. Bone can be stored frozen or, if

623

freeze-dried to a low residual moisture content (6% or less by gravimetric analysis or
8% by nuclear magnetic resonance spectrometry), at room temperature for 5 years
(Table 26-4). Frozen bone is processed
aseptically, some without a microbial inactivation step. Such tissue carries a greater
risk of bacterial contamination. A series of
infection cases, including one death, linked
to aseptically processed musculoskeletal allografts underscores the importance of recovery cultures of donated tissue.24 Freezedried tissue has undergone extensive processing to remove blood and marrow and
may have been exposed to alcohol. Thus,
the risk of disease transmission is reduced.
Most bone allografts used in the United
States are freeze-dried to simplify storage.
Some grafts, whether frozen or freeze-dried,
may be treated with gamma irradiation or
ethylene oxide to reduce the risk of infectious disease transmission. Demineralization of bone is believed to make its proteins
and growth factors more readily available,
thereby enhancing its capacity to promote
healing and bone formation.
The implantation of frozen bone allografts has stimulated blood group antibodies that have been implicated in hemolytic
disease of the newborn. Frozen, unprocessed bone allografts contain sufficient red
cells to stimulate production of Rh and
other red cell antibodies. When indicated,
the risk of blood group sensitization can be
avoided by using processed frozen or freezedried bone allograft, both of which are devoid of blood cells and marrow. With these
grafts, matching blood groups of donors
and recipients is not necessary. When using
unprocessed frozen bone in Rh-negative
females with childbearing potential, it is
advisable to use bone from Rh-negative donors to prevent alloimmunization or administer Rh immune globulin prophylactically. There is no evidence to date that ABO
or Rh incompatibility between the bone

624

AABB Technical Manual

Table 26-4. Recommended Preservation Conditions and Dating Periods for
Human Tissue and Organs
Storage Condition

Dating Period

–40 C

5 years*

–20 C

6 months

1-10 C

5 days

Liquid nitrogen

Not defined

Lyophilized, room temperature

5 years*

Tendon

–40 C

5 years*

Fascia lata

Lyophilized, room temperature

5 years*

–40 C

5 years*

–40 C

5 years*

1-10 C

5 days

Liquid nitrogen, immersed

Not defined

1-10 C

14 days

–40 C

Not defined

Lyophilized, room temperature

Not defined

Cornea

2-6 C

14 days

Hematopoietic progenitor
cells

Liquid nitrogen, immersed

Not defined

Liquid nitrogen, vapor phase

Not defined

Liquid nitrogen, immersed

Not defined

Liquid nitrogen, vapor phase

Not defined

Heart valve, vein, artery

–100 C

Not defined

Dura mater

Lyophilized, room temperature

Not defined

Kidney

Refrigerated

48-72 hours

Liver

Refrigerated

8-24 hours

Heart

Refrigerated

3-5 hours

Heart-lung

Refrigerated

3-5 hours

Pancreas

Refrigerated

12-24 hours

Tissue
Bone

Articular cartilage

Skin

Semen

Organ

*Unless a longer dating period has been validated by the processor

Chapter 26: Tissue and Organ Transplantation

donor and recipient has an adverse effect
on the success of the bone graft.30

Skin Banking
A human skin allograft is the dressing of
choice for deep burn wounds if sufficient
amounts of skin for autografting are unavailable. A skin allograft provides temporary coverage; speeds reepithelialization;
acts as a metabolic barrier against loss of
water, electrolytes, protein, and heat; and
provides a physical barrier to bacterial infection. Skin allografts are replaced periodically until sufficient autograft skin can
be obtained. A skin allograft may also be
used for donor sites for pedicle flaps and
skin autografts, and for traumatically denuded areas or unhealed areas of chronic
injury, such as decubitus ulcers.
Following preparation, skin donation involves removing a layer of skin approximately 0.015 inch thick. After collection, refrigerated skin can be stored at 1 to 10 C for
up to 14 days. For refrigerated storage, standard tissue-culture nutrient media are
used, with added antibiotics. Skin can be
frozen soon after collection, usually with
10% to 15% glycerol, although dimethyl
sulfoxide (DMSO) is an acceptable alternative.31 Skin is often cryopreserved on finemesh gauze in flat cryopreservation bags.
Cryogenic damage is minimized by controlled-rate freezing at about 1 C per minute, or by freezing, using a validated heat
sinking method, followed by storage in liquid nitrogen or in a mechanical freezer at a
temperature colder than –40 C. Alternatively,
skin placed in aluminum plates inside insulated boxes can be placed directly into a
–40 C mechanical freezer. This simple process also provides a slow, predictable freezing rate and maintains cellular viability. The
optimal freezing procedure and the maximal storage period that maintain viability

625

and structural integrity in the frozen state
have not been determined. Skin should be
transported to the operating room on wet
ice if stored at 4 C, or on dry ice if cryopreserved. A variety of bioengineered skin
substitutes, such as cultured keratinocyte
allografts and autografts, are available for
treatment of acute and chronic wounds.
Keratinocytes extracted from foreskin can
be seeded onto biodegradable platforms to
create bioengineered tissue products used
in wound healing or as cadaveric skin substitutes for burns.32

Heart Valves
Human heart valve allografts provide longterm function for valve replacement—superior to that of mechanical or porcine
valves. Recipients of human heart valve
allografts do not require anticoagulation
and the incidence of thromboembolism is
low. These allografts may rarely transmit
bacterial or fungal infection. They are the
graft of choice for children, to avoid longterm anticoagulation; for pregnant women,
to avoid teratogenic risks of anticoagulants;
and for patients with infection at the aortic root. Despite their advantages, widespread use of human valve allografts has
been slow because implantation is technically difficult and appropriate size valve
allografts are not always readily available.
To obtain allograft valves, hearts are
aseptically collected in an operating room,
autopsy room, or morgue. Subsequently, in
the tissue bank, the pulmonic and aortic
valves are dissected out, cryopreserved with
DMSO, and stored in liquid nitrogen. Compared with valves stored at 5 C in antibiotics and culture medium, cryopreserved heart
valves are associated with increased cell viability; reduced incidence of valve degeneration, rupture, and leaflet perforation; and
reduced occurrence of valve-related death.33

626

AABB Technical Manual

Records of Stored Tissue
Allografts
In two cases in which HIV and HCV were
transmitted (through unprocessed frozen
bone or organs and tendon allografts, respectively) from two deceased donors to
multiple recipients,22,23 investigations revealed that several hospitals had insufficient records to identify recipients of other
tissue from the same infected donors. Voluntary standards of national professional
associations and government regulations
require tissue banks to have a recordkeeping system that identifies the donor
and allows tracking of any tissue from the
donor (or supplier source) to the consignee.6(pp12,47,79),10,14,34 Records must show the
source facility, the identification number
of the donor or lot, storage temperatures,
and final disposition of each tissue. These
records must be retained at least 10 years
beyond the distribution date, transplantation date, disposition date, or expiration
date, whichever is latest. Donor eligibility
records for dura mater must be retained
indefinitely. Hospitals should also have
records that identify recipients who received tissue from a specific donor or tissue lot. Hospitals should have procedures
in place to recognize adverse outcomes of
tissue use and to report them to the tissue
bank supplying the tissue.

FDA Regulation of Tissue
The FDA regulates human tissue collected
for transplantation.7,8 There are requirements for infectious disease testing, donor screening, and record-keeping. A tissue establishment must have and follow
written, validated procedures to prevent
contamination and cross-contamination
during processing.7,35 The FDA has pub-

lished rules for determining eligibility for
donation of human cells, tissues, and cellular and tissue-based products (HCT/
Ps)36 that became effective May 25, 2005.
Federal rules also require all facilities that
recover, process, store, or distribute HCT/Ps
or screen or test the donor to register with
the agency and list their products by mail37
ing or faxing Form FDA 3356. Information may be submitted electronically at
the FDA website www.fda.gov/cber/tissue/tisreg.htm. Products not covered by
these regulations include xenogeneic tissue, vascularized organs, transfusable
blood products, products used in the
propagation of cells or tissues, and products that are secreted or extracted from
cells or tissues. Minimally manipulated
marrow, such as marrow that undergoes
cell separation, the relevant characteristics of which are not altered by the processing, is not covered.
Under the federal regulations, infectious
disease testing is required for allogeneic tissues, except reproductive tissue from sexually intimate partners. Current good tissue
practice rules to address concerns about
proper handling, storage and processing of
34
tissue have been finalized. Until such time
that the comprehensive regulatory framework for human cells, tissues, and cellular
and tissue-based products is effective, including donor eligibility requirements,
good tissue practice regulations, and appropriate enforcement provisions, human
dura mater and human heart valves will remain subject to the medical devices requirements under the Federal Food, Drug,
38
and Cosmetic Act. Federal regulation of
tissue banks, formerly under the purview of
the FDA’s Center for Biologics Research and
Review, Office of Blood Research and Review, is now the responsibility of the Center
for Biologics Evaluation and Research, Office of Cellular, Tissue, and Gene Therapies’
Division of Human Tissues.

Chapter 26: Tissue and Organ Transplantation

The Importance of ABO
Compatibility
The ABO antigens are important in transplantation practice because they constitute
very strong histocompatibility antigens that
are expressed on vascular endothelium.
Major ABO mismatching can cause rapid
graft rejection due to endothelial damage
by ABO antibodies and subsequent widespread thrombosis within the graft. ABO
matching is important to the success of
vascularized grafts (ie, kidney, 39 heart,
liver, and pancreas), but ABO matching is
not important in tissue grafts (ie, fascia,
bone, heart valves, skin, and cornea).40
The definition of an ABO-compatible
graft is the same as that for a red cell transfusion. A group O donor of tissue or organ
is a universal donor whose graft can be
transplanted into recipients of all blood
groups. Case reports document rare successful organ transplants with major ABO
incompatibility, but these are few in number.41 In some cases, A2 donor kidneys can
be successfully transplanted into group O
recipients with survival comparable to that
of group O donor kidneys.42 ABO-incompatible transplants have occurred, often with
fatal results, due to errors of record-keeping
or labeling. It has been estimated that inadvertent ABO-incompatible heart or kidney
transplants occur with a frequency of 1 per
1000.43 This underscores the importance of
a final ABO check of donor and recipient
blood at the transplant facility to reduce
this risk.

The Role of Transfusion in
Kidney Transplants

627

pretransplant blood transfusions. The association between fewer transfusions and
declining renal allograft survival led
transplant centers to initiate deliberate
pretransplant transfusion protocols in the
mid-1970s. Subsequent studies have supported the theory that pretransplant blood
transfusions enhance renal allograft survival through mechanisms of inducing
tolerance that remain imperfectly under45,46
However, with the availability of
stood.
cyclosporine and other immunosuppressive agents, interest in this approach has
waned.47 If the controversial practice of
transfusion to induce tolerance in patients
before transplantation is begun, nonleukocyte-reduced Red Blood Cells (RBCs) should
be used.48,49 The introduction of erythropoietin has reduced the need for red cell
transfusions in patients awaiting a renal
transplant.

Liver Transplants
A liver transplant program presents one of
the greatest challenges to the donor center and hospital transfusion service, demanding maximal support in terms of
preparedness, supply, and responsiveness. Massive blood loss and hypocoagulability due to preexisting liver disease
and/or the anhepatic interval during the
procedure create complex problems for the
transfusion service. The liver is the major
site for synthesis of clotting factors and
other essential proteins and is a prime regulator of acid-base, electrolyte, and glucose
homeostasis. The three surgical phases of
the procedure—recipient hepatectomy,
anhepatic interval, and biliary reconstruction—seriously derange these functions.

In 1973, Opelz et al noted decreasing kidney allograft survival when hemodialysis
staff attempted to avoid priming the dialysis equipment with blood and to limit

Support Required
To achieve a successful liver transplant
program, a major commitment to this sup-

628

AABB Technical Manual

port is required. A successful program requires cooperation and communication
among the hospital administration; operating room and intensive care unit; respiratory therapy, radiology, gastroenterology, and anesthesiology services; the
coagulation and transfusion laboratories;
and the regional donor center. The institutional commitment must extend 24
hours a day, 365 days a year, because there
may be no more than a few hours’ advance notice of a liver transplant. Consistent availability of blood bank staff on
short notice is essential to meet the transfusion requirements. The surgical procedure frequently takes place at night or on
weekends because of the availability of the
donor organ and in order to avoid disrupting the operating room schedule. The
surgical procedure takes an average of 6
to 8 hours but may take up to 24 hours
and involve massive blood use and several surgical teams.
The blood bank should be notified as
soon as the donor organ becomes available
and the decision for transplantation is
made. The blood bank obtains a generous
blood sample from the recipient for crossmatching, but there may be more than one
patient waiting for a liver and the surgeons
may be undecided about the specific recipient. Therefore, the blood bank may have to
perform numerous crossmatches for patients who may have different ABO groups
and/or Rh types. Liver transplant programs
initially used hundreds of units of blood
and components per patient. Although
blood use has steadily declined over the
years, liver transplant procedures frequently use a volume of blood components
equal to one whole-body blood volume and
sometimes several blood volumes. Intraoperative blood recovery frequently plays a
major role in the conservation of red cells
in such cases.

Considerations of ABO and Rh
Except in emergencies, donor livers
should be ABO-compatible with the recipient. ABO-identical RBCs and Fresh
Frozen Plasma (FFP) are generally used
for transfusion support of group O and
group A recipients. Group B recipients
who need large quantities of red cells can
be switched to group O RBCs. Group AB
recipients needing massive transfusion are
often switched to group A RBCs to conserve group O RBCs for other patients. If
the supply of AB FFP is insufficient, early
use of group A RBCs followed by a switch
to group A FFP is appropriate. A general
rule for massive transfusions is to switch
red cells first, then switch plasma, and reverse the order when returning to the patient’s original blood group.46
Special considerations apply to the recipient of an out-of-group but ABO-compatible liver transplant. In a group A patient
receiving a group O liver, lymphocytes of
donor origin may produce ABO antibodies
that cause hemolysis that begins several
days after the procedure and may continue
for 2 weeks or longer.50 Although passenger
lymphocytes may produce antibodies in
any out-of-group but compatible combination, significant hemolysis is seen most
often in the recipient of a group O liver.
Transfusion support of Rh-negative patients not immunized to the D antigen is
not standardized. 46 Because successful
pregnancy has occurred after liver transplantation, most programs consider it preferable to provide D-negative units to
D-negative females with childbearing potential, if needs are expected to be moderate. Should massive blood loss occur, the
patient could be switched intraoperatively
to D-positive blood, if necessary. For premenopausal females without anti-D, some
programs reserve 10 units of D-negative
RBCs; if more than 10 units are required,

Chapter 26: Tissue and Organ Transplantation

they switch to D-positive blood. Production of anti-D occurs less frequently in
D-negative liver transplant patients exposed to the D antigen than in other
D-negative patients.51 In some programs,
patients without anti-D who are D-negative
postmenopausal females or D-negative
males are transfused exclusively with
D-positive blood.

Red Cell Alloantibodies
Liver transplant patients with clinically
significant red cell alloantibodies represent a special challenge to blood banks.
Sometimes, a sufficient quantity of antigen-negative blood can be secured before
surgery. Some programs reserve a limited
number of antigen-negative units for use
at the beginning of surgery, when alloantibody is present, and at the end of
massive blood loss, when transfused cells
are expected to remain in circulation. Antibody screening during the interval of
massive blood loss can help guide use of
antigen-positive units during surgery.

Coagulation Considerations
During surgery, hemodilution, platelet
consumption, disordered thrombin regulation, and fibrinolysis derange the hemostatic process. The coagulopathy is especially severe during the anhepatic and
early reperfusion stage. The following tests
are useful: the hematocrit guides the use
of red cells, colloids, and crystalloids; the
platelet count guides transfusion of platelets; the prothrombin time and activated
partial thromboplastin time guide FFP
use; and fibrinogen determinations guide
use of Cryoprecipitated AHF and antifibrinolytic agents.46,52,53

629

Other Organ Transplants
Blood bank support for cardiac transplantation is very similar to that routinely
used for other surgical procedures in
which cardiopulmonary bypass is employed. The blood bank may also provide
ABO testing and assist in the release
of ABO-compatible organs to prevent
ABO-mismatched organ transplantation.
Pancreatic transplants have comparatively low transfusion requirements, but a
specimen from the recipient should routinely be examined for clinically significant unexpected red cell antibodies; in
some institutions, the protocol calls for
crossmatching several units.

Transfusion Service Support
for Organ Transplantation
The blood bank provides vital support for
a clinical transplantation program. Close
communication with the surgeons and
other professionals involved in the program is essential. Transfusion practices in
the peritransplant period have a major effect on morbidity, mortality, and graft survival rates.
Potential recipients of solid organ transplants are generally available well before
the procedure, so there is ample time to obtain a history and perform laboratory tests.
It is important for the transfusion service to
know if there have been previous pregnancies, transplants, or transfusions.
Laboratory tests routinely performed include: ABO group and Rh type, the direct
antiglobulin test (DAT), a screen for unexpected red cell antibodies, and determination of CMV serostatus. HLA typing and
HLA antibody studies are routine for organ
recipients.
Passenger lymphocyte hemolysis (typically “ABO”-incompatible hemolysis), as

630

AABB Technical Manual

discussed previously in regard to liver
transplantation, can occur with other solid
organ transplants, such as lung, heart, and
kidney. In the case of a recipient receiving
an ABO-compatible but non-group-identical organ, prophylactic use of mutually
ABO-compatible erythrocytes (compatible
for the donor and the recipient) has been
suggested for intraoperative and postoperative infusions, during the first postoperative
month, or at the appearance of an antibody. At present, there is no consensus on
this issue. It is important to remember that
if immediate-spin or computer crossmatching is routinely performed after an
ABO-unmatched transplant, ABO incompatibility due to these IgG antibodies may
be missed. In such cases, the routine use of
a crossmatch with an antihuman globulin
phase or the use of a DAT (which may detect such cases earlier than a crossmatch) is
recommended. If ABO hemolysis is present,
the patient should be transfused with group
O RBCs.
CMV infection, a serious and often fatal
complication in transplant recipients, is related to the presence of CMV in the donor
and recipient and the degree to which the
recipient is immunosuppressed. The primary test used to determine CMV status is
the demonstration of circulating antibody.
CMV-seronegative recipients of CMV-seronegative transplants characteristically receive transfusion components processed to
reduce risk of CMV transmission, either by
preparation from seronegative donors or by
leukocyte reduction to 5 × 106 cells/component or below.

2.
3.

10.

11.

12.
13.

14.

15.

16.

References
17.
1.

Strong M, Eastlund T, Mowe J. Tissue bank
activity in the United States: 1992 survey of
AATB-inspected tissue banks. Tissue Cell Rep
1996;3:15-18.

18.

2002 EBAA statistical report. Washington, DC:
Eye Bank Association of America, 2002.
Yap PL. Viral transmission by blood, organs
and tissues. J Hosp Infect 1999;43(Suppl):S13744.
The Organ Procurement and Transplantation
Network data reports, 2003. Richmond, VA:
United Network for Organ Sharing, 2003.
Warwick RM, Eastlund T, Fehily D. Role of the
blood transfusion service in tissue banking.
Vox Sang 1996;71:71-7.
Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.
Food and Drug Administration. Human tissue
intended for transplantation. Fed Regist 1997;
62:40429-47.
Food and Drug Administration. Guidance for
industry: Screening and testing of donors of
human tissue intended for transplantation.
( July 1997) Rockville, MD: CBER Office of
Communication, Training, and Manufacturers Assistance, 1997.
Centers for Disease Control and Prevention.
Guidelines for preventing HIV transmission
through organ and tissue transplantation.
MMWR 1994;43(RR-8):1-17.
Woll J, Kasprisin D, eds. Standards for tissue
banking. McLean, VA: American Association
of Tissue Banks, 2002.
American Society for Reproductive Medicine.
Guidelines for gamete and embryo donation.
Fertil Steril 2002;77:Suppl 5.
Medical standards of EBAA. Washington, DC:
Eye Bank Association of America, 2002.
Articles of incorporation, by-laws, and policies of UNOS. Richmond, VA: United Network
of Organ Sharing, 2004 (revised at least annually).
New York State Department of Health. Tissue
banks and nontransplant anatomic banks,
Part 52. New York State Codes, Rules, and Regulations, Title 10. Albany, NY: New York State
Department of Health, 2004. (Available at
http://www.wadsworth.org/labert/blood_tissue.)
Eastlund T. Infectious disease transmission
through cell, tissue and organ transplantation. Reducing the risk through donor selection. Cell Transplant 1995;4:455-77.
Eastlund T, Strong DM. Infectious disease transmission through tissue transplantation. In:
Phillips GO, ed. Advances in tissue banking.
Vol. 7. Singapore: World Scientific, 2004:51-132.
CJD and the eye. Monograph 7. London:
Royal College of Ophthalmologists, 1998.
Hogan RN, Brown P, Heck E, Cavanagh HD.
Risk of prion disease transmission from ocular tissue transplantation. Cornea 1999;18:2-11.

Chapter 26: Tissue and Organ Transplantation

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.
29.

30.

31.

32.

Human immunodeficiency virus transmitted
from an organ donor screened for HIV antibody—North Carolina. MMWR Morb Mortal
Wkly Rep 1987;36:306-8.
Eastlund T. Hemodilution due to blood loss
and transfusion and reliability of cadaver tissue donor infectious disease testing. Cell and
Tissue Banking 2000;1:121-7.
Linden JV, Critser JK. Therapeutic insemination by donor II: A review of the known risks.
Reprod Med Rev 1995;4:19-29.
Simonds RJ, Holmberg SD, Hurwitz RL, et al.
Transmission of human immunodeficiency
virus type 1 from a seronegative organ and
tissue donor. N Engl J Med 1992;326:726-32.
Conrad EU, Gretch D, Obermeyer K, et al. The
transmission of hepatitis C virus by tissue
transplantation. J Bone Joint Surg 1995;77A:
214-23.
Kainer MA, Linden JV, Whaley DN, et al.
Clostridium infections associated with
musculoskeletal-tissue allografts. N Engl J
Med 2004;350:2564-71.
Centers for Disease Control and Prevention.
Investigation of rabies infections in organ donor and transplant recipients—Alabama, Arkansas, Oklahoma, and Texas, 2004. MMWR
Morb Mortal Wkly Rep 2004;53:586-9.
Centers for Disease Control and Prevention.
Lymphocytic choriomeningitis virus infection in organ transplant recipients—Massachusetts, Rhode Island, 2005. MMWR Morb
Mortal Wkly Rep 2005;54:537-9.
Food and Drug Administration. Guidance for
industry: Availability of licensed donor
screening tests for use with cadaveric blood
specimens. ( June 23, 2000) Rockville, MD:
CBER Office of Communication, Training, and
Manufacturers Assistance, 2000.
Choo SY, Eastlund T. Tissue transplantation
and HLA typing. Tissue Cell Rep 1995;2:3-4.
LeFor WM, McGonigle AF, Wright CE, Shires
DL. The frequency of false positive HBsAg
screening test results with cadaver tissue donors is dependent upon the assay procedure
used. Tissue Cell Rep 1996;3:6-170.
Eastlund T. Bone transplantation and bone
banking. In: Lonstein JE, Bradford DS, Winter
RB, Ogilvie JW, eds. Moe’s textbook of scoliosis
and other spinal deformities. 3rd ed. Philadelphia: WB Saunders, 1995:581-95.
Bravo D, Rigley TH, Gibran N, et al. Effect of
storage and preservation methods on viability of transplantable human skin allografts.
Burns 2000;26:367-78.
Phillips TJ. New skin for old: Developments
in biological skin substitutes. Arch Dermatol
1998;134:344-9.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

631

O’Brien MF, Stafford EG, Gardner MAH, et al.
Cryopreserved viable allograft aortic valves.
In: Yankoh AC, Hetzer R, Miller DC, et al, eds.
Cardiac valve allografts 1972-1987. New York:
Springer-Verlag, 1988:311-21.
Food and Drug Administration. Current good
tissue practice for human cell, tissue, and cellular and tissue-based product establishments;
inspection and enforcement; final rule. (November 4, 2004) Fed Regist 2004;69:68611-88.
Food and Drug Administration. Guidance for
Industry. Validation of procedures for processing human tissues intended for transplantation. (March 2002) Rockville, MD:
CBER Office of Communications, Training,
and Manufacturers Assistance, 2002.
Food and Drug Administration. Eligibility determination for donors of human cells, tissues,
and cellular and tissue-based products; final
rule. Fed Regist 2004;69:29786-834.
Food and Drug Administration. Human cells,
tissues, and cellular and tissue-based products; establishment registration and listing;
final rule. Fed Regist 2001;66:5447-69.
Food and Drug Administration. Human cells,
tissues, and cellular and tissue-based products; establishment registration and listing;
interim final rule; opportunity for public
comment. Fed Regist 2004;69:3823-6.
Alkhunaizi AM, de Mattos AM, Barry JM, et al.
Renal transplantation across the ABO barrier
using A2 kidneys. Transplantation 1999;67:
1319-24.
Eastlund T. The histo-blood group ABO system and tissue transplantation. Transfusion
1998;38:975-88.
Alexandre GPJ, Squifflet JP, DeBruyere M, et
al. ABO-incompatible related and unrelated
living donor renal allografts. Transplant Proc
1986;18:1090-2.
Breimer ME, Brynger H, Rydberg L, et al.
Transplantation of blood group A2 kidneys to
O recipients. Biochemical and immunological studies of group A antigens in human kidneys. Transplant Proc 1985;17:2640-3.
Terasaki PI. Red-cell crossmatching for heart
transplants (letter). N Engl J Med 1991;325:
1748-9.
Opelz G, Sengar DPS, Mickey MR, Terasaki P.
Effect of blood transfusion on subsequent
kidney transplants. Transplant Proc 1973;5:
253-9.
Blumberg N, Heal JM. Transfusion immunomodulation. In: Anderson KC, Ness PM, eds.
Scientific basis of transfusion medicine. 2nd
ed. Philadelphia:WB Saunders, 2000:427-43.
Dzik WH. Solid organ transplantation. In:
Petz LD, Swisher SN, Kleinman S, et al, eds.
Clinical practice of transfusion medicine. 3rd

632

47.

48.

49.

AABB Technical Manual

ed. New York: Churchill Livingstone, 1996:
783-806.
Lundgren G, Groth CG, Albrechtsen D, et al.
HLA matching and pretransplant blood transfusions in cadaveric renal transplantation—a
changing picture with cyclosporin. Lancet
1986;ii:66-9.
Iwaki Y, Cecka JM, Terasaki PI. The transfusion effect in cadaver kidney transplants, yes
or no. Transplantation 1990;49:56-9.
Opelz G, Vanrenterghem Y, Kirste G, et al.
Prospective evaluation of pretransplant
blood transfusions in cadaver kidney recipients. Transplantation 1997;63:964-7.

50.

51.

52.

53.

Triulzi DJ, Shirey RS, Ness PM, Klein AS. Immunohematologic complications of ABOunmatched liver transplants. Transfusion
1992;32:829-33.
Casanueva M, Valdes MD, Ribera MC. Lack of
alloimmunization to D antigen in D-negative
immunosuppressed liver transplant recipients. Transfusion 1994;34:570-2.
Triulzi DJ, Bontempo FA, Kiss JE, Winkelstein
A. Transfusion support in liver transplantation. Transfus Sci 1993;14:345-52.
Motschman TL, Taswell HF, Brecher ME, et al.
Blood bank support of a liver transplantation
program. Mayo Clin Proc 1989;64:103-11.

Chapter 27: Noninfectious Complications of Blood Transfusion

Chapter 27

Noninfectious
Complications of Blood
Transfusion
27

HIS CHAPTER ADDRESSES four
broad categories of transfusion reactions: 1) acute immunologic, 2)
acute nonimmunologic, 3) delayed immunologic, and 4) delayed nonimmunologic
complications, as shown in Table 27-1.1,2
For each individual type of reaction, the
pathophysiology, treatment, and prevention are discussed. More detailed coverage is available elsewhere. 1 Infectious
risks of transfusion are discussed in
Chapter 28.

Manifestations
All personnel involved in ordering and
administering transfusions must be able
to recognize a transfusion reaction so that
appropriate actions can be taken promptly.
Listed below are signs and symptoms that
are typically associated with acute trans-

fusion reactions and can aid in their recognition. In general, one should consider
any adverse manifestation occurring at
the time of the transfusion to be a transfusion reaction until proven otherwise.
■
Fever with or without chills [generally defined for surveillance purposes as a 1 C (2 F) increase in body
temperature] associated with transfusion. Fever is the most common
symptom of a hemolytic transfusion
reaction (HTR),3 but more frequently
it has other causes.
■
Shaking chills (rigors) with or without fever.
■
Pain at the infusion site or in the
chest, abdomen, or flanks.
■
Blood pressure changes, usually acute,
either hypertension or hypotension.
Circulatory shock in combination
with fever, severe chills, and highoutput cardiac failure suggests acute
633

634

Table 27-1. Categories and Management of Adverse Transfusion Reactions*

Incidence

Etiology

Presentation

Diagnostic Testing

Chills, fever,
hemoglobinuria,
hypotension, renal failure
with oliguria, DIC (oozing
from IV sites), back pain,
pain along infusion vein,
anxiety

■
■
■

Therapeutic/Prophylactic
Approach

Acute (<24 hours) Transfusion Reactions–Immunologic
Hemolytic

1:38,0001:70,000

Red cell incompatibility

■

Fever/chill,
nonhemolytic

RBCs:
1:200-1:17
(0.5-6%)
Plts:
1:100-1:3
(1-38%)

■
■

1:100-1:33
(1-3%)

Antibody to donor plasma
proteins

Antibody to donor WBCs Fever, chills/rigors, headAccumulated cytokines ache, vomiting
in platelet unit

■

■
■

Urticarial

Urticaria, pruritis, flushing

■

Clerical check
DAT
Visual inspection (free
Hb)
Repeat patient ABO, preand posttransfusion
sample
Further tests as
indicated to define
possible incompatibility
Further tests as
indicated to detect
hemolysis (LDH,
bilirubin, etc)
Rule out hemolysis
(DAT, inspect for
hemoglobinemia, repeat
patient ABO)
Rule out bacterial
contamination
WBC antibody screen
Rule out hemolysis
(DAT, inspect for
hemoglobinemia, repeat
patient ABO)

■

■
■

■

Keep urine output >100
mL/hr with fluids and IV
diuretic (furosemide)
Analgesics (may need
morphine)
Pressors for
hypotension (low-dose
dopamine)
Hemostatic components
(platelets, cryo, FFP) for
bleeding

Antipyretic
premedication
(acetaminophen, no
aspirin)
Leukocyte-reduced
blood
Antihistamine, treatment
or premedication (PO or
IV)
May restart unit slowly
after antihistamine if
symptoms resolve

AABB Technical Manual

Type

Anaphylactic

1:20,0001:50,000

Antibody to donor plasma
proteins (includes IgA,
haptoglobin, C4)

Hypotension, urticaria,
bronchospasm (respiratory distress, wheezing),
local edema, anxiety

■

■
■

Rule out hemolysis
(DAT, inspect for
hemoglobinemia, repeat
patient ABO)
Anti-IgA
IgA, quantitative

■
■
■

■

Transfusionrelated acute
lung injury

1:5,0001:190,000

WBC antibodies in donor Hypoxemia, respiratory
(occasionally in recipient), failure, hypotension, fever,
other WBC-activating
bilateral pulmonary edema
agents in components

■

■
■

■

Gram’s stain
Culture of component
Patient culture
Rule out hemolysis
(DAT, inspect for
hemoglobinemia, repeat
patient ABO)

■

Acute (<24 hours) Transfusion Reactions—Nonimmunologic
Transfusionassociated
sepsis

Varies by
Bacterial contamination
component
(see Chapter
28)

Fever, chills, hypotension

■
■
■
■

■

Broad spectrum
antibiotics (until
sensitivities completed)
Treat complications (eg,
shock)
(cont’d)

635

Rule out hemolysis
(DAT, inspect for
hemoglobinemia, repeat
patient ABO)
WBC antibody screen in
donor and recipient. If
positive, antigen typing
may be indicated
WBC crossmatch
Chest X-ray

Chapter 27: Noninfectious Complications of Blood Transfusion

■

Trendelenberg (feet up)
position
Fluids
Epinephrine (adult dose:
0.3-0.5 mL of 1:1000
solution SC or IM; in
severe cases, 1:10,000
IV)
Antihistamines,
corticosteroids, beta-2
agonists
IgA-deficient blood
components
Supportive care until
recovery
Defer implicated donors

636

Table 27-1. Categories and Management of Adverse Transfusion Reactions* (cont’d)

Incidence

Hypotension
Dependent
associated with on clinical
ACE inhibition setting

Etiology

Presentation

Diagnostic Testing

Inhibited metabolism of
Flushing, hypotension
bradykinin with infusion of
bradykinin (negatively
charged filters) or activators of prekallikrein

■

Rule out hemolysis
(DAT, inspect for
hemoglobinemia, repeat
patient ABO)

Therapeutic/Prophylactic
Approach
■
■

■

Withdraw ACE inhibition
Avoid albumin volume
replacement for
plasmapheresis
Avoid bedside leukocyte
filtration
Upright posture
Oxygen
IV diuretic (furosemide)
Phlebotomy (250-mL
increments)
Identify and eliminate
cause

Circulatory
overload

<1%

Volume overload

Dyspnea, orthopnea,
cough, tachycardia, hypertension, headache

■

Chest X-ray

■
■
■
■

Nonimmune
hemolysis

Rare

Hemoglobinuria,
hemoglobinemia

■

Rare

Rule out patient
hemolysis (DAT, inspect
for hemoglobinemia,
repeat patient ABO)
Test unit for hemolysis
X-ray for intravascular
air

■

Air embolus

Physical or chemical destruction of blood (heating, freezing, hemolytic
drug or solution added to
blood)
Air infusion via line

■

Place patient on left side
with legs elevated above
chest and head

Hypocalcemia
(ionized calcium)

Dependent
on clinical
setting

Ionized calcium
Prolonged Q-T interval
on electrocardiogram

■

Slow calcium infusion
while monitoring ionized
calcium levels in severe
cases
PO calcium supplement
for mild symptoms
during apheresis
procedures

Rapid citrate infusion
(massive transfusion of
citrated blood, delayed
metabolism of citrate,
apheresis procedures)

■

Sudden shortness of
breath, acute cyanosis,
pain, cough, hypotension,
cardiac arrythmia
Paresthesia, tetany,
arrhythmia

■

■
■

■

AABB Technical Manual

Type

Hypothermia

Dependent
on clinical
setting

Rapid infusion of cold
blood

Cardiac arrhythmia

Central body temperature

■

Employ blood warmer

Positive blood group
antibody screening test

■
■

■

Avoid unnecessary
transfusions
Leukocyte-reduced
blood
Avoid unnecessary
transfusions
Leukocyte-reduced
blood
Identify antibody
Transfuse compatible
red cells as needed

Delayed (>24 hours) Transfusion Reactions–Immunologic
1:100 (1%)

Alloimmunization, HLA
antigens

1:10 (10%)

Hemolytic

1:11,0001:5000

Graft-vs-host
disease

Rare

Immune response to
foreign antigens on
RBCs, or WBCs and
platelets (HLA)

Antibody screen
DAT

■

Platelet refractoriness,
delayed hemolytic
reaction, hemolytic disease of the newborn

■
■

Anamnestic immune
response to red cell
antigens

Fever, decreasing
hemoglobin, new
positive antibody screening test, mild jaundice

■
■
■

Donor lymphocytes engraft in recipient and
mount attack on host tissues

Erythroderma,
maculopapular rash, anorexia, nausea, vomiting,
diarrhea, hepatitis,
pancytopenia, fever

■
■

Platelet antibody screen
Lymphocytotoxicity test

■
■

Antibody screen
DAT
Tests for hemolysis
(visual inspection for
hemoglobinemia, LDH,
bilirubin, urinary
hemosiderin as clinically
indicated)
Skin biopsy
HLA typing

■
■

Corticosteroids,
cytotoxic agents
Irradiation of blood
components for patients
at risk (including related
donors and HLAselected components)

637

(cont’d)

Chapter 27: Noninfectious Complications of Blood Transfusion

Alloimmunization, RBC
antigens

638

Type

Incidence

Posttransfusion Rare
purpura

Immunomodulation

Unknown

Therapeutic/Prophylactic
Approach

Etiology

Presentation

Diagnostic Testing

■

Thrombocytopenic
purpura, bleeding, 8-10
days after transfusion

■

Platelet antibody screen
and identification

■
■
■

IGIV
HPA-1-negative platelets
Plasmapheresis

Increased renal graft survival, infection rate,
postresection tumor recurrence rate (controversial)

■

None specific

■

Avoid unnecessary
transfusions
Autologous transfusion
Leukocyte-reduced red
cells and platelets

Recipient platelet
antibodies (apparent
alloantibody, usually
anti-HPA-1) destroy
autologous platelets
Incompletely understood
interaction of donor WBC
or plasma factors with recipient immune system

■
■

Delayed (>24 hours) Transfusion Reactions–Nonimmunologic
Iron overload

Typically af- Multiple transfusions with Diabetes, cirrhosis,
ter >100 RBC obligate iron load in trans- cardiomyopathy
units
fusion-dependent patient

■
■
■

Serum ferritin
Liver enzymes
Endocrine function tests

■

Desferioxamine (iron
chelator)

*For platelet refractoriness, see Chapter 16; for septic transfusion reactions, see Table 28-1; for a recent summary of transfusion reactions, see Popovsky.1
ACE = angiotensin-converting enzyme; antibody screen = blood group antibody screening test; DAT = direct antiglobulin test; DIC = disseminated intravascular coagulation; FFP
= Fresh Frozen Plasma; Hb = hemoglobin; IV = intravenous; IGIV = intravenous immunoglobulin; IM = intramuscular; LDH = lactate dehydrogenase; PO = by mouth; RBC = Red
Blood Cell; SC = subcutaneous; WBC = White Blood Cell.

AABB Technical Manual

Table 27-1. Categories and Management of Adverse Transfusion Reactions* (cont’d)

Chapter 27: Noninfectious Complications of Blood Transfusion

■
■

■

sepsis but may also accompany an
acute HTR. Circulatory collapse without fever and chills may be the most
prominent finding in anaphylaxis.
Respiratory distress, including dyspnea,
tachypnea, wheezing, or hypoxemia.
Skin changes, including urticaria,
pruritis (itching), flushing, or localized edema (angioedema).
Nausea with or without vomiting.
Darkened urine or jaundice. Dark urine
may be the earliest indication of an
acute hemolytic reaction in anesthetized patients.
Bleeding or other manifestations of
a consumptive coagulopathy.

Acute Transfusion Reactions
Immune-Mediated Hemolysis

Pathophysiology and Manifestations
The most severe hemolytic reactions occur when transfused red cells interact
with preformed antibodies in the recipient. In contrast, the interaction of transfused antibodies with the recipient’s red
cells rarely causes symptoms. However,
there may be accelerated red cell destruction, and plasma-containing products with
high-titer ABO antibodies can cause acute
hemolysis. The interaction of antibody
with antigen on the red cell membrane
can initiate a sequence of complement
activation (see Chapter 11), cytokine and
coagulation effects, and other elements of
a systemic inflammatory response4 that
result in the clinical manifestations of a
severe acute HTR. Severe symptoms can
occur after the infusion of as little as 10 to
15 mL of ABO-incompatible red cells. In
anesthetized patients who cannot report
symptoms, the initial manifestations of
an acute HTR may be hemoglobinuria,

639

hypotension, or diffuse bleeding at the
surgical site.
Such severe acute HTRs today are usually caused by ABO incompatibility5 but occasionally may be caused by antibodies
with other specificities. 6 In contrast,
hemolysis of an entire unit of blood can occur in the virtual absence of symptoms7
and may be a relatively slow process. In
such cases, hemolysis is typically extravascular, without generation of significant
systemic levels of inflammatory mediators.
Complement Activation. The binding of
antibody to blood group antigens may activate complement, depending on the characteristics of both the antibody and the antigen, including antibody specificity, class,
subclass, titer, and antigen density (see
Chapter 11). C3 activation releases the anaphylatoxin C3a (see Chapter 11), and red
cells coated with C3b are removed by
phagocytes with complement receptors,
more rapidly than if antibody is present
alone. If the enzymatic cascade proceeds to
completion and a membrane attack complex is assembled, intravascular hemolysis
results, with the production of C5a, which
is 100 times as potent an anaphylatoxin as
3
C3a. This sequence is characteristic of ABO
incompatibility and causes the cardinal
manifestations of hemoglobinemia and, if
the renal threshold for hemoglobin is exceeded, hemoglobinuria.8(p182) Anaphylatoxins
interact with a wide variety of cells, including monocytes/macrophages, granulocytes,
platelets, vascular endothelial cells, and
smooth muscle cells, the latter leading to
hypotension and bronchospasm. Anaphylatoxins also cause the release or production of multiple local and systemic mediators, including granule enzymes, histamine
and other vasoactive amines, kinins, oxygen radicals, leukotrienes, nitric oxide, and
3
cytokines. These mechanisms may cause
manifestations that mimic allergy, such as
flushing and rarely urticaria, wheezing and

640

AABB Technical Manual

chest pain or tightness, and abdominal
pain, nausea, and vomiting.
With most non-ABO blood group antibodies, complement activation is usually
incomplete; hemoglobinemia is absent or
mild, but the consequences of complement
activation, most notably the release of anaphylatoxins and opsonization of red cells,
may still have adverse effects.
Cytokines. The role of cytokines in inflammatory responses (see Chapter 11), including acute HTRs, is increasingly recognized.9,10 The known activities of inflammatory
cytokines, such as tumor necrosis factor α
(TNFα), interleukin-1β and -6 (IL-1β, IL-6),
and chemokines such as IL-8 and monocyte chemoattractant protein (MCP), suggest that they mediate some of the effects of
alloimmune hemolysis. IL-1 and TNF cause
fever and hypotension (particularly in synergy), stimulation of endothelial cells to increase expression of adhesion molecules
and procoagulant activity, and recruitment
and activation of neutrophils and platelets,
perhaps through the induction of IL-8 and
MCP. Incubation of whole blood with
washed, ABO-incompatible red cells in vitro has been shown to cause dramatic increases in TNF, IL-8, and MCP. This cytokine response is complement dependent. A
similar model of hemolysis resulting from
anti-D (IgG) showed a different pattern of
cytokine production with “high-level” responses of IL-8 and MCP and “low-level”
responses of IL-1β, IL-6, and TNFα.9,10
The relevance of these in-vitro models to
HTRs in vivo is suggested by a case in
which TNFα and neutrophil elastase levels
were found to be elevated when a group O
patient received 100 mL of group A red
cells; elevation of neutrophil elastase is
consistent with IL-8 activity.11 These findings may lead to new therapeutic options
for patients. However, the complete role of
cytokines in the consequences of immune
hemolysis remains to be defined.

Coagulation Activation. Several mechanisms, including those listed above, may be
responsible for abnormalities of coagulation in HTRs.4 The antigen-antibody interaction may activate the “intrinsic” clotting
cascade through Hageman factor. In addition, activated Hageman factor (Factor XIIa)
acts on the kinin system to generate bradykinin; bradykinin increases capillary permeability and dilates arterioles, causing a
decrease in systemic arterial pressure. Several factors cited above may increase the
expression of tissue factor by leukocytes
and endothelial cells, including activated
complement components, TNFα, and
IL-1β. Tissue factor activates the “extrinsic”
coagulation pathway, and its release is associated with disseminated intravascular
coagulation (DIC), which may, in turn, cause:
1) formation of thrombi within the microvasculature and ischemic damage to tissues
and organs; 2) consumption of fibrinogen,
platelets, and other coagulation factors; 3)
activation of the fibrinolytic system and
generation of fibrin degradation products.
The outcome can be a hemorrhagic diathesis
characterized by generalized oozing or uncontrolled bleeding.
Shock and Renal Failure. Considering
the absolute mass of antigen and antibody—and the list of mediators that may
be involved in HTRs, including anaphylatoxins, vasoactive amines, kinins, and cytokines—it may not be surprising that shock
can occur. Hypotension provokes a compensatory sympathetic nervous system response that produces vasoconstriction in
organs and tissues with a vascular bed rich
in alpha-adrenergic receptors, notably, the
renal, splanchnic, pulmonary, and cutaneous capillaries, aggravating ischemia in
these sites.
Renal failure is another sequel of an
acute HTR. Although free hemoglobin, historically considered the cause of renal failure, does impair renal function,12 current

Chapter 27: Noninfectious Complications of Blood Transfusion

thought attributes renal failure largely to
hypotension, renal vasoconstriction, antigen-antibody complex deposition, and formation of thrombi in the renal vasculature,
all of which compromise renal cortical
blood supply.

Frequency
Clerical and other human errors leading
to mistaken identity are the most common
causes of ABO-incompatible transfusion,
occurring either at pretransfusion sample
collection, within the transfusion service,
or at the time of blood administration. A
study of reported transfusion errors in
New York State over a 10-year period (the
1990s) estimated the incidence of ABOincompatible red cell transfusions at
1:38,000. Correction for the expected rate
of fortuitously compatible transfusions
led to an estimate of the rate of mistransfusion of 1:14,000.7 A survey of 3601
institutions by the College of American
Pathologists found 843 acute HTRs reported over a 5-year period, of which 50
(6%) were fatal.13 The Serious Hazards of
Transfusion (SHOT) initiative in the United
Kingdom and Republic of Ireland reported 161 cases of ABO-incompatible
transfusion, with nine fatal cases (five definitely related deaths, one probably related death, and three possibly related
deaths) in 5 years.14 Although no precise
denominator is available for these confidential reports, it is believed that over
90% of the total transfusions were reviewed and approximately 2.5 million
RBC units were issued each year, for a rate
of no less than 1 in 78,000. These values
probably underestimate the true frequency, because even acute HTRs go unrecognized or unreported. Estimates of
mortality rates from acute HTRs are generally in the range of 1 in 1,000,000 transfusions.5,7,14

641

Treatment
The treatment of an acute HTR depends
4
on its severity. Vigorous treatment of hypotension and promotion of adequate renal
blood flow are the primary concerns. If
shock can be prevented or adequately
treated, progression to renal failure may
be avoided. Adequacy of renal perfusion
can be monitored by measurement of
urine output, with a goal of maintaining
urine flow rates above 100 mL/hour in
adults for at least 18 to 24 hours. The
usual first support is intravenous normal
saline, but underlying cardiac and/or renal disease may complicate therapy, and
it is important to avoid overhydration. Invasive monitoring of pulmonary capillary
wedge pressure is recommended in guiding fluid therapy in the face of hemodynamic instability. Diuretics help to improve blood flow to the kidneys and
increase urine output. Intravenous furosemide at a dose of 40 to 80 mg for an
adult or 1 to 2 mg/kg for a child not only
has a diuretic effect but also improves
blood flow to the renal cortex. This dose
may be repeated once, and the patient
should be adequately hydrated. Mannitol,
an osmotic diuretic, has been used in the
past, but furosemide is better for maintaining renal cortical blood flow. If no diuretic response occurs within a few hours
of instituting fluid and diuretic therapy,
there is a strong likelihood that acute tubular necrosis has occurred, and further
fluid administration may be harmful.
Treatment of hypotension with pressor
agents that decrease renal blood flow, such
as dopamine in higher doses, should be
avoided if possible. The use of low-dose dopamine (2-5 µg/kg/minute), as an agent to
protect renal function, has been recom4
mended in the management of acute HTRs.
However, evidence suggests that it is not ef-

642

AABB Technical Manual

fective in this role, and it has many toxicities.15
Consumptive coagulopathy, with resultant bleeding or generalized oozing, may be
a prominent clinical finding in some HTRs
and may be the initial presentation in an
anesthetized patient. Heparin has been recommended by some, both to forestall DIC
when an ABO incompatibility is first discovered and to treat the established coagulopathy. Others believe the dangers of
heparin outweigh its potential benefits, especially because the immune event that
provoked the DIC is self-limited. Administration of Platelets, Fresh Frozen Plasma
(FFP), and Cryoprecipitated AHF, a source
of fibrinogen and Factor VIII, may be necessary. Red cell exchange may be considered in patients with a significant load of
circulating incompatible red cells.
Acute hemolytic reactions are rare and
few clinicians have first-hand experience
with their treatment. Because medical
management of an acute HTR is often complicated and may require aggressive interventions such as hemodialysis, consultation
with physicians experienced in the organ
systems most damaged or specialists in
critical care medicine may be prudent when
treating a patient with a severe acute HTR.

Prevention
Because clerical errors cause the majority
of acute, immune-mediated HTRs, the
best hope for prevention lies in preventing or detecting errors in every phase of
the transfusion process. In each institution, there should be systems designed to
prevent and detect errors in patient and
unit identification at the time of phlebotomy (sample acquisition), at all steps in
laboratory testing, at the time of issue,
and when the transfusions are given. The
SHOT reports document multiple errors
in a majority of mistransfusion incidents

and particularly emphasize the importance of the bedside check at the time of
transfusion.14 Ensuring that all clinical staff
recognize the signs of acute reactions and
stop the transfusion before a critical volume of blood has been administered is essential to preventing harm to the patient.
Crucial in the prevention of transfusion
mishaps are training and assessment of
personnel performing transfusions. Active
participation by physicians and management, as well as by nursing, technical, and
clinical personnel, is essential.

Nonimmune-Mediated Hemolysis

Causes
Red cells may undergo in-vitro hemolysis
if the unit is exposed to improper temperatures during shipping or storage or is
mishandled at the time of administration.
Malfunctioning blood warmers, use of
microwave ovens or hot waterbaths, or inadvertent freezing may cause temperature-related damage. Mechanical hemolysis
may be caused by the use of roller pumps
(such as those used in cardiac bypass surgery), pressure infusion pumps, pressure
16
cuffs, or small-bore needles. Osmotic
hemolysis in the blood bag or infusion set
may result from the addition of drugs or
hypotonic solutions. Inadequate deglycerolization of frozen red cells may cause
the cells to hemolyze after infusion. Finally,
hemolysis may be a sign of bacterial
growth in blood units. In a patient with
transfusion-associated hemolysis for which
both immune and nonimmune causes
have been eliminated, the possibility might
be considered that the patient or donor has
an intrinsic red cell defect, such as glucose-6-phosphatase dehydrogenase deficiency, causing coincidental hemolysis.

Chapter 27: Noninfectious Complications of Blood Transfusion

Treatment
Treatment depends on the severity of the
reaction. If the patient develops a severe
reaction with hypotension, shock, and renal dysfunction, intensive clinical management is required even before the cause
of the mishap is investigated. If the patient exhibits only hemoglobinemia and
hemoglobinuria, supportive therapy may
be sufficient.

Prevention
There should be written procedures for all
aspects of procuring, processing, and issuing blood, and administering transfusions.
All staff should be trained in the proper
use of equipment, intravenous solutions,
and drugs used during the administration
of blood and blood components. Equipment must be properly maintained and
records kept of how and when items are
used. Intravenous medications shall not
be injected into blood bags, unless approved by the Food and Drug Administration (FDA) or documented to be safe for
that purpose,17(p48) and care must be exercised in the selection and use of intravenous access devices. Chapter 22 discusses
the details of administering transfusions.

Transfusion-Associated Sepsis
Bacterial contamination of transfused blood
should be considered if the patient experiences severe rigors, especially if they are
accompanied by cardiovascular collapse
18
and/or fever over 40 C. For a more detailed discussion of this potentially lifethreatening transfusion complication, see
Chapter 28.

Febrile Nonhemolytic Reactions

Pathophysiology and Manifestations
A febrile nonhemolytic transfusion reaction (FNHTR) is often defined as a tem-

643

perature increase of >1 C associated with
transfusion and without any other explanation. Such reactions are often associated
with chills or rigors. The 1 C definition is
arbitrary; the same events might cause
smaller temperature increments. Indeed,
some authors discuss reactions characterized by rigors or other symptoms, in the
absence of fever, as FNHTRs because of a
presumed common mechanism.2 In one
study of 108 reactions characterized by
chills, cold, or rigors, only 18 involved a
rise in temperature. 19 Febrile reactions
complicate 0.5% to 6% of nonleukocytereduced red cell transfusions. Previous
opportunities for alloimmunization, especially pregnancies and multiple transfusions, increase the frequency of FNHTRs
to red cells. The rate of such reactions is
much higher after platelet transfusion
(1-38%). Most FNHTRs are benign, although some may cause significant discomfort and hemodynamic or respiratory
changes. The temperature increase may
begin early in the transfusion or be delayed in onset for hours after completion
of the transfusion.
Many febrile reactions are thought to result from an interaction between antibodies in the recipient’s plasma and antigens
present on transfused lymphocytes, granulocytes, or platelets, most frequently HLA
antigens. There is also evidence that febrile
reactions, particularly those due to platelets, may be caused by the infusion of biologic response modifiers, including cytokines, that accumulate in the blood bag
during storage. Cytokine release in the recipient undoubtedly contributes to those
reactions that begin with recipient antibody
against donor leukocytes.2,20-22 Because fever
may be an initial manifestation of an acute
HTR or a reaction to transfusion of blood
contaminated with bacteria, any observation of an increase in temperature associated with transfusion warrants prompt at-

644

AABB Technical Manual

tention. The diagnosis of an FNHTR is
made after excluding other possible explanations for the fever, particularly a
hemolytic or septic reaction. Guidelines for
evaluating a suspected acute transfusion
reaction are presented later in this chapter.

Treatment
Traditionally, transfusion was discontin23
ued when an FNHTR occurred. However,
some clinicians believe that fever should
not routinely cause discontinuation of a
transfusion,2,24 depending on whether the
patient has symptoms, signs, or laboratory data that suggest hemolysis, transfusion-related acute lung injury (TRALI), or
bacterial contamination. The fever of an
FNHTR usually responds to antipyretics.
Acetaminophen is preferred to the use of
salicylates because the former drug does
not affect platelet function. Meperidine
injection may be useful in patients with
severe shaking chills. Antihistamines are
not indicated because most FNHTRs do
not involve histamine release.

Prevention
Febrile reactions in an alloimmunized
individual can often be prevented by
transfusion of leukocyte-reduced blood
components. Prevention of reactions
caused by cytokine accumulation during
storage requires that the leukocyte reduc2,19
tion be performed before storage, but
some patients will still react. With nonleukocyte-reduced platelets, cytokinemediated reactions may be less frequent
when the component(s) are less than or
equal to 3 days old. Plasma removal may
also decrease febrile reactions. Acetaminophen is commonly given before
transfusion, but there is no evidence that
the premedication lessens the incidence
of FNHTR symptoms due to prestorage
leukocyte-reduced platelets.25

Allergy; Urticaria (Hives) to Anaphylaxis

Pathophysiology and Manifestations
Allergic reactions to transfusion form a
continuum, with the vast majority clustered at the mild end, in the form of urticaria or “hives”—erythematous, sharply
circumscribed raised lesions, most often
present over the upper trunk and neck,
which may itch and which are not usually
accompanied by fever or other adverse
findings. At the other end of the spectrum
are anaphylactic reactions, in which there
are systemic symptoms including hypotension, loss of consciousness, shock,
and, in rare cases, death. The latter may
begin after infusion of only a few milliliters, but less severe reactions tend to take
longer to develop. The term “anaphylactoid” is used in transfusion medicine to
denote reactions in between these extremes, but it is also used to denote reactions that have clinical similarities to
anaphylaxis but different mechanisms.
Manifestations of these reactions may involve one or several systems, notably, the
skin (urticaria, generalized flushing or
rash, localized swelling or “angioedema”),
respiratory tract (upper or lower respiratory tract obstruction with cough, hoarseness, stridor, wheezing, chest tightness or
pain, dyspnea), the gastrointestinal tract
(cramps, nausea, vomiting, diarrhea), or
the circulatory system (tachycardia and
other arrhythmias including cardiac arrest).26 Fever is characteristically absent, a
feature that aids in differentiating these
reactions from hypotension due to a hemolytic reaction or bacterial contamination,
and from respiratory compromise caused
by TRALI (see below). The severity of allergic transfusion reactions may increase
with successive transfusions.
Allergic reactions are attributed to exposure to a soluble substance in donor plasma
that binds to preformed IgE antibodies on

Chapter 27: Noninfectious Complications of Blood Transfusion

mast cells, resulting in the activation and
release of histamine. This presumption is
based on the facts that reactions tend to recur in an affected recipient and that they
can be prevented by removal of the plasma
from cellular components or, in the case of
urticaria, by antihistamines. Anaphylactic
and anaphylactoid reactions are sometimes
associated with class, subclass, and allotype-specific antibodies against IgA, partic27
ularly in IgA-deficient patients. IgE antiIgA has been demonstrated in two patients
with common variable immunodeficiency
having reactions to immunoglobulin preparations.28 However, most of the IgA antibodies to which anaphylactic reactions are
27
attributed are of the IgG or IgM class, and
these antibodies are demonstrable in only a
minority of the anaphylaxis cases referred
for study (17.5% in the series of Sandler et
al27). Moreover, IgA antibodies are common
but anaphylactic reactions are not. Therefore, demonstration of anti-IgA in an individual who has not been transfused does
not predict anaphylaxis. Other allergens or
other mechanisms are likely.
Severe allergic reactions have been reported in patients with antibodies directed
against C4 determinants,29,30 haptoglobin,31
and elements of nonbiologic origin such as
ethylene oxide used for sterilizing tubing
sets.32 However, the causative antigens have
not been identified in the vast majority of
cases. Reactions caused by passively transferred donor antibody have rarely been
33,34
documented.
Hypotensive reactions mimicking anaphylaxis have been observed in patients
taking angiotensin-converting enzyme
(ACE) inhibitors who receive albumin during plasma exchange.35 They were thought
to be due to inhibition of bradykinin catabolism by the ACE inhibitors combined with
bradykinin activation by low levels of prekallikrein activator (a Hageman factor fragment) in the albumin used for replacement.

645

Similarly, bradykinin activation by prekallikrein activity in plasma protein fraction
has also been implicated in hypotensive reactions,36 and a similar mechanism is probably responsible for the many patients
taking ACE inhibitors reported to have
hypotensive reactions when receiving
blood components via bedside leukocyte
reduction filters.37-39 Similar reactions have
been observed in association with the contact of plasma with charged dialysis membranes, low-density lipoprotein adsorption
columns, and staphylococcal protein A
immunoadsorption columns. Other mechanisms that have been proposed include the
infusion of complement-derived anaphyla26
toxins and histamine. The differentiation
and appropriate classification of these different reactions will require additional research and refined diagnostic tools.

Frequency
Urticaria may complicate as many as 1%
to 3% of transfusions, the observed frequency depending on how vigorously it is
sought. The incidence of anaphylactic reactions fortunately is low, estimated to be
1 in 20,000 to 50,000 units. The SHOT data
suggest that anaphylaxis is much more
common as a complication of plasma and
platelet transfusions, than of red cells14;
although these reactions may have contributed to the death of a few severely ill
patients, they were not a primary cause of
death. The mortality rate reported to the
FDA is about 1 per year.26

Treatment
If urticaria is the only adverse event noted,
the transfusion may be temporarily interrupted while an antihistamine (eg, diphenhydramine, 25-50 mg) is administered orally or parenterally. If symptoms

646

AABB Technical Manual

are mild and promptly relieved, the transfusion may be resumed, provided the
interrupted infusion can be completed
within the acceptable time (see Chapter
22). If the patient develops severe urticaria,
a significant local swelling, respiratory or
gastrointestinal symptoms, or hypotension,
26
the transfusion should be discontinued.
The immediate treatment of an anaphylactic transfusion reaction should be to stop
the transfusion and treat hypotension by
placing the patient in the Trendelenberg
(feet up) position and administering a fluid
challenge. If the blood pressure does not
improve immediately, epinephrine should
be given. In mild to moderate cases, epinephrine (1:1000) should be delivered subcutaneously or intramuscularly in a starting
dose of 0.3 to 0.5 mL in adults, or 0.01
mL/kg in children. This dose may be repeated a second and third time at 5- to
15-minute intervals. In severe reactions (eg,
systolic blood pressure below 80 mm Hg,
laryngeal edema with upper airway compromise, or respiratory failure), the drug
should be given intravenously (1:10,000) for
the most rapid effect because drug absorption is unreliable in hypotensive patients.
Aerosolized or intravenous beta-2 agonists
and theophylline may be required in selected patients in whom bronchospasm is
unresponsive to epinephrine treatment, or
in whom epinephrine is ineffective because
of pre-existing beta-blocker therapy. Oxygen therapy should be administered as required, with endotracheal intubation if
there is significant upper airway obstruction. Continued hemodynamic instability
may require invasive hemodynamic monitoring. Under no circumstances should the
transfusion be restarted. Coincidental occurrence of myocardial infarction, pulmonary embolism, or other medical catastrophes could present with hypotension and
respiratory compromise and should be
considered.26

Prevention
Recipients who have frequent transfusionassociated urticarial reactions may respond well to administration of antihistamine (eg, 25-50 mg of diphenhydramine)
one-half hour before transfusion. However,
diphenhydramine should not be given
routinely without a history of previous allergic reactions, particularly to elderly pa40
tients. If antihistamine administration is
insufficient, 100 mg of hydrocortisone given
1 hour before transfusion may be useful.
If reactions are recurrent and severe or associated with other allergic manifestations
in spite of adequate premedication, transfusion of washed red cell or platelet components, or red cells that have been frozen,
thawed, and deglycerolized will usually be
tolerated.
Patients who have had a prior life-threatening anaphylactic reaction and who are
IgA-deficient or have a demonstrated IgA
antibody should receive blood components
that lack IgA, either by washing or preparation of components from IgA-deficient
blood donors. Severe reactions that are not
caused by anti-IgA can be prevented only
by maximal antiallergy immunosuppression or washing. A need for red cells may be
met by the use of washed or frozen, thawed,
26
and deglycerolized units. Washed platelets
are generally not readily available and may
result in decreased platelet recovery, func41
tion, and survival ; therefore, after the first
reaction, if there is no evidence that the reaction is mediated by IgA, some centers
elect to rechallenge the patient under
closely controlled circumstances. Prevention of anaphylactoid reactions in patients
such as those with thrombotic thrombocytopenic purpura who absolutely require
plasma components may be a tremendous
challenge if IgA-deficient donors will not
suffice. Pretreatment with antihistamines,
corticosteroids (starting with 100 mg of hy-

Chapter 27: Noninfectious Complications of Blood Transfusion

drocortisone), and ephedrine may help. Finally, it may be possible to collect and store
autologous blood components from patients
known to have experienced anaphylactic
reactions.

Transfusion-Related Acute Lung Injury

Pathophysiology and Manifestations
TRALI should be considered whenever a
transfusion recipient experiences acute
respiratory insufficiency and/or X-ray
findings are consistent with bilateral pulmonary edema but has no other evidence
of cardiac failure or a cause for respiratory
failure. The severity of the respiratory distress is usually disproportionate to the
volume of blood infused. The reaction
typically includes fever, chills, and hypotension, usually occurring during or within 1 to 2 after transfusion, often with an
immediate and dramatic onset. Implicated
components always have contained plasma,
but the volume may be as small as that of
a unit of cryoprecipitate or RBCs in an additive solution.42,43
Because the manifestations of TRALI are
variable and may overlap with those of the
patient’s underlying medical problems, it is
useful to define the syndrome, particularly
for the purpose of conducting studies of its
epidemiology and pathogenesis. A consensus conference of the blood services in
Canada developed such a definition.44 The
panel defined acute lung injury (ALI) as a
syndrome of: 1) acute onset; 2) hypoxemia
(PaO2/FIO2 <300 mm Hg, or O2 saturation
<90% on room air, or other clinical evidence); 3) bilateral lung infiltrates on a
chest x-ray; and 4) no evidence of circulatory overload. TRALI is then defined as: 1)
new ALI occurring during transfusion or
within 6 hours of completion; and 2) no
other temporally associated ALI risk factors.
If the latter are present, the case is considered “Possible TRALI.” Risk factors for ALI

647

include aspiration, pneumonia, toxic inhalation, lung contusion, near drowning,
severe sepsis, shock, multiple trauma, burn
injury, acute pancreatitis, cardiopulmonary
bypass, and drug overdose. It was noted
that such a definition will not include cases
of mild respiratory embarrassment having
a similar pathogenesis, cases of ALI in patients with circulatory overload, and cases
in which a transfusion-related process causes
worsening of pre-existing ALI.44
TRALI may result from multiple mechanisms. Donor antibodies directed against
recipient HLA class I or II antigens, or
neutrophil antigens of the recipient, have
been demonstrated45-47 and are thought to
cause a sequence of events that increase the
permeability of the pulmonary microcirculation so that high-protein fluid enters
the interstitium and alveolar air spaces. Infrequently, antibodies in the recipient’s circulation against HLA or granulocyte antigens initiate the same events.45,46,48 Although
one would expect causative antibodies to
be far more common in recipients than donors, the rarity of TRALI due to recipient
antibody might be due to the fact that the
pool of target leukocytes is much smaller in
a cellular blood component than in a recipient’s circulation. Monocyte activation, with
expression of cytokines including IL-1β,
TNFα, and tissue factor, has been demonstrated, and these reactions were highly
specific for cells bearing the target antigens.48
Perfusion of neutrophils, complement, and
neutrophil-specific antibody into an ex-vivo
rabbit lung preparation causes severe edema,49
and autopsy studies demonstrate neutrophil aggregation in the lungs of patients
who have died of TRALI.50 These and other
observations suggest that pulmonary edema
in TRALI is caused by neutrophil-mediated
endothelial damage, initiated by antibodies
activating neutrophils directly or via activation of monocytes, pulmonary macrophages,
and/or endothelial cells.

648

AABB Technical Manual

As the spectrum of antibodies implicated
in cases of TRALI broadens, more cases will
appear to be antibody-mediated. However,
other mechanisms have been proposed as
causes for transfusion-related respiratory
failure. Severe pulmonary reactions are reported after granulocyte transfusions, particularly in patients with known or unapparent lung infections or with conditions
likely to promote prompt complement acti51
vation. Other factors may include anaphylatoxins C3a and C5a, aggregation of granulocytes into leukoemboli that lodge in the
pulmonary microvasculature, or transfusion
of cytokines that have accumulated in
stored blood components. Recently, reactive lipid products from donor blood cell
membranes have been implicated as potential granulocyte activators in the pathogenesis of TRALI.52 These substances accumulate during blood bank storage and
prime neutrophils to produce vasoactive
mediators in response to a second stimulus
such as infection. One nested case-control
study found that component age and levels
of bioactive lipids, but not leukocyte antibodies, were associated with TRALI.53
The incidence rate of TRALI is not known,
but data from one institution in the 1980s
suggest that this complication may occur as
frequently as 1 in 5000 transfusions.45 The
passive surveillance data of 5 years of SHOT
reports14 include 70 TRALI cases (approximately 1 per 250,000 total components), of
which 18 cases were fatal (includes six definite, two probable, and 10 possible TRALIrelated fatalities). In this series, TRALI was
the most common cause of morbidity and
mortality, ahead of transfusion-associated
graft-vs-host disease (TA-GVHD), ABO incompatibility, and bacterial contamination.
The SHOT data suggest that the rate of
TRALI is higher after plasma and platelet
transfusion. Conclusions regarding incidence
and fatality rates will, of course, depend on
the definition of TRALI used.

Treatment
If any kind of acute pulmonary reaction is
suspected, the transfusion should be
stopped immediately and the same unit
should not be restarted even if symptoms
abate. Clinical management focuses on
reversing progressive hypoxemia with oxygen therapy and ventilatory assistance, if
necessary. The role of intravenous steroids is unproved. Unlike other forms of
acute respiratory distress syndrome, most
patients recover adequate pulmonary
function within 2 to 4 days,42,45 and the observed mortality is less than 6% to 23%
(Holness L, personal communication).

Prevention
If antibody in donor plasma can be shown
to have caused an acute pulmonary reaction, blood from that donor should not be
used for plasma-containing components.
No special precautions are needed for the
patient if the problem was donor-specific
and components from other donors are
available. Current policy in the United
Kingdom is not to prepare plasma from
female donors.

Circulatory Overload

Pathophysiology and Manifestations
Transfusion therapy may cause acute pulmonary edema due to volume overload,
and this can have severe consequences,
including death. Few data are available on
the incidence rate of transfusion-induced
circulatory overload in the general population, but young children and the elderly
are considered most at risk, and incidence
rates of up to 1% have been observed in a
study of elderly orthopedic patients. 54
Rapid increases in blood volume are especially poorly tolerated by patients with
compromised cardiac or pulmonary status and/or chronic anemia with expanded

Chapter 27: Noninfectious Complications of Blood Transfusion

plasma volume. The infusion of 25% albumin, which shifts large volumes of extravascular fluid into the vascular space, may
also cause circulatory overload. Hypervolemia must be considered if dyspnea,
cyanosis, orthopnea, severe headache, hypertension, or congestive heart failure occur during or soon after transfusion. Elevated levels of brain natriuretic peptide
may be seen in cases of circulatory over55
load, and this test may be useful in separating such cases from cases of TRALI.

Treatment
Symptoms usually improve when the infusion is stopped, and it should not be restarted until volume overload has been
addressed. Placing the patient in a sitting
position may help. Diuretics and oxygen
are often indicated and, if symptoms are
not relieved, multiple medical interventions may be required, including phlebotomy.

Prevention
Except in conditions of ongoing, rapid
blood loss, anemic patients should receive blood transfusions slowly, with attention to total fluid input and output.
The administration of diuretics before
and during the transfusion may be helpful.

Complications of Massive Transfusion
Among the numerous complications that
may accompany massive transfusion,
metabolic and hemostatic abnormalities
are matters of particular concern. Some
or all of the following metabolic derangements can depress left ventricular function: hypothermia from refrigerated blood,
citrate toxicity, and lactic acidosis from
systemic underperfusion and tissue
ischemia, often complicated by hyperkalemia. Metabolic alkylosis due to me-

649

tabolism of citrate can occur after massive transfusion but is probably not clinically significant. Patients who are losing
blood rapidly may have pre-existing or
coexisting hemostatic abnormalities or
develop them during resuscitation.
Hemostatic abnormalities may include
dilutional coagulopathy, DIC, and liver
and platelet dysfunction.

Citrate Toxicity
Pathophysiology and Manifestations.
When large volumes of FFP, Whole Blood,
or Platelets are transfused rapidly, particularly in the presence of liver disease,
plasma citrate levels may rise, binding
ionized calcium and causing symptoms.
Citrate is rapidly metabolized, however,
56
so these manifestations are transient.
Hypocalcemia is more likely to cause clinical manifestations in patients who are in
shock or are hypothermic. Prolonged
apheresis procedures put patients, and
occasionally blood donors, at some risk.
Exchange transfusion, especially in tiny
infants who are already ill, requires careful attention to all electrolytes.
A decrease in ionized calcium increases
neuronal excitability, leading, in the awake
patient or apheresis donor, to symptoms of
perioral and peripheral tingling, shivering,
and lightheadedness, followed by a diffuse
sense of vibration, tetanic symptoms such
as muscle cramps, fasciculations and
spasm, and nausea. In the central nervous
system, hypocalcemia is thought to increase the respiratory center’s sensitivity to
CO2, causing hyperventilation. Because
myocardial contraction is dependent on
the intracellular movement of ionized calcium, hypocalcemia also depresses cardiac
function.57
Treatment and Prevention. Massively
transfused patients, particularly those with
severe liver disease or those undergoing

650

AABB Technical Manual

rapid apheresis procedures such as peripheral blood progenitor cell collections, may
benefit from calcium replacement. It should
be noted, however, that empiric replacement therapy in the era before accurate
monitoring of ionized calcium was available was associated with iatrogenic mortality.58 Usually, however, unless a patient or
donor has a predisposing condition that
hinders citrate metabolism, hypocalcemia
due to citrate overload requires no treatment other than slowing the infusion. Calcium must never be added directly to the
blood container because the blood will clot.

Hypothermia
Pathophysiology and Manifestations.
Ventricular arrhythmias may occur in patients who receive rapid infusions of large
volumes of cold blood, and they can be
prevented by blood warming.59 The effect
of cold blood is presumed to be more
likely if the blood is administered via central catheters positioned close to the cardiac conduction system.60 Hypothermia
increases the cardiac toxicity of hypocalcemia or hyperkalemia and can result
in poor left ventricular performance.
Other complications of hypothermia include impaired hemostasis 6 1 and increased susceptibility to wound infections.62 Blood warming is a must during
massive transfusion of cold blood.
Treatment and Prevention. Hypothermia-induced arrhythmias are reduced by
avoiding rapid infusion of cold blood into
the cardiac atrium. Generalized effects of
hypothermia can be prevented by using
blood warmers. AABB Standards for Blood
Banks and Transfusion Services mandates
that warmers have a temperature monitor
and a warning system to detect malfunction and prevent hemolysis.17(p6) Attention to
proper protocol is critical during the use of
blood warming devices because overheat-

ing destroys red cells and has caused fatalities.5

Hyperkalemia and Hypokalemia
Pathophysiology. When red cells are stored
at 1 to 6 C, the potassium level in the supernatant plasma or additive solution increases. Although the concentration in the
plasma/anticoagulant portion of an RBC
unit may be high (see Chapter 8), because
of the small volume, the total extracellular
potassium load is less than 0.5 mEq for
fresh units and only 5 to 7 mEq for units at
their outdate. This rarely causes hyperkalemic problems in the recipient because
rapid dilution, redistribution into cells, and
excretion blunt the effect. Hypokalemia is
probably more often observed63 because
potassium-depleted red cells reaccumulate
this intracellular ion, and citrate metabolism causes movement of potassium into
the cells in response to the consumption
of protons. Hyperkalemia may be a problem in patients with renal failure and in
premature infants and newborns receiving relatively large transfusions, such as in
cardiac surgery or exchange transfusion;
otherwise, it can be demonstrated only as
a transient effect in very rapid transfusion.
Treatment and Prevention. No treatment or preventive strategy is usually necessary, provided the patient is adequately
resuscitated from whatever condition required the massive transfusion.63 For largevolume transfusion to sick infants or adults
at risk, many professionals prefer red cells
that are no more than 5 to 14 days old or
washed units. However, for infants receiving small-volume transfusions infused
slowly, units may be used safely until their
expiration date.64 There is no evidence that
routine red cell transfusions require manipulation to lower potassium levels, even in
patients with no renal function.

Chapter 27: Noninfectious Complications of Blood Transfusion

Coagulopathy in Massive Transfusion
Pathophysiology. Of greater concern is the
occurrence of coagulopathy during massive transfusion. Classically, this coagulopathy is ascribed to dilution of platelets
and clotting factors, which occurs as patients lose hemostatically active blood. The
lost blood is initially replaced with red
cells and asanguinous fluids. Classic stud65
66
ies of military and civilian trauma
patients receiving stored Whole Blood
demonstrated a progressive increase in the
incidence of “microvascular bleeding” (MVB)
characteristic of a coagulopathy with increasing transfusion, typically occurring after replacement of two to three blood volumes (20 to 30 Whole Blood units). Although
platelet counts, coagulation times, and levels of selected clotting factors all correlated
with volume transfused, contrary to expectations from a simple dilutional model,
the relationship was marked by tremendous variability. Inspection of laboratory
parameters in the patients developing a
bleeding diathesis, as well as the response
to various hemostatic components, suggested that platelet deficits were more important in causing the bleeding than were
coagulation factor deficiencies. MVB typically occurred when the platelet count fell
below 50,000 to 60,000/µL. On the other
hand, no simple relationship could be determined between a patient’s coagulation
tests and the onset of bleeding.
Subsequent studies have refined these
observations. Significant platelet dysfunction has been demonstrated in massively
67,68
transfused trauma patients. In the stud66,69
ies of Counts and coworkers, low fibrinogen and platelet levels were better predictors of hemostatic failure than elevations
of prothrombin time (PT ) and partial
thromboplastin time (PTT), suggesting that
consumption coagulopathy was an important factor in addition to dilution. A similar

651

conclusion was reached by Harke and
Rahman,70 who showed that the degree of
platelet and clotting abnormalities correlated with the length of time the patient
was hypotensive, in groups of patients receiving similar transfusion volumes, also
suggesting that the most important cause
was DIC due to shock. Taking these data to71
gether, Collins concluded that “...coagulopathy in heavily transfused patients was due
to hypoperfusion, not transfusion.”
These data may not be generalizable to
patients undergoing massive transfusion in
the “clean” setting of the operating room,
where hypotension due to volume loss is
prevented. In this setting, coagulation factor levels may indeed have priority over
platelet problems.72
Treatment and Prevention. The dilutional model of coagulopathy in massive
transfusion would suggest that prophylactic
replacement of hemostatic components
based on the volume of red cells or whole
blood transfused would prevent development of a bleeding diathesis. However, prospective studies have consistently shown
that such regimens do not work,73 perhaps
due to patient variability. Instead, replacement of platelets and coagulation factors in
the massively transfused trauma or surgical
patient should be based on characterization of the specific abnormality by use of
platelet counts, the PT (international normalized rate), aPTT, and fibrinogen levels.
Thromboelastography may also be useful.
It is imperative that the laboratory rapidly
complete testing. Empiric therapy with
platelets and/or plasma may be initiated
immediately after specimens are obtained.

Air Embolism
Air embolism can occur if blood in an open
system is infused under pressure or if air
enters a central catheter while containers
or blood administration sets are being

652

AABB Technical Manual

changed. It has been reported in association with intraoperative and perioperative
blood recovery systems that allow air into
the infusion bag.7 The minimum volume
of air embolism that is potentially fatal for
an adult is approximately 100 mL.74 Symptoms include cough, dyspnea, chest pain,
and shock.
If air embolism is suspected, the patient
should be placed on the left side with the
head down, to displace the air bubble from
the pulmonic valve. Aspiration of the air is
sometimes attempted. However, proper use
of infusion pumps, equipment for blood recovery or apheresis, and tubing couplers is
still essential to prevent this complication.

Evaluation of a Suspected
Acute Transfusion Reaction
The Role of Clinical Personnel Attending
the Patient
Medical personnel attending the patient
are generally the first to suspect that a
transfusion reaction has occurred and the
first to take action. The appropriate actions
should be specified in the institution’s patient care procedures manual, and transfusion service personnel should be prepared to act as consultants.
1.
If a transfusion reaction is suspected,
the transfusion should be stopped to
limit the volume of blood infused.
2.
All labels, forms, and patient identification should be checked to determine whether the transfused component was intended for the recipient.
3.
An intravenous line should be maintained with normal saline (0.9% sodium chloride), at least until a medical evaluation of the patient has been
completed.
4.
The transfusion service and the patient’s physician should be notified

immediately. A responsible physician
should evaluate the patient to determine whether a transfusion reaction
is a possibility, what kind it might be,
and what immediate actions should
be undertaken. The possibilities of acute
hemolytic reaction, anaphylaxis, transfusion-induced sepsis, and TRALI
should be kept in mind because these
conditions require aggressive medical
management and must be reported
promptly to the laboratory.
If the observed events are limited to
urticaria or circulatory overload, the
transfusion service need not evaluate postreaction blood samples. If there
are signs and symptoms other than
urticaria or circulatory overload,
particularly if there is any possibility
of acute HTR, anaphylaxis, TRALI,
transfusion-induced sepsis, or other
serious problem, a postreaction blood
sample(s) should be sent to the laboratory for evaluation. The specimen(s) must be carefully drawn to
avoid mechanical hemolysis and
must be properly labeled. In addition, the transfusion container with
whatever contents remain, the administration set (without the needle), and the attached intravenous
solutions should be sent to the laboratory, following standard precautions. In some cases, a postreaction
urine sample will be useful.

The Role of the Laboratory
Whenever hemolysis is a possibility, the
laboratory should perform three steps as
soon as possible after receiving notification and the clinical material: check for
clerical errors; perform a visual check for
hemolysis; and check for evidence of blood
group incompatibility by performing a direct antiglobulin test (DAT) and a recon-

Chapter 27: Noninfectious Complications of Blood Transfusion

firmation of the recipient’s ABO type. Some
laboratories do not follow this sequence
when the only manifestations are urticarial or febrile reactions to ABO-compatible platelets.

Check for Identification Errors
The identification of each patient’s sample and the blood component(s) must be
checked for errors. If an error is discovered, the patient’s physician or other responsible health-care professional must
be notified immediately, and a search of
appropriate records should be initiated to
determine whether misidentification or
incorrect issue of other specimens or
components has put other patients at risk.
Once the acute crisis has passed, each step
of the transfusion process should be reviewed to find the source of error.

653

oration. An increase in bilirubin may begin
as early as 1 hour after the reaction, peak
at 5 to 7 hours, and disappear within 24
hours if liver function is normal.
In examining a postreaction urine specimen, it is important to differentiate among
hematuria (intact red cells in the urine),
hemoglobinuria (free hemoglobin in the
urine), and myoglobinuria (free myoglobin
in the urine). In acute HTRs, free hemoglobin released from damaged cells can cross
the renal glomeruli and enter the urine, but
hematuria and myoglobinuria would not
be expected. Urine examination should be
done on the supernatant fluid after centrifugation of a freshly collected specimen;
misinterpretation can occur if free hemoglobin is released when previously intact
red cells in a specimen undergo in-vitro
hemolysis during transportation or storage.

Visual Check for Hemolysis

Serologic Check for Incompatibility

The serum or plasma in a postreaction
blood specimen must be inspected for evidence of hemolysis and compared with a
prereaction sample, if available. Pink or
red discoloration after, but not before, the
reaction suggests destruction of red cells
and release of free hemoglobin. Intravascular hemolysis of as little as 2.5 mL of red
cells may produce visible hemoglobinemia.75
Hemolysis resulting from poor collection
technique or other medical interventions
can cause hemoglobinemia; if faulty sampling is suspected, examination of a second specimen should resolve the question.
Myoglobin, released from injured muscle,
may also cause pink or red plasma and
might be suspected if a patient has suffered severe trauma or muscle injury.76(p369)
If the sample is not drawn until 5 to 7
hours after an episode of acute hemolysis,
hemoglobin degradation products, especially bilirubin, may be in the bloodstream and cause yellow or brown discol-

A DAT must be performed on a postreaction specimen, preferably one anticoagulated with a chelating agent (such as EDTA)
to avoid in-vitro coating of red cells by
complement proteins. If the postreaction
DAT is positive, a DAT should be performed
on red cells from the pretransfusion specimen (unless this was already done as part
of pretransfusion testing) and compared.
If transfused incompatible cells have been
coated with antibody but not immediately
destroyed, the postreaction specimen DAT
is likely to be positive, often with a mixedfield agglutination pattern. If the transfused cells have been rapidly destroyed,
the postreaction DAT may be negative,
particularly if the specimen is drawn several hours later. If both the pre- and postreaction DATs are positive, further workup is required to rule out incompatibility.
Comparison of the graded strength of these
two tests is not a reliable method to rule
this out. Nonimmune hemolysis (eg, from

654

AABB Technical Manual

thermal damage or mechanical trauma)
causes hemoglobinemia but not a positive DAT. The recipient’s ABO type must
also be confirmed on the postreaction
specimen.

Additional Laboratory Evaluation
If any of the three initial checks and tests
(error check, visual inspection for hemoglobinemia, DAT and ABO confirmation)
gives positive or suspicious results, the diagnosis of an acute HTR should be vigorously pursued. Even if no error or apparent
incompatibility is found, the possibility of
an acute HTR should still be considered if
the patient’s clinical presentation is consistent with such a reaction. The tests
listed below help characterize the cause
of the HTR, if one has occurred, or help
clarify the immunologic and serologic status of patients in whom the diagnosis is
unclear. Some or all may be performed
following a written institutional protocol
or at the discretion of the physician in
charge of the transfusion service.
1.
If ABO and Rh typing on the prereaction and postreaction samples do not
agree, there has been an error in patient or sample identification, or in
testing. If sample mix-up or mislabeling has occurred, another patient’s specimen may also have been
incorrectly labeled; it is important to
check the records of all specimens
received at approximately the same
time.
2.
Perform ABO and Rh testing on blood
from the unit or an attached segment. If blood in the bag is not of the
ABO type noted on the bag label,
there has been an error in unit labeling.
3.
Perform antibody detection tests on
the prereaction and postreaction
samples and on the donor blood. If a

previously undetected antibody is
discovered, it should be identified.
Once the antibody has been identified, retained samples from transfused
donor units should be tested for the
corresponding antigen. If a previously undiscovered antibody is present in a postreaction specimen but
not in a prereaction sample, the reason may be 1) a sample identification error, 2) anamnestic antibody
production after a recent transfusion,
or, less likely, 3) passive transfer of
antibody from a recently transfused
component. It may be desirable to use
enhancement techniques, such as an
increased serum-to-cell ratio, lowionic-strength saline, Polybrene, polyethylene glycol, or enzyme techniques, when retesting the prereaction
specimen.
Repeat crossmatch tests, with prereaction and postreaction samples in
parallel using the antiglobulin technique. A positive crossmatch in the
face of a negative antibody screening test may indicate the presence of
an antibody directed against a lowincidence blood group antigen.
Perform DAT and antibody detection
tests on additional specimens obtained at intervals after the transfusion reaction. A first postreaction
sample may have serologically undetectable levels of a significant alloantibody, especially if all the antibody molecules have attached to the
incompatible transfused cells. In this
event, antibody levels would rise
rapidly, and antibody detection and
identification would become possible within a few days.
Perform frequent checks of the patient’s hemoglobin values, to see
whether the transfused cells produce
the expected therapeutic rise, or

Chapter 27: Noninfectious Complications of Blood Transfusion

whether a decline occurs after an
initial increase. In patients with sickle
cell anemia, the survival of transfused red cells can be followed by
evaluation of the levels of hemoglobin A. In complex cases, phenotypic
differences between autologous and
transfused cells quantitated by flow
cytometry have been used to follow
survival.77
In-vivo red cell survival studies have
been used to demonstrate the rare
occurrence of an acute HTR in the
absence of detectable alloantibody.78
When the patient is phenotyped in
preparation for such studies, it is important that the sample be one that
contains only the patient’s red cells.
This may be difficult if the patient
has received transfusions within the
previous several weeks. Method 2.15
gives a technique for obtaining autologous red cells from a patient who
has been transfused. If an antigen is
present on the donor’s red cells and
absent from those of the patient, its
presence or absence in postreaction
samples indicates whether the transfused cells have survived and remained in the circulation.
Markers of hemolysis, including lactate dehydrogenase, unconjugated
bilirubin, and haptoglobin levels,
may be useful, particularly if preand multiple postreaction measurements are available.
Examine the blood remaining in the
unit and the administration tubing
for evidence of hemolysis, especially
if no immune explanation for hemolysis can be demonstrated. Depending
on how the blood was handled and
administered, hemolysis may be
present in the container and the administration tubing, or only in the
administration tubing. For example,

655

if the unit had been inappropriately
heated in the container, both the
blood in the container and in the
administration tubing would be
hemolyzed. If a faulty infusion device had been used during blood
administration, hemolysis might be
present in the administration tubing,
but not in the container.
Additional testing may also be useful for
significant nonhemolytic reactions.
1.
If the presentation suggests an anaphylactic reaction, test the patient’s
serum for the presence of anti-IgA.
Preliminary information can be obtained by quantitation of IgA because
most patients with IgA-related anaphylaxis have been IgA deficient.27
Note, however, that subclass- or allotype-specific antibodies may develop in patients with normal IgA
levels.26 If additional transfusions are
required, cellular components can
be washed; if plasma (or platelets;
see above) is required, IgA-deficient
plasma can be used.
2.
Examine the returned unit for any
abnormal appearance, including clots
or any brownish, opaque, muddy, or
purple discoloration. If the clinical
presentation suggests bacterial sepsis, a Gram stain and bacterial cultures of the contents should be performed, even if the unit looks normal.
Blood cultures should also be performed on the patient’s blood. 7 9
Treatment for suspected bacterial
contamination should be based on
clinical considerations because a delay in therapy may result in severe
morbidity or death. Treatment includes prompt intravenous administration of broad-spectrum antibiotics after blood and other appropriate
cultures have been obtained, combined
with therapy for shock, if present.

656

AABB Technical Manual

If the clinical presentation suggests
TRALI, test the patient’s pretransfusion sample and a sample of the donor’s plasma for antibodies to HLA and
neutrophil antigens. Crossmatching
recipient lymphocytes or granulocytes
with implicated donor sera can provide supportive evidence for TRALI.

Delayed Consequences of
Transfusion
Alloimmunization to Red Cell Antigens

Pathophysiology
Primary alloimmunization, evidenced by
the appearance of newly formed antibodies to red cell antigens, becomes apparent
weeks or months after transfusion. It has
been estimated that alloimmunization
occurs in unselected immunocompetent
recipients with a risk of 1% to 1.6% per
RBC unit, provided that D-negative recipients receive D-negative cellular components.80 Hemolysis has been reported in
cases of primary immunization, but these
reports are controversial,81 and, even if it
occurs, the phenomenon must be very
rare and usually subclinical.
Serologic Observations. Once alloimmunization has occurred, blood group
antibodies can become undetectable, especially those of the Kidd system. One investigator reported that this occurred to 29% of
82
antibodies after a median of 10 months
and to 41% of antibodies after 5 or more
years.83 If red cells that express the antigen
are subsequently transfused, however, an
anamnestic response may cause the appearance, within hours or days, of IgG antibodies that react with the transfused red
cells. In a prospective study, previously undetected alloantibodies were found in 58 of
2082 (2.8%) recipients (37% known previously transfused, 36% previously pregnant)

within 7 days of transfusion.84 In two of the
58, only the DAT was positive, but, in all
others, repeat antibody screening would
have detected the new antibody. Regardless, characterization of an eluate is necessary because alloantibodies may be present
on the red cells that are not in the serum. If
the clinical laboratory discovers an anamnestic response, both the transfusion service director and the patient’s clinician
should be notified and the possibility of a
delayed HTR (DHTR) should be investigated.
Delayed Reactions. In most cases, anamnestic antibody production does not cause
detectable hemolysis, leading to the designation “delayed serologic transfusion reac85
tion” (DSTR). However, in some patients,
clinically apparent hemolysis will result
from the combination of significant levels
of antibody with hemolytic potential and
large numbers of transfused red cells in the
circulation; in the study cited above, only
one of the 58 recipients with a new antibody within 7 days of transfusion had clinically evident hemolysis.84 This translated
into a DHTR rate of one per 2082 recipients,
or one for every 11,328 units transfused. As
would be expected, retrospective studies,
which would be similar to the routine experience of a transfusion service, yield a lower
rate of DSTRs, but the rate of clinically detectable hemolysis may be roughly equivalent.85-87 The most common presentation of
a DHTR is a declining hemoglobin and a
newly positive antibody screen, but fever,
leukocytosis, and mild jaundice may be
present. Some DHTRs present as the absence of the anticipated increase in hemoglobin after transfusion. Other clinical
problems are infrequent; hemoglobinuria is
occasionally noted, but acute renal failure
is uncommon. However, DHTRs may be
particularly problematic in patients with
sickle cell disease. In these patients,
hemolysis may include autologous red
cells, a phenomenon termed sickle cell

Chapter 27: Noninfectious Complications of Blood Transfusion

hemolytic transfusion reaction syndrome
(see Chapter 24).88
If a DHTR is suspected, a freshly obtained blood sample may be tested for unexpected alloantibodies, both in the serum
and, by DAT, on the red cells. Discovery of a
new red cell alloantibody in a recently
transfused patient with hemolysis strongly
suggests a DHTR, and the diagnosis is supported by demonstration of the corresponding antigen on the red cells from a
retained segment from one or more transfused units. Antigen typing of the red cells
circulating in the patient may also suggest
whether the newly incompatible cells have
been eliminated, or whether some are still
circulating. Repeat antibody screening on
the patient’s previous specimen will rule
out technical errors.

Treatment

657

mandates permanent preservation of records of clinically significant antibodies,
and review of previous records before red
cells are issued for transfusion.17(pp39,72) Prospective antigen matching may prevent
DHTRs in selected patients, particularly
those with sickle cell disease (see Chapters 21 and 24).89

Posttransfusion Autoantibody
Occasionally, transfusion of allogeneic red
cells and platelets stimulates production
of autoantibodies; in some of these patients, hemolytic anemia or thrombocytopenia may occur.90 See Chapter 20 for more
details.

Alloimmunization to Leukocyte Antigens
and Refractoriness to Platelet
Transfusions
See Chapter 16 and Chapter 17.

Specific treatment is rarely necessary, although it may be prudent to monitor the
patient’s urine output and renal function
and observe for changes in coagulation
function. If transfusion is still necessary,
donor red cells should lack the antigen
corresponding to the newly discovered
antibody. Passenger lymphocyte hemolysis,
seen after solid organ transplantation, is a
variant of DHTR and is covered in Chapter 26.

Prevention
Future transfusions for the patient should
lack the antigen(s) responsible for the
anamnestic response, even if the antibody
again becomes undetectable. Some facilities issue a medical alert card with this information for the patient to carry and
present at the time of hospitalization or
transfusion in a different facility. It is to
prevent these problems that Standards for
Blood Banks and Transfusion Services

Transfusion-Associated Graft-vs-Host
Disease
Transfusion-associated graft-vs-host disease is a usually fatal immunologic transfusion complication caused by engraftment and proliferation of donor lymphocytes in a susceptible host.91 The engrafted
lymphocytes mount an immunologic attack against the recipient tissues, including hematopoietic cells, leading to refractory pancytopenia with bleeding and
infectious complications, which are primarily responsible for the 90% to 100%
mortality rate in afflicted patients. TAGVHD is rare in US transfusion recipients
and has been observed almost exclusively
in immunocompromised patients. In contrast, over 200 cases of TA-GVHD have
been described in Japan,92 with incidence
rates reaching 1:660 in patients undergoing cardiovascular surgery.93 Greater genetic homogeneity of the Japanese population and frequent use of fresh Whole

658

AABB Technical Manual

Blood from related donors are thought to
be the primary reasons for the surprisingly frequent occurrence of TA-GVHD in
that country. The SHOT data14 demonstrate a significant decline in the incidence of TA-GVHD since the introduction
of universal leukocyte reduction, but two
cases occurred despite leukocyte reduction.
In the first 3 years of this study, the rate of
TA-GVHD was approximately 1 per 600,000
cellular components transfused and, in
the first 5 years, accounted for a greater
number of deaths than acute HTRs and
only slightly fewer than did TRALI.

Pathophysiology and Manifestations
The pathophysiology of TA-GVHD is complex and incompletely understood. The
overall mechanism includes the escape of
donor T lymphocytes present in cellular
blood components from immune clearance
in the recipient and subsequent proliferation of these cells, which then mount an
immune attack on host tissues. Manifestations include fever, enterocolitis, rash,
hepatitis, and pancytopenia. The rash
typically begins as a blanching, maculopapular erythema of the upper trunk,
neck, palms, soles, and earlobes, which
becomes confluent with additional findings ranging from edema to widespread
blistering. Skin biopsy reveals infiltration
of the upper dermis by mononuclear cells
and damage to the basal layer of epithelial cells. Hepatitis manifests as elevations
in alanine and aspartate aminotransferases, alkaline phosphatase, and bilirubin. Enterocolitis causes anorexia, nausea,
and up to 3 to 4 liters per day of secretory
diarrhea. Pancytopenia is associated with
a hypocellular marrow. Symptoms typically appear within 8 to 10 days of the
transfusion but may occur as early as 3
days and as late as 30 days. The diagnosis
is proven by demonstration of donor-de-

rived lymphocytes in the recipient’s peripheral blood or tissues by HLA typing.91
Factors that determine an individual patient’s risk for TA-GVHD include whether and
to what degree the recipient is immunodeficient, the degree of HLA similarity between donor and recipient, and the number and type of T lymphocytes transfused
that are capable of multiplication.91 TA-GVHD
may occur in an immunologically normal
recipient if the donor is homozygous for an
HLA haplotype for which the recipient is
heterozygous, a so-called “one-way” HLA
match, and if the component contains viable T cells (the fresher the unit, the higher
the risk). Cytokine dysfunction, recruitment
of host cells into the immune reaction, and
release of biologic mediators, in particular
nitric oxide, all play a role in the pathogenesis.94 Of interest is the fact that TA-GVHD
has not been reported in an AIDS patient.

Treatment and Prevention
Treatment of TA-GVHD with immunosuppressive agents has been attempted but
rarely succeeds, so prevention is necessary. Irradiation of cellular blood components is the accepted standard method to
prevent TA-GVHD. The dose mandated by
the FDA is a minimum of 25 Gy targeted
to the midline of the container and a minimum dose of 15 Gy delivered to all other
parts of the component.95 This renders T
lymphocytes incapable of replication
without substantially affecting the function
of red cells, platelets, and granulocytes.
AABB Standards for Blood Banks and
Transfusion Services requires routine irradiation of cellular components from units
collected from the recipient’s blood relatives, and donors selected for HLA compatibility by typing or crossmatching.17(p43) Policies should be in place to define the other
groups of patients who should receive irradiated cellular components, and there must

Chapter 27: Noninfectious Complications of Blood Transfusion

be a process for ensuring that once a patient has been determined to be at risk for
TA-GVHD, all cellular components will be irradiated as long as clinically indicated.
Published guidelines96 additionally recommend component irradiation for: 1)
hematopoietic progenitor cell (HPC) transplant recipients (this includes allogeneic
and autologous HPC transplants), 2) patients with hematologic disorders who will
be undergoing allogeneic HPC transplantation imminently, 3) intrauterine transfusions, 4) neonates undergoing exchange
transfusion or use of extracorporeal membrane oxygenation, 5) patients with Hodgkin’s disease, and 6) patients with congenital cellular immunodeficiencies. TA-GVHD
has also been reported in patients with
acute lymphoid and myeloid leukemias,
chronic lymphocytic leukemia particularly
in patients receiving fludarabine phos91
phate, patients with B-cell malignancies
including non-Hodgkin’s lymphoma,
myeloma, and Waldenstrom’s macroglobulinemia,14 premature or low-birthweight infants without specific immunodeficiency
disorders, and children being treated for
neuroblastoma and rhabdomyosarcomas.91

Posttransfusion Purpura

Pathophysiology and Manifestations
Posttransfusion purpura (PTP) is an uncommon event, although over 200 cases
have been published. It is characterized by
the abrupt onset of severe thrombocytopenia (platelet count usually <10,000/µL)
an average of 9 days after transfusion
(range, 1-24 days).97 Components provoking the reaction have usually been RBCs
or Whole Blood, but PTP has also been
reported after platelet and plasma transfusion, and after transfusion of frozen
deglycerolized RBCs. Most patients have
previously been pregnant or transfused.
“Wet purpura” is common, and fatal intra-

659

cranial hemorrhage can occur. The ratio
of affected patients is five women to one
man, and the median age is 51 years
(range, 16-83). Most cases (68%) involve
patients whose platelets lack the HPA-1a
(PlA1) antigen (<2% of the population) and
who form the corresponding antibody.
However, immunization to HPA-1b is reported in 10%, and other platelet antibodies, including HLA antibodies, have been
associated with the syndrome as well. PTP
is usually self-limited, with full recovery
within 21 days. Historically, 10% to 15% of
patients have been reported to die from
PTP, typically from intracranial bleeding,
so treatment is desirable.
The reason for destruction of the patient’s own platelets by what appears to be
a platelet alloantibody is controversial.
Three mechanisms have been proposed,
including: 1) formation of immune complexes of patient antibody and soluble donor antigen that bind to Fc receptors on the
patient’s platelets and mediate their destruction, 2) conversion of antigen-negative
autologous platelets to antibody targets by
soluble antigen in the transfused component, and 3) cross-reactivity of the patient’s
antibodies with autologous platelets (ie, the
presence of an autoantibody component).
The last of these theories has received the
most support.

Treatment
Because PTP remits spontaneously, treatment may appear falsely efficacious. Steroids are frequently given but their role is
controversial. Plasma exchange can achieve
platelet counts of 20,000/µL in 1 to 2 days,98
but the use of high-dose Immune Globulin Intravenous (IGIV) is now supplanting
this therapy.98,99 With the use of IGIV, recovery to platelet counts of 100,000/µL is
typically achieved within 3 to 5 days. As it
does in other disorders such as immune

660

AABB Technical Manual

thrombocytopenic purpura, IGIV appears
to block antibody-mediated clearance of
the target cells, although the mechanism
of action has not been established. If randomly selected platelets are transfused,
patients may experience a febrile transfusion reaction, and, in the vast majority of
cases, such transfusions have not been efficacious. Antigen-negative platelets can
be of benefit in PTP, and, in conjunction
with IGIV, reversal of this disorder in 1 day
is now possible.100,101 Unfortunately, the
time necessary to procure such platelets
often limits their usefulness. After recovery, some authors have suggested that future transfusions should be from donors
lacking the offending antigen, or, if they
97
are not available, washed platelet units.

Immunomodulatory Effects of Transfusion
Transfusion has been known to modulate
immune responses since the 1973 observation by Opelz and coworkers102 of improved renal allograft survival in transfused patients. This beneficial toleranceinducing effect of transfusion raised concerns that transfusion may have other adverse effects in different clinical settings,
including increased rates of postoperative
solid tumor recurrence and bacterial infection.103 Despite numerous retrospective
and several large prospective studies, the
clinical significance of transfusion-associated immunomodulation and the usefulness of preventive strategies, such as leukocyte reduction of transfused components,
remain controversial.104,105

Iron Overload
Every RBC unit contains approximately
200 mg of iron. Chronically transfused
patients, especially those with hemoglobinopathies, have progressive and continuous accumulation of iron and no physiologic means of excreting it. Storage oc-

curs initially in reticuloendothelial sites,
but when they are saturated, there is deposition in parenchymal cells. The
threshold for clinical damage is lifetime
exposure to greater than 50 to 100 RBC
units in a nonbleeding person.106 Iron deposition interferes with function of the
heart, liver, and endocrine glands (eg,
pancreatic islets, pituitary); hepatic failure and cancer, diabetes mellitus, and cardiac toxicity cause most of the morbidity
and mortality. Elevated ferritin levels
demonstrate increased iron stores, and tissue damage can be shown with organ-specific assays such as liver enzyme levels or
endocrine function tests (eg, glucose, thyroid-stimulating hormone).
Treatment is directed at removing iron
without reducing the patient’s circulating
hemoglobin. Metered subcutaneous infusion of desferoxamine, an iron-chelating
agent, can reduce body iron stores in such
patients, but the regimen of nightly subcutaneous infusion by pump is arduous and
expensive, and compliance is often poor. In
transfusion-dependent patients with hemoglobinopathies, red cell exchange can minimize additional iron loads and can reduce
the total iron burden.107 An oral iron chelator has been studied but is not yet available in the United States.

Records of Transfusion
Complications
Each transfusion service must maintain
indefinitely the records of patients who
have had transfusion complications or evidence of alloimmunization. Possible cases
of contaminated blood must be reported
to the institution where the blood was drawn.
Records must be kept, and consulted, to
prevent patients who have had a transfusion reaction from having a recurrence with

Chapter 27: Noninfectious Complications of Blood Transfusion

subsequent transfusions. For example, patients with a history of IgA-related anaphylactic reactions should be transfused with
plasma products that lack IgA. A history of
repeated or severe FNHTRs might prompt
the use of leukocyte-reduced cellular blood
components. Red cell alloantibodies may
become undetectable over time as discussed above, 82,83 so records should be
checked and compatible blood issued in
order to prevent a DHTR. Routine checking
of previous results of ABO and Rh testing
may disclose an error in testing or in the
identification of a current sample.

661

In the absence of such errors as administration of ABO-incompatible blood or of
physiologic events clearly attributable to
acute hemolysis, anaphylaxis, TRALI, or
sepsis, transfusion is highly unlikely to be
acutely responsible for death. The review
should include all available medical and
laboratory records and the results of an autopsy, if performed. On the other hand, if
an investigation does reveal evidence or the
possibility of hemolysis, anaphylactic or
pulmonary events, unexplained sepsis, or
ambiguous identification records, the case
may warrant more extensive inquiry.

Records of Patients with Special Needs
In addition to records of transfusion reactions, transfusion services should maintain
records of patients who need specially
prepared or manipulated components.
This is especially important in institutions where physicians rotate frequently,
and the need for irradiated, leukocyte-reduced, or IgA-deficient components may
not be known to a particular physician
writing an individual order.

Reporting Transfusion Fatalities
When a complication of blood transfusion
has been confirmed to be fatal, it must be
reported to the Director, Office of Compliance, Center for Biologics Evaluation and
Research, FDA, as soon as possible, with a
written report within 7 days (see Chapter
28 for reporting information). Patients
who are critically ill and near death often
receive transfusions in close temporal
proximity to death, and clinical suspicion
of cause and effect may occasionally be
raised. The overwhelming majority of
such deaths are unrelated to transfusion,
but if there is a suggestion that a transfusion might have contributed to death, it
may be prudent to pursue an investigation.

References
1. Popovsky MA, ed. Transfusion reactions. 2nd
ed. Bethesda, MD: AABB Press, 2001.
2. Heddle NM, Kelton JG. Febrile nonhemolytic
transfusion reactions. In: Popovsky MA, ed.
Transfusion reactions. 2nd ed. Bethesda, MD:
AABB Press, 2001:45-82.
3. Davenport RD. Hemolytic transfusion reactions. In: Popovsky MA, ed. Transfusion reactions. 2nd ed. Bethesda, MD: AABB Press, 2001:
1-44.
4. Capon SM, Goldfinger D. Acute hemolytic
transfusion reaction, a paradigm of the systemic inflammatory response: New insights
into pathophysiology and treatment. Transfusion 1995;35:513-20 [Erratum in Transfusion
1995;35:794].
5. Sazama K. Report of 355 transfusion-associated deaths: 1976-1985. Transfusion 1990;30:
583-90.
6. Del Greco F, Kurtides ES. Kell incompatibility
with acute renal failure. Arch Intern Med 1963;
112:727-30.
7. Linden JV, Wagner K, Voytovich AE, Sheehan
J. Transfusion errors in New York State: An
analysis of 10 years’ experience. Transfusion
2000;40:1207-13.
8. Jandl JH. Blood: Textbook of hematology.
2nd ed. Boston: Little, Brown & Co, 1996.
9. Davenport RD, Kunkel SL. Cytokine roles in
hemolytic and non-hemolytic transfusion
reactions. Transfus Med Rev 1994;8:157-68.
10. Davenport RD. Inflammatory cytokines in
hemolytic transfusion reactions. In: Davenport RD, Snyder EL, eds. Cytokines in transfusion medicine: A primer. Bethesda, MD:
AABB Press, 1996:85-97.

662

AABB Technical Manual

11. Butler J, Parker D, Pillai R, et al. Systemic release of neutrophil elastase and tumour necrosis factor alpha following ABO incompatible blood transfusion. Br J Haematol 1991;79:
525-6.
12. Savitsky JP, Doczi J, Black J, Arnold JD. A clinical safety trial of stroma-free hemoglobin.
Clin Pharmacol Ther 1978;23:73-80.
13. Simon T. Proficiency testing program. CAP
Survey 1991 J-C. Northfield, IL: College of
American Pathologists, 1991.
14. Asher D, Atterbury CLJ, Chapman C, et al. Serious Hazards of Transfusion (SHOT). Annual
Report, 2000-2001. Manchester, UK: Serious
Hazards of Transfusion Steering Group, 2002.
15. Marik PE. Low-dose dopamine: A systematic
review. Intensive Care Med 2002;28:877-83.
16. Beauregard P, Blajchman MA. Hemolytic and
pseudo-hemolytic transfusion reactions: An
overview of the hemolytic transfusion reactions and the clinical conditions that mimic
them. Transfus Med Rev 1994;8:184-99.
17. Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.
18. Blajchman MA. Transfusion-associated bacterial sepsis: The phoenix rises yet again.
Transfusion 1994;34:940-2.
19. Heddle NM, Blajchman MA, Meyer RM, et al.
A randomized controlled trial comparing the
frequency of acute reactions to plasma-reduced
platelets and prestorage WBC-reduced platelets. Transfusion 2002;42:556-66.
20. Ferrara JLM. The febrile platelet transfusion
reaction: A cytokine shower. Transfusion 1995;
35:89-90.
21. Davenport RD, Burdick M, Moore SA, Kunkel
SL. Cytokine production in IgG-mediated red
cell incompatibility. Transfusion 1993;33:1924.
22. Brand A. Passenger leukocytes, cytokines, and
transfusion reactions. N Engl J Med 1994;331:
670-1.
23. Widmann FK. Controversies in transfusion
medicine: Should a febrile transfusion response occasion the return of the blood component to the blood bank? Pro. Transfusion
1994;34:356-8.
24. Oberman HA. Controversies in transfusion
medicine: Should a febrile transfusion response occasion the return of the blood component to the blood bank? Con. Transfusion
1994;34:353-5.
25. Wang SE, Lara PN, Lee-Ow A, et al. Acetaminophen and diphenhydramine as premedication for platelet transfusions: A prospective
randomized double-b lind placebo-controlled trial. Am J Hematol 2001;70:191-4.

26. Vamvakas EC, Pineda AA. Allergic and anaphylactic reactions. In: Popovsky MA, ed.
Transfusion reactions. 2nd ed. Bethesda, MD:
AABB Press, 2001:83-128.
27. Sandler SG, Mallory D, Malamut D, Eckrich R.
IgA anaphylactic transfusion reactions. Transfus Med Rev 1995;9:1-8.
28. Burks AW, Sampson HA, Buckley RH. Anaphylactic reactions after gamma globulin administration in patients with hypogammaglobulinemia. N Engl J Med 1986;314:560-4.
29. Lambin P, LePennec PY, Hauptmann G, et al.
Adverse transfusion reactions associated with
a precipitating anti-C4 antibody of antiRodgers specificity. Vox Sang 1984;47:242-9.
30. Westhoff CM, Sipherd BD, Wylie DE, Toalson
LD. Severe anaphylactic reactions following
transfusions of platelets to a patient with
anti-Ch. Transfusion 1992;32:576-9.
31. Shimada E, Tadokoro K, Watamabe Y, et al.
Anaphylactic transfusion reactions in haptoglobin-deficient patients with IgE and IgG
haptoglobin antibodies. Transfusion 2002;42:
766-73.
32. Leitman SF, Boltansky H, Alter HJ, et al. Allergic reactions in healthy plateletpheresis donors caused by sensitization to ethylene oxide gas. N Engl J Med 1986;315:1192-6.
33. Ramirez MA. Horse asthma following blood
transfusion; report of case. JAMA 1919;73:985.
34. Routledge RC, De Kretser DMH, Wadsworth
LD. Severe anaphylaxis due to passive sensitization to donor’s blood. Br Med J 1976;1:434.
35. Owen HG, Brecher ME. Atypical reactions associated with use of angiotensin-converting
enzyme inhibitors and apheresis. Transfusion
1994;34:891-4.
36. Alving BM, Hojima Y, Pisano JJ, et al. Hypotension associated with pre-kallikrein activator (Hageman factor fragments) in plasma
protein fraction. N Engl J Med 1978;299:66-70.
37. Shiba M, Tadokoro K, Sawanobori M, et al.
Activation of the contact system by filtration
of platelet concentrates with a negatively
charged white cell-removal filter and measurement of venous blood bradykinin level in
patients who received filtered platelets.
Transfusion 1997;37:457-62.
38. Hume HA, Popovsky MA, Benson K, et al. Hypotensive reactions: A previously uncharacterized complication of platelet transfusion?
Transfusion 1996;36:904-9.
39. Mair B, Leparc GF. Hypotensive reactions associated with platelet transfusions and angiotensin-converting enzyme inhibitors. Vox
Sang 1998;74:21-30.
40. Agostini JV, Leo-Summers LS, Inonye SK.
Co g n i t i v e a n d o t h e r a d v e r s e e f f e c t s o f

Chapter 27: Noninfectious Complications of Blood Transfusion

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

diphenhydramine use in hospitalized older
patients. Arch Intern Med 2001;161:2091-7.
Pineda AA, Zylstra VW, Clare DE, et al. Viability and functional integrity of washed platelets. Transfusion 1989;29:524-7.
Popovsky MA. Transfusion-related acute lung
injury (TRALI). In: Popovsky MA, ed. Transfusion reactions. 2nd ed. Bethesda, MD: AABB
Press, 2001:155-70.
Zoon KC. Transfusion related acute lung injury (“Dear Colleague” letter). (October 19,
2001) Rockville, MD: CBER Office of Communication, Training, and Manufacturers
Assistance, 2001.
Kleinman S, Caulfield T, Chan P, et al. Toward
an understanding of transfusion-related acute
lung injury: Statement of a consensus panel.
Transfusion 2004;44:1774-89.
Popovsky MA, Moore SB. Diagnostic and pathogenetic considerations in transfusion-related acute lung injury. Transfusion 1985;25:
573-7.
Popovsky MA, Haley NR. Further characterization of transfusion-related acute lung injury: Demographics, clinical and laboratory
features, and morbidity. Immunohematology
2000;16:157-9.
Kao GS, Wood IG, Dorfman DM, et al. Investigations into the role of anti-HLA class II
antibodies in TRALI. Transfusion 2003;43:
185-91.
Kopko PM, Paglieroni TG, Popovsky MA, et
al. TRALI: Correlation of antigen-antibody and
monocytes activation in donor-recipient pairs.
Transfusion 2003;43:177-84.
Seeger W, Schneider U, Kreusler B, et al. Reproduction of transfusion-related acute lung
injury in an ex vivo lung model. Blood 1990;
76:1438-44.
Dry SM, Bechard KM, Milford EL, et al. The
pathology of transfusion-related acute lung
injury. Am J Clin Pathol 1999;112:216-21.
McCullough J. Granulocyte transfusion. In:
Petz LD, Swisher SN, Kleinman S, et al, eds.
Clinical practice of transfusion medicine. 3rd
ed. New York: Churchill Livingstone, 1996:41332.
Silliman CC, Paterson AJ, Dickey WO, et al.
The association of biologically active lipids
with the development of transfusion-related
acute lung injury: A retrospective study.
Transfusion 1997;37:719-26.
Silliman CC, Boshkov LK, Mehdizadehkashi
Z, et al. Transfusion-related acute lung injury:
Epidemiology and a prospective analysis of
etiologic factors. Blood 2003;101:454-62.
Audet AM, Popovsky MA, Andrzejewski C.
Transfusion-associated circulatory overload
in orthopedic surgery patients: A multi-insti-

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

663

tutional study. Immunohematology 1996;12:
87-9.
Bowman RJ, Laudi N, Aslan D, Burgher A. Use
of BNP to evaluate TRALI (abstract). Transfusion 2004;44(Suppl):24A.
Dzik WH, Kirkley SA. Citrate toxicity during
massive blood transfusion. Transfus Med Rev
1988;2:76-94.
Olinger GN, Hottenrott C, Mulder DG, et al.
Acute clinical hypocalcemic myocardial depression during rapid blood transfusion and
postoperative hemodialysis: A preventable
complication. J Thorac Cardiovasc Surg 1976;
72:503-11.
Howland WS, Jacobs RG, Goulet AH. An evaluation of calcium administration during rapid
blood replacement. Anesth Analg 1960;39:
557-63.
Boyan CP, Howland WS. Cardiac arrest and
temperature of bank blood. JAMA 1963;183:
58-60.
Iserson KV, Huestis DW. Blood warming: Current applications and techniques. Transfusion
1991;31:558-71.
Valeri CR, Feingold H, Cassidy G, et al. Hypothermia-induced reversible platelet dysfunction. Ann Surg 1987;205:175-81.
Sessler DI. Current concepts: Mild perioperative hypothermia. N Engl J Med 1997;336:
1730-7.
Collins JA. Problems associated with the
massive transfusion of stored blood. Surgery
1974;174:274-95.
Liu EA, Manino FL, Lane TA. Prospective,
randomized trial of the safety and efficacy of
a limited donor exposure transfusion program for premature neonates. J Pediatr 1994;
125:92-6.
Miller RD, Robbins TO, Tong MJ, Barton SL.
Coagulation defects associated with massive
blood transfusion. Ann Surg 1971;174:794-801.
Counts RB, Haisch C, Simon TL, et al. Hemostasis in massively transfused trauma patients.
Ann Surg 1979;190:91-9.
Lim RC, Olcott C, Robinson AJ, Blaisdell FW.
Platelet response and coagulation changes
following massive blood replacement. J
Trauma 1973;13:577-82.
Harrigan C, Lucas CE, Ledgerwood KAM, et
al. Serial changes in primary hemostasis after massive transfusion. Surgery 1985;98:
836-43.
Ciavarella D, Reed RL, Counts RB, et al. Clotting factor levels and the risk of microvascular bleeding in the massively transfused patient. Br J Haematol 1987;67:365-8.
Harke H, Rahman S. Haemostatic disorders
in massive transfusion. Bibl Haematol 1980;
46:179-88.

664

AABB Technical Manual

71. Collins JA. Recent developments in the area
of massive transfusion. World J Surg 1987;11:
75-81.
72. Murray DJ, Pennell BJ, Weinstein SL, Olson
JD. Packed red cells in acute blood loss: Dilutional coagulopathy as a cause of surgical
bleeding. Anesth Analg 1995;80:336-42.
73. Reed RL, Ciavarella D, Heimbach DM, et al.
Prophylactic platelet administration during
massive transfusion: A prospective, randomized, double-blind clinical study. Ann Surg 1986;
203:40-8.
74. O’Quin RJ, Lakshminarayan S. Venous air
embolism. Arch Intern Med 1982;142:2173-6.
75. Elliott K, Sanders J, Brecher, ME. Transfusion
medicine illustrated. Visualizing the hemolytic transfusion reaction. Transfusion 2003;43:
297.
76. Henry JB. Clinical diagnosis and management
by laboratory methods. 20th ed. Philadelphia: WB Saunders, 2001.
77. Nance ST. Flow cytometry in transfusion
medicine. In: Anderson KC, Ness PM, eds.
Scientific basis of transfusion medicine. Philadelphia: WB Saunders, 1994:707-25.
78. Baldwin ML, Barrasso C, Ness PM, Garratty
G. A clinically significant erythrocyte antibody
detectable only by 5 1 Cr survival studies.
Transfusion 1983;23:40-4.
79. Sazama K. Bacteria in blood for transfusion:
A review. Arch Pathol Lab Med 1994;118:35065.
80. Lostumbo MM, Holland PV, Schmidt PJ. Isoimmunization after multiple transfusions. N
Engl J Med 1966;275:141-4.
81. Issitt PD, Anstee DJ. Applied blood group serology. 4th ed. Durham, NC: Montgomery
Scientific Publications, 1998.
82. Ramsey G, Larson P. Loss of red cell antibodies over time. Transfusion 1988;28:162-5.
83. Ramsey G, Smietana SJ. Long-term follow-up
testing of red cell alloantibodies. Transfusion
1994;34:122-4.
84. Heddle NM, Soutar RL, O’Hoski PL, et al. A
prospective study to determine the frequency
and clinical significance of alloimmunization
post-transfusion. Br J Haematol 1995;91:
1000-5.
85. Ness PM, Shirey RS, Thoman SK, Buck SA.
The differentiation of delayed serologic and
delayed hemolytic transfusion reactions: Incidence, long-term serologic findings, and
clinical significance. Transfusion 1990;30:
688-93.
86. Pinkerton PH, Coovadia AS, Goldstein J. Frequency of delayed haemolytic transfusion reactions following antibody screening and immediate-spin crossmatching. Transfusion 1992;
32:814-7.

87. Vamvakas EC, Pineda AA, Reisner R, et al. The
differentiation of delayed hemolytic and serologic transfusion reactions: Incidence and
predictors of hemolysis. Transfusion 1995;35:
26-32.
88. Garratty G. Severe reactions associated with
transfusions of patients with sickle cell disease. Transfusion 1997;37:357-61.
89. Vichinsky EP, Luban NL, Wright E, et al. Pros p e c t i ve R B C p h e n o t y p e m a t c h i n g i n a
stroke-prevention trial in sickle cell anemia:
A multicenter transfusion trial. Transfusion 2001;
41:1086-92.
90. Petz LD, Garratty G. Immune hemolytic
anemias. 2nd ed. Philadelphia: Churchill
Livingstone, 2004:335-40.
91. Webb IJ, Anderson KC. Transfusion-associated
graft-vs-host disease. In: Popovsky MA, ed.
Transfusion reactions. 2nd ed. Bethesda, MD:
AABB Press, 2001:171-86.
92. Ohto H, Anderson KC. Survey of transfusionassociated graft-versus-host disease in immunocompetent recipients. Transfus Med
Rev 1996;10:31-43.
93. Juji T, Takahashi K, Shibata Y. Post-transfusion graft versus host disease as a result of directed donations from relatives (letter). N
Engl J Med 1989;321:56.
94. Ferrara JL, Krenger W. Graft-vs-host disease:
The influence of type 1 and type 2 cell cytokines. Transfus Med Rev 1998;12:1-17.
95. Food and Drug Administration. Memorandum.
Recommendations regarding license amendments and procedures for gamma irradiation
of blood products. ( July 22, 1993) Rockville,
MD: CBER Office of Communication, Training, and Manufacturers Assistance, 1993.
96. Przepiorka D, LeParc GF, Stovall MA, et al. Use
of irradiated blood components. Practice parameter. Am J Clin Pathol 1996;106:6-11.
97. McFarland JG. Postransfusion purpura. In:
Popovsky MA, ed. Transfusion reactions. 2nd
ed. Bethesda, MD: AABB Press, 2001:187-212.
98. McLeod BC, Strauss RG, Ciavarella D, et al.
Management of hematological disorders and
cancers. J Clin Apheresis 1996;11:211-30.
99. Mueller-Eckhardt C, Kiefel V. High-dose IgG
for post-transfusion purpura revisited. Blut 1988;
57:163-7.
100. Brecher ME, Moore SB, Letendre L. Posttransfusion purpura: The therapeutic value of
PlA1-negative platelets. Transfusion 1990;30:
433-5.
101. Win N, Matthey F, Slater NGP. Blood components—Transfusion support in post-transfusion purpura due to HPA-1a immunization.
Vox Sang 1996;71:191-3.
102. Opelz G, Senger DP, Mickey MR, Terasaki PI.
Effect of blood transfusions on subsequent

Chapter 27: Noninfectious Complications of Blood Transfusion

kidney transplants. Transplant Proc 1973;5:
253-9.
103. Blumberg N, Heal JM. Effects of transfusion
on immune function: Cancer recurrence and
infection. Arch Pathol Lab Med 1994;118:
371-9.
104. Blajchman MA. Allogeneic blood transfusions,
immunomodulation, and postoperative bacterial infection: Do we have the answers yet?
Transfusion 1997;37:121-5.
105. Vamvakas EC, Blajchman MA, eds. Immunomodulatory effects of blood transfusion.
Bethesda, MD: AABB Press, 1999.

665

106. Sharon BI, Honig GR. Management of congenital hemolytic anemias. In: Simon TL,
Dzik WH, Snyder EL, et al, eds. Rossi’s principles of transfusion medicine. 3rd ed. Baltimore, MD: Lipincott Williams and Wilkins,
2002:463-82.
107. Adams DM, Schultz WH, Ware RE, Kinney
TR. Erythrocytapheresis can reduce iron
overload and prevent the need for chelation
therapy in chronically transfused pediatric
patients. J Pediatr Hematol Oncol 1996;18:
46-50.

Chapter 28: Transfusion-Transmitted Diseases

Chapter 28

Transfusion-Transmitted
Diseases

ANY ADVANCES HAVE been
made in the testing of blood donations for infectious diseases.
However, the risk of transmitting viral,
bacterial, and parasitic diseases via transfusion still exists, and new agents may
appear at any time. Thus, infectious complications of transfusion remain an important area of concern in transfusion
medicine.

Hepatitis
Hepatitis is inflammation of the liver that
can be caused by many different toxins,
immunologic processes, or infectious agents.
Hepatitis linked to transfusion is almost
exclusively caused by viruses. These viruses
include hepatitis viruses A-E (HAV, HBV,
HCV, HDV, HEV), cytomegalovirus (CMV),
Epstein-Barr virus (EBV ), and possibly

newly described putative hepatitis viruses
(such as TTV and SEN-V). Infectious agents
pose a serious threat to transfusion recipients if they persist in the circulation of
asymptomatic blood donors and can
cause clinically significant acute or chronic
disease manifestations in recipients.
The vast majority of posttransfusion
hepatitis in the past was attributable to
HBV and HCV, both of which can establish
prolonged carrier states in donors characterized by high-titer viremia in the absence
of symptoms. HBV and HCV also cause significant long-term liver-related morbidity
1,2
and mortality. These viruses are considered in detail below.
HAV and HEV, which are enterically transmitted viruses, circulate only transiently
during the acute phase of infection. Because the viremic individual is usually clinically ill and not a candidate for donation,
HAV and HEV are not serious threats to transfusion recipients. However, HAV viremia
667

668

AABB Technical Manual

may be present for up to 28 days before
symptoms develop, and isolated cases have
been reported associated with transfusion
of cellular components3 and outbreaks with
4
Factor VIII concentrate. Because HAV lacks
a lipid envelope, it is not inactivated by solvent/detergent treatment; additional inactivation methods are under development to
prevent recurrence of such outbreaks. HEV
is rare in the United States, and there have
been no documented cases of transfusion
transmission in this country.
HDV, formerly called the delta agent, can
cause infection and serious hepatitis after
transfusion or other parenteral exposure.
However, because HDV is a defective virus
found only in HBV carriers, screening donors for HBV infection simultaneously
eliminates the risk of HDV.5 HGV, also called
GBV-C, is distantly related to HCV and has
a high prevalence rate (>1%) among asymptomatic donors. Although HGV is unequivocally transfusion-transmissible,6 a causal
relationship has not been established between HGV infection and hepatitis or any
other disease manifestation, despite intensive study.
TTV appears similar to HGV with respect
to prevalence, transmissibility, and the lack
of clinical disease significance. Thus,
screening blood donors for HGV or TTV is
not currently recommended. Hepatitis associated with CMV or EBV is generally mild
in the absence of severe immunosuppression. The frequency and severity of such
hepatitis cases do not justify routine
screening measures.7 SEN-V has been associated with transfusion-associated non-A
through non-E hepatitis in one study,8 but a
causal association has not been established, nor has SEN-V been significantly associated with chronic non-A through non-E
hepatitis. SEN-V appears to be distantly related to TTV and to be a member of a family of small, circular DNA viruses called
Circoviridae. Screening for these agents is

not currently recommended because disease
associations have not been established.

Clinical Manifestations of Hepatitis
Most individuals who acquire HBV or
HCV infection have a subclinical primary
infection without obvious symptoms or
physical evidence of disease. Some develop overt hepatitis with jaundice, nausea, vomiting, abdominal discomfort, fatigue, dark urine, and elevation of liver
enzymes. Signs and symptoms usually resolve spontaneously. Acute hepatitis C
tends to be milder than hepatitis B. Uncommonly, the clinical course of HBV and,
rarely, HCV infections may be complicated by fulminant hepatitis. Of greater
concern is a propensity of hepatitis C to
evolve to chronic hepatitis (75% to 85% of
affected individuals), with a significant
number demonstrating long-term progression to cirrhosis, liver failure, or hepatocellular carcinoma. Hepatitis A tends to
be clinically mild in otherwise healthy
hosts and is not known to progress to
chronic hepatitis or a chronic carrier
state.9 HEV infection may lead to severe
10
disease in pregnant women. Vaccination
is available for hepatitis A and for hepatitis B, and hepatitis B immune globulin
(HBIG) has proven useful for post-exposure prophylaxis for hepatitis B and immune serum globulin (ISG) for hepatitis
A.

Chronic Carriers of HBV
After initial HBV infection, a proportion of
patients fail to clear infectious virus from
the bloodstream and become chronic carriers for years or life. HBV carriers produce,
in addition to the infectious viral particle,
large amounts of noninfectious envelope
protein detected by the assay for hepatitis
B surface antigen (HBsAg). The risk of becoming an HBsAg carrier is strongly age-

Chapter 28: Transfusion-Transmitted Diseases

dependent; ≤5% of those infected with
HBV as adults become chronic HBsAg
carriers, whereas ≥95% recover completely
and develop protective antibody against
HBsAg (anti-HBs). In contrast, before routine prophylactic immune globulin infusion and immunization, 90% or more of
infants infected perinatally became carriers and became at risk for progressing to
cirrhosis and hepatocellular carcinoma.
According to World Health Organization
estimates, the number of HBsAg carriers is
approximately 400 million worldwide,11
with a prevalence of up to 10% in some
Asian countries, 0.1% to 0.5% in the general US population, and 0.02% to 0.04% in
US blood donors. Small proportions (<10%)
of HBsAg carriers develop clinical manifestations, such as hepatic insufficiency,
cirrhosis, or hepatocellular carcinoma.

Chronic Carriers of HCV
Most people who are initially infected with
HCV become chronic HCV carriers, with
75% to 85% having persistent HCV RNA in
the serum and liver for years to decades.
At least 50% of such HCV carriers have
biochemical and histologic evidence of
chronic liver inflammation.12 Despite this
chronic inflammatory process, most HCVinfected individuals remain asymptomatic. During the first 20 years after infection, HCV is usually indolent and associated with low mortality and morbidity.
Those with clinical liver disease represented about 10% of the entire infected
13
cohort in one study. The risk that and the
rate at which chronic hepatitis by itself
will progress to cirrhosis is unknown. It is
thought that alcohol may play a synergistic role in exacerbating chronic hepatitis
C. Posttransfusion HCV in children infected after cardiac surgery appears to resolve more frequently than that in adults

669

and to be a mild infection at almost 20
years of follow-up.14
Recommendations for clinical management of persons with chronic HCV infection were developed by a National Institutes of Health consensus development
conference15 and have been updated and
16
expanded by others.

Markers of Viral Infection
Laboratory tests can identify markers of
previous exposure and probable current
infectivity for HBV and HCV, which are
useful for screening and diagnostic applications. Table 28-1 lists the molecular and
serologic markers commonly used in the
diagnosis of hepatitis. Figure 28-1 illustrates the sequence of test results typical
of individuals with acute HBV infection
that completely resolves.
The period between exposure to HBV
and emergence of circulating markers of infection (HBV DNA or HBsAg) is usually
3,9
about 6 weeks. HBV DNA, detectable by
pooled nucleic acid amplification testing
(NAT) techniques, is the first marker to appear, followed by detectable HBsAg. Individual donor NAT (ID-NAT) is able to detect
HBV DNA approximately 19 days before
minipooled NAT (MP-NAT).17 Antibody to
the HBV core protein (anti-HBc) usually appears several weeks later, first as IgM and
then as IgG. The clearance of HBsAg and
appearance of anti-HBs signal resolution of
infection. However, there has been a report
of a fatality, after reactivation of HBV in a
patient treated with rituximab, who was
previously anti-HBs-reactive.18 The specific
virus showed multiple mutations in major
antigenic sites and was thought to escape
the patient’s endogenous immunity.
Two additional HBV markers, HBeAg or
its antibody (anti-HBe), are useful diagnostic and prognostic markers but are not employed in donor screening. An asymptom-

670

Table 28-1. Molecular and Serologic Tests in the Diagnosis of Viral Hepatitis

HBV

HDV

HCV

Test Reactivity

Interpretation

DNA

HBsAg

Anti-HBc
Total

IgM

Anti-HBs

HBeAg Anti-HBe

–

Window period

+/–

–

+/–

–

Early acute HBV infection/chronic carrier

–

Acute infection

+/–

–

+/–

Early convalescent infection/possible early
chronic carrier

+/–

–

+/–

Chronic carrier*

–

+/–

Recovered infection

–

Vaccinated or recovered infection

–

Recovered infection? False positive?

RNA

HBsAg

Anti-HBc

Anti-HBs

Anti-Delta

–

Acute or chronic HDV infection

–

Recovered infection

RNA

Anti-HCV
(Screening EIA)

Recombinant Antigens (RIBA)
5-1-1

c100-3

+/–

Not available

–

c33c

c22-3
Probable acute or chronic HCV infection (if RNA
is positive)

–

False positive

AABB Technical Manual

Virus

HEV

–

Probable false positive (if RNA is negative); possible acute infection (if RNA is positive)†

+/–

–

Early acute or chronic infection (if RNA is positive); false positive or late recovery (if RNA is
negative)†

Acute or chronic infection

–

+/–

Recovered HCV†

RNA

Anti-HAV
Total

IgM

Acute HAV

–

Recovered HAV/vaccinated

RNA

Anti-HEV
Total

IgM

Acute HEV

–

Recovered HEV

*Those with HBeAg are more infectious and likely to transmit vertically.
†
Anti-5-1-1 and anti-c100-3 generally appear later than anti-c22-3 and anti-c33c during seroconversion and may disappear spontaneously, during immunosuppression or after
successful antiviral therapy.
HBsAg = hepatitis B surface antigen; anti-HBc = antibody to hepatitis B core antigen; anti-HBs = antibody to HBsAg; HBeAg = hepatitis B e antigen; anti-delta = antibody to delta
antigen; anti-HAV = antibody to hepatitis A virus; anti-HCV = antibody to hepatitis C virus; anti-HEV = antibody to hepatitis E virus.

Chapter 28: Transfusion-Transmitted Diseases

HAV

+/–

671

672

AABB Technical Manual

Figure 28-1. Serologic markers in hepatitis B virus infection that resolved without complications. In the
acute phase, markers often appear before onset of liver function test (LFT) abnormalities and symptoms
(SYMP). Anti-HBs and anti-HBc persist after recovery and indicate immunity. In chronic carriers (not
shown), HBsAg persists and anti-HBc is usually present, but anti-HBs is absent (HBeAg and anti-HBe
may be present, see Table 28-1). HBV DNA (not shown) may be detected approximately 1 to 2 weeks before HBsAg.

atic HBsAg-positive individual may either
be in the early phase of acute HBV infection
(without anti-HBc or with IgM anti-HBc) or
a chronic HBV carrier (with IgG anti-HBc).
HBsAg particles are produced in excess
during acute and chronic infection; blood
from individuals with circulating HBsAg
can infect others. Current screening immunoassays detect approximately 0.2 to 0.7
ng/mL HBsAg or ≥3 × 107 particles.3The
number of HBsAg particles in most acute
and chronic infections exceeds this level,
but transmission of HBV from HBsAg
seronegative donors has been described.
NAT testing allows the detection of as few
as 10 genomic copies of HBV DNA.3 The
value of NAT for the detection of seronegative donors infected with HBV is under
study but may be reduced by high sensitivity of current assays for HBsAg and the relatively slow rise in HBV DNA levels contrasted with HIV and HCV. It is possible that

effective detection of seronegative HBV infected donors by NAT will require testing at
the single donation level.19 HBV vaccines
contain noninfectious HBsAg protein, which
may result in false-positive HBsAg screening test results for a few days after the inoculation. Resulting protective antibodies are
directed against HBsAg; vaccination does
not produce anti-HBc.
Tests for antibodies to HCV are enzyme
immunoassays (EIAs) using recombinant
antigens of HCV coated on a solid phase as
the capture reagent. Current assays detect
antibodies to c200 (including c33c and
c100-3), c22-3, and NS-5. Anti-HCV is detectable by third-generation EIAs approximately 10 weeks after infection. HCV RNA
is present at high concentrations in plasma
during most of the period from exposure to
antibody seroconversion. Anti-HCV is detected in 40% to 50% of samples from patients at initial diagnosis of acute hepatitis,

Chapter 28: Transfusion-Transmitted Diseases

either transfusion-transmitted or communityacquired.20
The clinical significance of a positive
screening test for anti-HCV in healthy blood
donors is unclear without supplemental
testing. Approximately 0.21% of US blood
donors have repeatedly reactive EIA results.21 Several generations of recombinant
immunoblot assays (RIBA) have been licensed by the Food and Drug Administration (FDA) for further elucidation of repeatedly reactive EIA results. An individual who
is positive by RIBA is considered to have true
HCV antibody; in 70% to 90% of these
cases, HCV nucleic acid is detectable by
NAT methods. The infectivity of units that
are positive for HCV RNA approaches
22
100%. In contrast, EIA repeatedly reactive
donors with negative or indeterminate
RIBA 3.0 results, representing 37% of EIA
repeatedly reactive donors, are rarely infected or infectious. Regardless of RIBA results, a donation with a repeatedly reactive
EIA result cannot be used for transfusion.
Donors with negative RIBA results may be
considered for reentry (Table 28-2).
In 1999, NAT for HCV RNA was implemented as a donor screening assay under
FDA-sanctioned investigational new drug
(IND) protocols. The testing was performed
in minipools of samples from 16 to 24 whole
blood donations. The rapid increase in
viremia and high viral load of seronegative,
acutely infected donors allows sensitive detection of HCV RNA even in these diluted
pools. The window period for HCV detection with pooled NAT is reduced to 10 to 30
days.17 After testing over 39 million donors,
approximately 1:270,000 donors had been
identified as being in the seronegative window with a positive NAT result.23
NAT results can be used in lieu of supplemental testing in specific circumstances.
An FDA variance is required.24 It is anticipated that NAT HCV testing will be used in
the future for reentry.

673

Surrogate Markers
Before HCV was identified and anti-HCV
testing became feasible, two nonspecific
or “surrogate” tests on donor blood were
introduced to reduce the risk of non-A, non-B
(NANB) hepatitis after transfusion. In 1986
and 1987, the AABB called for testing of
whole blood donations for alanine aminotransferase (ALT) and anti-HBc as surrogates for the direct detection of the NANB
agent. Current very sensitive tests for antiHCV have essentially eliminated the value
of surrogate tests in preventing hepatitis,25
but anti-HBc testing continues as recommended by FDA to prevent HBV transmission. HBV from liver transplant donors with
reactive anti-HBc and negative HBsAg test
results has been transmitted to their recipients, and reports in the literature
show that transfusions of blood reactive
for anti-HBc and negative for HBsAg have
been associated with development of
hepatitis B in some recipients. There may
also be a small number of potential donors infected with HBsAg mutants of HBV
that may not be optimally detected by
currently licensed HBsAg tests.26 Donors
who test repeatedly reactive for anti-HBc
on two occasions or who test repeatedly
reactive on tests from two different manufacturers should be deferred.

Current Risk of Posttransfusion Hepatitis
The risk of posttransfusion HBV or HCV
infection decreased dramatically, to an estimated 1 in 60,000 to 1 in 100,000 risk before implementation of HCV NAT.27 Not a
single new case of transfusion-associated
HCV has been detected by the CDC Sentinel Counties Viral Hepatitis Surveillance
System since 1994 (M. Alter, personal communication, 3/04).1 The development of
progressively improved HCV antibody
tests and stringent selection measures for
donors have contributed to this remark-

674

AABB Technical Manual

Table 28-2. Reentry of Donors with Repeatedly Reactive Screening Tests
Reentry
Status

Repeatedly Reactive for
Anti-HIV-1
or -1/2

Anti-HIV-2

HIV-1-Ag

HBsAg

Anti-HCV

Initial sample
Licensed West- Different HIV-2
ern blot posiEIA RR
tive or indeterminate or
IFA reactive

Confirmed by Confirmed by RIBA indeter- Not eligible
neutralizaneutralizaminate or
for reentry
tion or
positive
tion
anti-HBc
RR

Licensed West- Different HIV-2 Not confirmed HBsAg speci- RIBA negative Evaluate for
by neutralficity not
reentry
ern blot or IFA
EIA NR and liization
confirmed
NR
censed Western blot or IFA
by neutralNR
ization and
anti-HBc
NR
Follow-up sample
(Drawn 6
months
later)

(Drawn 6
months
later)

(Drawn 8
weeks
later)

(Drawn 6
months
later)

EIA RR or West- RR HIV-1 or dif- HIV-1-Ag RR, HBsAg RR or EIA RR or
Not eligible
ferent HIV-2
ern blot posineutralizafor reentry
anti-HBc
RIBA indetive or indeEIA RR or a lition conRR
terminate
censed Westterminate or
firmed or
or positive
IFA reactive
ern blot or IFA
not conreactive or infirmed
determinate
Screening test
HIV-1-Ag and HBsAg NR
Original EIA
and a differanti-HIV-1
and
method NR
ent HIV-2 EIA
EIA NR or
anti-HBc
and whole viNR and liHIV-1-Ag
NR
rus lysate
censed WestRR, not
anti-HIV-1 EIA
ern blot or
confirmed
NR and liIFA NR
(temporary
censed Western blot or IFA
deferral for
NR
additional
8 weeks)

Licensed
Eligible for
multiantigen
reentry
EIA
method NR
and RIBA
negative

NR = nonreactive; RR = repeatedly reactive; RIBA = recombinant immunoblot assay; IFA = immunofluorescence assay;
EIA = enzyme immunoassay; anti-HIV-1 = antibody to human immunodeficiency virus, type 1; anti-HIV-2 = antibody to
human immunodeficiency virus, type 2; HIV-1-Ag = HIV-1 antigen; HBsAg = hepatitis B surface antigen; anti-HCV = antibody to hepatitis C virus; anti-HBc = antibody to hepatitis B core antigen.

Chapter 28: Transfusion-Transmitted Diseases

able decline, and transfusion is no longer
considered a major risk factor for HCV
transmission.28 Nucleic acid screening assays for HCV were implemented by blood
centers in 1999. In 3 years of NAT testing,
170 HCV NAT-positive, seronegative donations were identified in the United
States among 39.7 million screened donations.23 NAT has probably reduced the residual risk for HCV transmission to ≤1 in
2,000,000 components transfused.29,30

Quarantine and Recipient Tracing
Donations with repeatedly reactive screening test results (HBsAg, anti-HBc, and/or
anti-HCV) cannot be used for transfusion.
In addition, in-date components from
collections preceding the current unsuitable donation may need to be quarantined as follows, and consignee notification for the purpose of recipient tracing
(ie, look-back) may be required31,32:
For HCV:
■
Extending back indefinitely, to the
extent that computerized electronic
records exist for anti-HCV repeatedly reactive and confirmed donations or repeatedly reactive donations
for which supplemental tests were
not performed.
■
Extending back to January 1, 1988 if
computerized electronic records are
not available.
For HBsAg and anti-HBc:
■
In-date components, extending back
5 years, or 12 months from the most
recent negative test result for units
that were repeatedly reactive or confirmed, or for which confirmatory
testing was not performed.
Depending on the results of licensed
supplemental tests and prior screening
tests, the quarantined units may be released for transfusion or further manufacture, or may have to be destroyed. Recipi-

675

ents notified as a result of the HCV look-back
should be counseled regarding the nature
of the subsequent donor test results and offered appropriate testing. If they test positive, life style changes (eg, abstinence from
alcohol consumption) and evaluation for
chronic liver disease justifying antiviral
therapy to reduce the likelihood of disease
progression may be warranted. Earlier experiences from Canada and several European countries indicate that the number of
transfusion recipients who ultimately bene33
fit from look-back efforts is small. A survey
of US blood collection facilities and hospital transfusion services after implementation of targeted HCV look-back resulted in
an estimate that notification of the recipients of 98,484 components would result in
the identification of 1520 infected persons
who were previously unaware of their in34
fection. This would represent less than 1%
of the 300,000 still-living recipients who
may have acquired infection by blood
transfusion. More recently, 0.9% to 5.0% of
patients tested for hepatitis C were found to
be positive in a review of look-back studies
in Canada that notified recipients of any
previous transfusions of the risk of HCV
and then provided testing; 42% to 58% of
the cases were newly identified.35

Human Immunodeficiency
Viruses
The human immunodeficiency viruses type
1 (HIV-1) and type 2 (HIV-2) are the
etiologic agents of AIDS. The AIDS syndrome was recognized in 1981, well before the discovery of the causative virus in
1984. Wider implications of the immune
disorder were noted when, in 1982, AIDS
was reported in three patients with hemo36
philia, and in a 17-month-old infant
whose multiple transfusions at birth included a unit of platelets from a donor

676

AABB Technical Manual

who subsequently developed AIDS. Within a few years, studies established that well
over 50% of patients with hemophilia who
received clotting factor concentrates in
the early 1980s developed HIV-1 infection.38 In some regions of the United States,
up to 1% of single-donor unit transfusions
were infected with HIV in the early 1980s.39

Clinical Manifestations of HIV Infection
HIV is a cytopathic retrovirus that preferentially infects CD4-positive T lymphocytes (helper T cells) in lymph nodes and
40
other lymphoid tissue. After primary infection, HIV replicates and disseminates
initially as cell-free virions, and 10 days to
3 weeks after infection, viremia is first de-

tectable in the plasma. During this time,
about 60% of acutely infected persons develop an acute retroviral syndrome, characterized by a flu-like illness with fever,
enlarged lymph nodes, sore throat, rash,
joint and muscle pain—with or without
headache, diarrhea, and vomiting. As HIV-1
antibodies appear, the disease enters a
clinically latent stage; however, viral replication and dissemination continue. During this phase, the virus can be transmitted by blood or genital secretions (Fig
28-2).
Persistent infection with an asymptomatic clinical status has been estimated to
last a median of 10 to 12 years in the absence of treatment.41 After years of asymptomatic infection, both plasma viremia and
the percentage of infected T lymphocytes

Figure 28-2. Virologic events during primary HIV infection. After initial infection and propagation of
HIV in lymph nodes, a blood donor becomes infectious (defined as day 0), with HIV RNA being detectable in plasma on days 14-15, HIV DNA detectable in leukocytes at days 17-20, and HIV antibodies detectable between days 20 and 25. Anti-HIV persists indefinitely but may be lost in the preterminal
stage of the disease, in parallel with a surge in viral burden, indicating collapse of the immune system. HIV = human immunodeficiency virus; RT-PCR = reverse transcriptase polymerase chain reaction;
PBMC = peripheral blood mononuclear cells.

Chapter 28: Transfusion-Transmitted Diseases

increase. Loss of the immune functions
served by helper T cells impairs immune
reactivity, and there may be inappropriate
immune activation and cytokine secretion.
Eventually, there is a sharp decline in the
number of CD4+ T lymphocytes, and the
vast majority of infected individuals succumb to opportunistic illnesses fostered by
profound immunosuppression.
Enumeration of viral load and CD4+ cells
is used to guide clinical and therapeutic
management of HIV-infected persons. The
AIDS classification system devised by the
Centers for Disease Control and Prevention
(CDC) is based on the number of CD4+ T
cells (<200/µL defines AIDS), the presence
or absence of systemic symptoms, and existence of any of the 26 clinical conditions
considered to be AIDS-defining illnesses.42
Among these conditions are otherwise unusual malignancies, such as Kaposi’s sarcoma, central nervous system lymphoma,
and a wide array of devastating, potentially
lethal opportunistic infections with fungi
and parasites, the most common being
Pneumocystis carinii pneumonia.
Advances in treatment of HIV and opportunistic infections have dramatically enhanced the survival of infected persons.
Unfortunately, worldwide, the disease is
still spreading rapidly and, for the majority
of HIV-infected individuals in developing
countries, effective therapy is either not
available or not affordable.

Risk Factors for HIV Infection
HIV can be transmitted through sexual
contact, childbirth, breast-feeding, and
parenteral exposure to blood. Those identified early as being at highest risk were
men who had sex with other men; commercial sex workers and their contacts;
needle-sharing drug users; patients with
hemophilia who received human-derived
clotting factor concentrates; and, to a les-

677

ser extent, recipients of blood transfusions.
By 1989, the rate of infection in each
group was no longer increasing exponentially and appeared to have reached a plateau in the populations most at risk.43 HIV
seroprevalence had stabilized in most US
cities. Heterosexual transmission of HIV
represented a progressively larger proportion of US HIV infections and AIDS cases
reported in the 1990s.44 This is of importance in transfusion medicine because
screening for heterosexual high-risk behavior is more problematic than screening for male-to-male sex and parenteral
45
drug use.

HIV-2 and HIV-1, Group O
First discovered in 1985, HIV-2 causes endemic infection in many countries in
West Africa. Although HIV-2 was initially
restricted to West Africa, recent studies in
European countries such as Great Britain
and France (which have significant immigration from West Africa) have observed
increasing rates of HIV-2 and other HIV
subtypes.46,47
The first case of HIV-2 infection in the
United States was reported in March 1988
in a young West African who had recently
immigrated to the United States.48 The
spectrum of disease attributable to HIV-2 is
similar to that caused by HIV-1; however,
there appears to be a longer incubation period and lower incidence of progression to
AIDS. HIV-2 is spread both sexually and
from mother to child, but transmission is
less efficient than for HIV-1.
Tests in the United States on parenteral
drug users, persons with sexually transmitted diseases, newborn infants, and homosexual men confirm the very limited prevalence and transmission of the agent. HIV-1/
HIV-2 combination tests were implemented
in the United States in 1992. Since then,
three HIV-2-infected donors have been

678

AABB Technical Manual

identified; none appeared to have been infected in the United States.49
To date, three groups of HIV-1 viruses
have been identified: group M (major
group); group O (outlier group); and, most
recently, group N. Further, there are 10 subtypes (A-J) of group M. None of 97 donors
retrospectively identified as being HIV-1 infected in 1985 and three (1%) of 383 donors
prospectively identified between 1993 and
1996 were found to have non-B subtypes
(two subtype As and one subtype C).50 Of
note, this study did find an increase in env
gene diversity among HIV-1 group B strains
over time and called for continued surveillance for emergence of non-B subtypes and
development of test systems for their detection. A follow-up study from these same
investigators documented characterized
HIV subtypes in 291 infected US donors
identified from 1997 through 2000 and
identified that six (2%) were non-B sub51
types of HIV-1 and one was HIV-2. In
Cameroon and surrounding West African
countries, an estimated 1% to 2% of HIV infections are caused by group O viral strains.52
As with HIV-2, group O isolates have rarely
been seen outside this geographic area.
Concern arose when studies demonstrated
that some group O viral isolates were not
reliably detected by several EIA tests used
for blood donor screening. Of the two
FDA-licensed tests for NAT, both were evaluated by the manufacturer for the detection
of non-B subtypes including group O and
N, using a limited number of specimens.
(although neither test detects HIV-2). However, until reliable detection of group O infections is established, the FDA recommends indefinite deferral of blood and
plasma donors who were born, resided, or
traveled in West Africa since 1977, or had
sexual contact with someone identified by
these criteria.53 The risk of group O infection in the United States is very low. In a
survey of HIV subtypes in US blood donors

over a period spanning two decades, only
three non-B subtypes were identified. Two
50
of these three donors were born in Africa.

Transfusion Considerations

Transfusion-Transmitted HIV-1
All blood components can transmit HIV-1.
Although approximately 1% of all AIDS
exposures have resulted from transfusion
or organ or tissue transplantation, the introduction of MP-NAT in 1999 virtually
eliminated the risk of transfusion-transmitted HIV.54 The few cases of HIV infection that have been documented since
1999 were attributed to low-level viremic
units that likely would have been detected
by ID-NAT.55-57
Most but not all recipients of HIV-infected blood transfusions become infected.
In one large study, HIV infection developed
in 89.5% of recipients who received blood
from anti-HIV-positive donors.58 Transmission rates correlated with component type
and viral load in the donation. With the exception of coagulation factor concentrates,
plasma derivatives (such as albumin and
immune globulins) have not been reported
to transmit HIV infection. No transmission
of HIV attributable to coagulation factors has
been documented in the United States
since implementation of full donor screening
and virus inactivation techniques in 1987.59

Transfusion-Transmitted HIV-2 and HIV-1,
Group O
There have been two reports of possible
HIV-2 transmission through blood component use, both in Europe. Two women
were infected by Whole Blood obtained from
a donor who developed AIDS at least 16
years after becoming infected with HIV-2;
both women were asymptomatic 14 years
after transfusion.60 Two hemophilia patients who received clotting factors were
also infected. Because of their extremely

Chapter 28: Transfusion-Transmitted Diseases

low prevalence, no HIV-2 or HIV-1 group
O transmissions have been reported in
the United States by blood transfusion or
any other transmission route.

Current Risk of Posttransfusion HIV
With screening tests available before 1992,
the seronegative interval (“window period”) averaged 45 days. More sensitive
screening tests for HIV antibody closed
the antibody-negative window to approximately 22 days.17 Introduced in 1996, p24
antigen screening further reduced the potentially infectious window by an estimated 6 days,61 although it appears that
fewer HIV-infected units were intercepted
by the introduction of this test than had
been expected based on the calculated reduction of the infectious window period.
Risk from seronegative donations will
vary in proportion to the incidence of HIV
infection in the donor community. Overall
estimates of posttransfusion HIV risk in the
United States since the implementation of
HIV NAT are approximately 1 in 2 million
screened donations.23,30

HIV Testing of Blood Donors
AABB Standards for Blood Banks and Transfusion Services62(pp33,34) and FDA regulations63
require that all units of blood and components be nonreactive for anti-HIV-1 and
anti-HIV-2 before they are issued for
transfusion. HIV-1-antigen (HIV-1-Ag)
testing is no more required by the FDA or
AABB as long as licensed HIV-1 NAT is in
place. Figure 28-3 shows the sequence of
screening and confirmatory testing for antiHIV-1/2.
Because the consequence of missing even
one true positive is great, screening tests
are designed to have high sensitivity both
to immunovariant viruses and to low-titer
antibody during seroconversion. EIA-de-

679

tectable antibody develops 2 to 4 weeks
after exposure,58 days to a week after the onset of symptoms in those who have any recognized acute illness,64 and about 12 days
after detectable viremia by ID-NAT or 9 to
10 days by MP-NAT.17,65
A few days later, HIV-1 antibodies become
detectable by the HIV-1 Western immunoblot. With very rare exceptions, all persons
infected with HIV develop anti-HIV reactivity detectable by EIA and Western blot that
persists for life.
More sensitive tests using NAT technologies have been shown to detect additional
potentially infectious donors. In February
2002, the FDA approved the first NAT system for screening whole blood donors and
issued draft guidance for blood establishments; the final guidance was issued in
66,67
October 2004. It has been estimated that
HIV NAT has reduced the window period
for HIV from 16 days to 10 days.17 The use
of NAT for donor testing has not only increased sensitivity, but has also decreased
the number of false-positive tests, increasing specificity. During the 3 years of investigational HIV NAT, 12 confirmed HIV-1
RNA-positive antibody-negative donors
were detected in 37 million donations
screened, or 1 in 3.1 million, of which only
two were detected by HIV-1 p24 antigen.23

Confirmatory Testing for Antibodies to
HIV-1/2
If a disease has low prevalence in the
tested population, the likelihood is high
that most positive screening test results will
be false positive. More specific supplemental tests are then required to confirm the
screening test results. The most commonly
used of these tests for antibodies to HIV-1/2
is the Western blot (see Chapter 7).
According to current FDA and CDC criteria, a sample is defined as anti-HIV-positive if at least two of the following bands are

680

AABB Technical Manual

Figure 28-3. Decision tree for anti-HIV-1/HIV-2 testing of blood donors. IFA = immunofluorescence assay; WB = Western blot. If EIA testing is nonreactive, NAT testing must also be nonreactive before release of a donation.

present: p24, gp41, and/or gp120/160.
Negative Western blot results have no
bands present. Western blot results classified as indeterminate have some bands
present but do not have the pattern defining HIV positivity. Individuals infected with
HIV may have indeterminate patterns when
initially tested but develop additional bands
within 6 weeks. Healthy individuals with
initial indeterminate patterns continue to
have negative or indeterminate results on
repeat samples and are negative on clinical
examination and additional tests, including
viral cultures and NAT. Healthy donors who
continue to show the same indeterminate
pattern for more than 3 months can be reassured that they are unlikely to have HIV in-

fection, but they are not currently eligible
to donate blood. Several groups have identified Western blot patterns in blood donors
that were identified as false-positive results;
for these, testing (using NAT) is recommended to resolve the infectious status of
the donor.69
Approximately 50% of all HIV screening
EIA repeatedly reactive donors test indeterminate by licensed HIV-1 Western blot assays. However, when these donations were
tested by ID-NAT, less than 0.1% were
shown to contain HIV-1 RNA (1:1450 RNApositive, indeterminate donors). When combined with those donations that tested
Western blot negative, the frequency of a
donor testing HIV repeatedly reactive and

Chapter 28: Transfusion-Transmitted Diseases

then Western blot indeterminate or negative and demonstrating RNA was 1:4.27
million (S. Stramer, personal communication, 3/17/04).
The FDA has approved reentry protocols
to qualify donors with negative confirmatory test results as eligible for subsequent
donations (see Table 28-2).70 Reentry currently
requires retesting at least 6 months later, to
detect delayed seroconversion; the use of
EIA tests based on whole-virus lysate; and
use of either a licensed Western blot to ensure appropriate sensitivity of the methods
or an FDA-licensed immunofluorescence
assay. 71 The later sample must also be
nonreactive in an EIA test for anti-HIV-2, if
standard testing does not include HIV-2.
The FDA recently published draft reentry
guidelines that will allow reinstatement of
donors with indeterminate Western blot results, eliminate the need for viral lysate EIA
testing, and include testing by HIV-1 NAT.
NAT results can be used in lieu of supplemental testing in specific circumstances.
An FDA variance is required.24 It is anticipated that NAT HIV-1 testing will be used in
the future for reentry.

Positive Tests in Autologous Donors
Whether HIV EIA repeatedly reactive or
NAT-positive autologous donations should
be withheld from transfusion is controversial.72 These units may be supplied for
autologous use if the following conditions
are met: 1) there is a written, signed, and
dated request from the patient’s physician
authorizing this shipment, 2) there is a
written statement from the transfusion
service indicating willingness to receive this
product, and 3) the transfusion service
takes responsibility for ensuring that there
is documented verification of the accurate
identity of the transfusion recipient. These
units must be labeled “BIOHAZARD” and
“FOR AUTOLOGOUS USE ONLY.”

681

Whether a facility elects to offer autologous services is an internal decision. Institutions should consider, however, that
where feasible for a patient, it is generally
accepted that the patient should have the
option to use his or her blood. In addition,
a US Supreme Court decision in the Bragdon v Abbott case ruled that HIV-positive
individuals are protected under the Americans with Disabilities Act.73 Whether this
would apply to HIV-positive donations that
could represent a risk to hospitalized transfused patients remains controversial.

Recipient Tracing (Look-Back)
Identification of persons who have received
seronegative or untested blood from a donor later found to be infected by HIV is
referred to as “look-back.” Because the interval between receipt of an infected
transfusion and onset of AIDS can be very
long, recipients are usually unaware of their
infection and may be infectious to others.
To identify these individuals, blood centers must have procedures to notify recipients of previous donations from any donor later found to have a confirmed
positive test for anti-HIV or a confirmed
positive test for HIV using licensed NAT. If
a patient with AIDS is known to have donated previously, recipients of blood or
blood components from these donations
should be traced and notified. Recipient
tracing and testing are usually accomplished through the patient’s physician, not
through direct contact with the patient. In
companion rules, the FDA and the Centers for Medicare and Medicaid Services
established timelines and standards de74-77
If recipients of units
fining look-back.
that were donated at least 12 months before the last known negative test are tested
and found negative, earlier recipients are
probably not at risk because infectivity

682

AABB Technical Manual

earlier than 12 months before a negative
screening test is extremely unlikely.

Human T-Cell Lymphotropic
Viruses
HTLV, Type I
Human T-cell lymphotropic virus, type I
(HTLV-I) was the first human retrovirus
isolated and the first to be causally associated with a malignant disease of humans,
adult T-cell lymphoma-leukemia (ATL).58
HTLV-I is also associated with the neurologic condition HTLV-associated myelopathy (HAM), often called tropical spastic
paraparesis (TSP). ATL was described before HAM. Both these conditions occur in
a small minority (no more than 2-4%) of
persons harboring the virus. Infection
during childhood is an important aspect
of, and possibly a requirement for, developing ATL many years later, whereas
childhood or adult infection can cause
HAM, with a variable latent period.
Prevalence of HTLV-I infection shows
striking geographic clustering, with pockets
of high endemicity in parts of southern
Japan and certain Pacific Islands; sub-Saharan Africa; and the Caribbean basin, Central America, and South America. Transmission is by mother to child through breast
milk, by sexual contact (predominantly
male-to-female), and by exposure to blood.

HTLV, Type II
Human T-cell lymphotropic virus, type II
(HTLV-II) was described several years after HTLV-I. There is at least 60% similarity
of genetic sequences to those of HTLV-I;
antibodies to either show strong cross-reactivity in tests with viral lysates. HTLV-II
also shows clustering, but in different
populations. High prevalence has been

noted among some Native American populations and in intravenous drug users in
the United States, in whom seroprevalence is 1% to 20%. Rare disease associations with HTLV-II include HAM; its
occurrence seems to be somewhat less
frequent than with HTLV-I.58
Epidemiologic data suggest that there is
an excess of infectious syndromes (eg,
bronchitis, urinary infections, and pneumonia) among blood donors infected with
78-81
HTLV-I or -II.

Clinical Observations
For both HTLV-I and -II, infection persists
lifelong, as does the presence of antibody.
Studies of prevalence and transmission
use seroconversion as the endpoint for diagnosis. Infection does not cause any recognizable acute events, and with the exception of those developing ATL or HAM,
infected individuals experience few, if
any, health consequences. Most carriers
are asymptomatic and completely unaware of the infection.

Transmission
Both viruses are very strongly cell-associated. Contact with infected viable lymphocytes can cause infection, but plasma
appears to be not, or much less, infective.
Cellular components from infected donors
cause seroconversion in at least 50% of
recipients in Japan, but apparently in a
much smaller proportion of US recipients. 58 After refrigerated storage for 10
days or more, red cells transfused from an
infected donor are far less likely to result
in seroconversion, presumably due to degradation of lymphocytes that transmit
the virus. 8 2 , 8 3 Transfusion-transmitted
HTLV-I infection has been associated with
HAM of rather rapid onset and at least
one case of ATL.

Chapter 28: Transfusion-Transmitted Diseases

Donor Tests
Donor screening for anti-HTLV-I began in
the United States in late 1988; at that time,
the rate of confirmed positive tests was
approximately 0.02%, or 1 in 5000 units
collected, a figure that has since declined
at least 10-fold as seropositive persons
have been removed from the donor pool.84
The combined risk of transfusion-transmitted HTLV-I/II infection has been esti84
mated as about 1 in 641,000 units. Further risk reduction can be expected from
the implementation of combination
HTLV-I/II EIA tests in blood donor screening. The first such test was licensed in
1998 in the United States. By using viral
lysates from both HTLV-I and HTLV-II viruses, the test offers sensitive detection of
both anti-HTLV-I and anti-HTLV-II. The
originally licensed anti-HTLV-I EIA
screening tests might have missed up to
50% of HTLV-II infections.85
A donation that is repeatedly reactive on
EIA may not be used for transfusion. If a
donor tests repeatedly reactive by the EIA
screening assay on two or more occasions,
he or she should be notified and indefinitely deferred.85 Another recommended
approach is to test donor serum with a second manufacturer’s EIA test kit.86 If that test
is also repeatedly reactive, then the donor is
indefinitely deferred on the basis of that
single donation. Further testing of serum
that is repeatedly reactive for anti-HTLV-I/II
against antigen preparations specific for
the two agents (HTLV-I or HTLV-II), or by
NAT on material from peripheral blood
mononuclear cells, can characterize the infecting agent. Half or more of US blood donors confirmed infected after EIA screening
prove to have HTLV-II infections. Although
there is no FDA requirement to perform additional testing (no confirmatory test for
HTLV is licensed), most centers do so using
an investigational supplemental test or se-

683

ries of tests in an algorithm if the donation
tests repeatedly reactive by both the testof-record and second HTLV-I/II EIAs; if
supplemental tests are positive, the donor
is indefinitely deferred (see Table 28-3). Regardless of the results of these investigational supplemental tests, a donor meeting
the EIA criteria for indefinite deferral described above must still be indefinitely deferred.

Quarantine and Look-Back
In-date prior collections of blood or components from donors who subsequently
are found repeatedly reactive for antiHTLV-I/II need to be quarantined. Because recipients of units from seropositive donors do not consistently seroconvert and because many seropositive
donors have lifelong infection, the time
frame for look-back is not self-evident. No
requirement for recipient tracing and notification has been established.85 Screening, in place since 1988, has probably removed from the active donor pool most
donors with lifelong infection.

West Nile Virus
West Nile virus (WNV) is a flavivirus primarily transmitted in birds through mosquito bites; humans are incidental hosts.
In humans, symptoms range from a mild
febrile illness to encephalitis, coma, and
death, although about 80% of infected individuals remain asymptomatic. WNV
outbreaks have been reported in Europe,
the Middle East, and Russia over the past
decade and have been associated with
human encephalitis and meningitis (in
<1%), although no transfusion-associated
cases were reported. WNV was observed
in the United States, in the metropolitan

684
AABB Technical Manual

Table 28-3. Recommended Actions for HTLV-I/II Testing
First Donation to Be Tested for HTLV-I/II Antibodies

Subsequent Donation(s)

Second
Manufacturer

EIA

WB/RIPA

Donation

Donor

EIA

WB/RIPA

Donation

Donor

Repeatedly
reactive

Positive

Destroy all
components*

Defer and
counsel

Not applicable/Donor deferred

Repeatedly
reactive

Negative or Destroy all
indeterm.
components*

No action

Repeatedly
reactive

Pos

Any result

Destroy all
components*

Defer and
counsel

Nonreactive†

Neg

Not done

All compo- No action
nents acceptable

*Destroyed unless appropriately labeled as positive for HTLV-I/II antibodies, and labeled for laboratory research use or further manufacture into in-vitro diagnostic reagents.
†
Assuming that separate prior donations have been repeatedly reactive for HTLV-I/II antibody no more than once. If separate prior donations had been repeatedly reactive for
HTLV-I/II antibodies on two or more occasions, the donor should have been indefinitely deferred.
HTLV-I = human T-cell lymphotropic virus, type I; HTLV-II = human T-cell lymphotropic virus, type II; EIA = enzyme immunoassay; WB = Western blot; RIPA = recombinant
immunoprecipitation assay.

Chapter 28: Transfusion-Transmitted Diseases

New York City area, in 1999. Subsequently,
it dispersed rapidly westward throughout
the country, spread by infected birds. In
2001, 66 human cases occurred in 10
states. In 2002, 4161 cases of WNV illness
were reported in 39 states, including 284
deaths. In 2002, 23 cases were associated
with transfusion (with an infected donor
identified); Red Blood Cells (RBCs), Fresh
Frozen Plasma, and Platelets were impli87
cated. The implicated donations occurred between July 22 and October 6,
2002. Statistical resampling of data available regarding case onset dates during the
2002 epidemic was used to generate estimates of the mean risk of transfusion-associated WNV transmission (per 10,000
donations) for six states and six selected
metropolitan areas, with results ranging
from a mean of 2.12 to 4.76 and 1.46 to
12.33, respectively.88
An FDA guidance document in May
2003 recommended deferral of donors with
a diagnosis of WNV infection for 28 days after onset of symptoms or 14 days after resolution, whichever is later, and recommended inquiring whether donors had
experienced a fever with headache in the
week before donation.89 However, because
most infected persons are asymptomatic,
the yield of such measures would be expected to be modest, and testing was
sought as the best method to identify infected donors. During the summer and fall
of 2003, almost 5 million donations constituting over 95% of collections in the United
States were tested for WNV by NAT in
minipools of six or 16 samples under one of
two IND protocols—one using a polymerase chain reaction method and one using a
transcription-mediated amplification
method. During 2003, approximately 1000
donors were confirmed as viremic for WNV
and approximately 1500 likely infected
components were interdicted.90 However,
six probable or confirmed transfusion-as-

685

sociated WNV cases were reported in 2003;
four recipients had WNV encephalitis, one
had West Nile fever, and one critically ill
patient did not have discernible WNV-compatible illness despite confirmed WNV in91
fection. Some of the donors were identified through retrospective testing of
individual samples, and it appears that all
were related to specimens with very low viral titers. The number of transfusion-associated cases undoubtedly would have been
much higher had widespread testing under
IND protocols not been initiated. There
were 9862 WNV cases and 264 deaths in the
United States overall during this time.
The persistent low-level transmission of
WNV by transfusion in 2003 led to the implementation of targeted ID-NAT in high
epidemic regions in 2004. This effort successfully interdicted low-level viremic units
that would have been missed by MPNAT.92,93
In 2004, the virus appeared predominantly in western states, with California accounting for 31% of cases.94 Only three
states (Hawaii, Alaska, and Washington) remained free of reports of infection in either
humans or animals (mammalian and
avian). A total of 2470 cases of human WNV
infection were reported in 40 states plus the
District of Columbia, including 88 fatal
cases—far fewer than the previous year. A
total of 192 WNV-positive donors were
identified, almost all from states west of the
Mississippi River.94 Of these donors, three
subsequently reported neuroinvasive WNV
illness and 55 subsequently developed
WNV fever.94 One probable transfusion-as95
sociated case was reported.
The experience to date indicates that
blood screening for WNV has improved
blood safety. However, a small risk of WNV
transfusion-associated transmission remains. If the number of WNV cases continues to dramatically decline, the need for
WNV testing will be reassessed. The FDA

686

AABB Technical Manual

has agreed that asking the question concerning fever with headache in the week
before donation may be discontinued (K
Gregor y, personal communication,
3/30/05).

Herpesviruses and Parvovirus
Cytomegalovirus
CMV, a member of the human herpesvirus family, is a ubiquitous DNA virus
that causes widespread infection; transmission can occur through infectious
body secretions, including urine, oropharyngeal secretions, breast milk, blood,
semen, and cervical secretions. About 1%
of newborns are infected, transplacentally
or through exposure to infected cervical
secretions at delivery or by breast milk. In
early childhood, CMV is often acquired
through close contact, especially in daycare settings; in adulthood, through sexual intercourse. The prevalence of CMV
antibodies ranges from 50% to 80% in the
general population.96 The rate increases
with age and is generally higher in lower
socioeconomic groups, in urban areas,
and in developing countries.

Clinical Observations
In persons with an intact immune system,
CMV infection may be asymptomatic and
remain latent in tissues and leukocytes
for many years. Infection, either primary
or reactivation of latent infection, can be
associated with a mononucleosis-like
syndrome of sore throat, enlarged lymph
nodes, lymphocytosis, fever, viremia,
viruria, and hepatitis. Intrauterine infection may cause jaundice, thrombocytopenia, cerebral calcifications, and motor
disabilities; the syndrome of congenital
infection causes mental retardation and
deafness and may be fatal.

CMV infection can progress to CMV disease and cause serious morbidity and mortality in premature infants, recipients of
organ, marrow, or peripheral blood progenitor cell transplants, and in AIDS patients.96
Pneumonitis, hepatitis, retinitis, and multisystem organ failure are manifestations of
CMV disease. CMV infection can result from
blood transfusions. Other sources of infection, however, such as organ transplants
from CMV-positive donors or reactivation
of latent virus, may be as much or more of a
risk than transfusion.

Transfusion-Transmitted CMV
Infection with CMV varies greatly according to socioeconomic status and geographic region. Although approximately
50% of blood donors can be expected to
be CMV seropositive, it has been estimated that, currently, less than 1% of seropositive cellular blood components are
able to transmit the virus.96,97 Rarely, posttransfusion hepatitis may be due to CMV.
The postperfusion mononucleosis syndrome that first focused attention on
CMV in transfused components in the
early 1960s is now rarely seen. Posttransfusion CMV infection is generally of no
clinical consequence in immunocompetent recipients, and intentional selection
of CMV-reduced-risk blood (see below) is
not warranted.
In light of the potential for severe CMV
disease in immunocompromised patients,
several categories of recipients have been
identified who should be protected from
transfusion-transmitted CMV.7 These include low-birthweight premature infants
born to seronegative mothers; seronegative
recipients of hematopoietic progenitor cells
from CMV-negative donors; seronegative
pregnant women, because the fetus is at
risk of transplacental infection; and recipi-

Chapter 28: Transfusion-Transmitted Diseases

ents of intrauterine transfusions. In some
cases, seronegative recipients of organ
transplants from a seronegative donor;
seronegative individuals who are candidates for autologous or allogeneic hematopoietic progenitor cell transplants; and
those few patients with AIDS who are free
of CMV infection are also included.

Preventive Measures
Blood from donors who test negative for
CMV antibody has very little risk of transmitting CMV, but the supply of seronegative blood is limited.96,97 Another approach
to reduce risk is to remove leukocytes
from donated blood (because leukocytes
are the principal reservoir for CMV).98-100
Although the precise leukocyte population
that harbors the virus has not been defined, leukocyte removal with high-efficiency filters, to 5 × 106 leukocytes per
component or fewer, can significantly reduce, if not prevent, posttransfusion CMV
in high-risk neonates and transplant recipients. Effectively leukocyte-reduced
cellular components are considered equivalent to serologically screened components by many experts, although this is
controversial.98-100 The incremental benefit
of serologic testing when added to leukocyte reduction has not been established.
Prophylactic therapy with CMV immune
globulin and prophylactic use of antiviral
agents are being investigated as options
for high-risk immunosuppressed organ
transplant recipients.96,97

Epstein-Barr Virus
EBV causes most cases of infectious mononucleosis and is closely associated with
the endemic form of Burkitt’s lymphoma
in Africa and with nasopharyngeal carcinoma. Most persons have been infected

687

by the time they reach adulthood; although usually asymptomatic, infection
persists. Infection is spread by contact
with infected saliva. Primary infection in
children is either asymptomatic or is
characterized by a sore throat and enlarged lymph nodes. Primary infection in
older, immunologically mature persons
usually causes a systemic syndrome, infectious mononucleosis, with fever; tonsillar
infection, sometimes with necrotic ulcers;
enlarged lymph nodes; hematologic and
immunologic abnormalities; and sometimes hepatitis or other organ involvement.
EBV infection targets B lymphocytes,
which undergo polyclonal proliferation and
then induce a T-lymphocyte response, observed as “atypical lymphocytes.”
Transfusion-transmitted EBV infection is
usually asymptomatic, but it has been a
rare cause of the postperfusion syndrome
that follows massive transfusion of freshly
drawn blood during cardiac surgery and is
a rare cause of posttransfusion hepatitis.101
EBV plays a role in the development of nasopharyngeal carcinoma and at least one
form of Burkitt’s lymphoma and has the
in-vitro capacity to immortalize B lymphocytes. EBV contributes to the development
of lymphoproliferative disorders in immunosuppressed recipients of hematopoietic and organ transplants. Given a 90%
seropositivity rate for EBV among blood donors and essentially no risk for clinical disease from transfusion-transmitted EBV in
immunocompetent recipients, serologic
screening for this virus has not been considered helpful. As is the case for CMV, leukocyte reduction of cellular blood components would be expected to reduce the risk
of EBV infection in severely immunosuppressed seronegative patients who may be
at risk for clinical disease. However, there
have been no studies to verify such a
reduced risk.

688

AABB Technical Manual

Human Herpesviruses 6 and 8
As with CMV and EBV, human herpesvirus
6 (HHV-6) is a cell-associated virus that
integrates with the genome of lymphocytes. The seroprevalence in some adult
populations approaches 100%. Primary
infection in immunocompetent children
is recognized as exanthem subitum, a febrile illness characterized by a rash; it is
rarely complicated by involvement of other
organ systems. Immunocompromised patients (eg, those with transplants or AIDS)
may experience manifestations of reactivation of HHV-6 infection in multiple organ systems. Studies have sought an association between multiple sclerosis and
HHV-6 infection, but this remains controversial. With the ubiquity of antibodies to
HHV-6 and absence of disease associations
after transfusion transmission, no recommendations have been made for protection of seronegative blood recipients from
transmission by blood components.102
HHV-8 (also known as Kaposi’s sarcomaassociated herpesvirus or KSHV) is causally
associated with both Kaposi’s sarcoma and
body cavity-based lymphomas. It has been
found in apparently healthy blood donors,
but spread appears to be primarily by the
venereal route. Low titers of HHV-8 antibodies
were found in 11% of 91 healthy US blood
donors.103 Among HHV-8-seropositive women,
injection drug use and indices of sexual activity were independent risk factors for
HHV-8 infection.104 In this study, the association with injection drug use suggests that
transmission by infected blood is possible.
Transmission by organ donation has been
documented.105-107 Epidemiologic studies
suggest that blood transfusion is associated
with a small risk of HHV-8 transmission.108

Parvovirus
Parvovirus B19 is the cause of erythema
infectiosum or “fifth disease,” a contagious

febrile illness of early childhood. Infections
in adults may be associated with arthritis
but are generally benign. More ominously, parvovirus B19 can infect and lyse red
109
cell precursors in the marrow. This may
result in sudden and severe anemia in patients with underlying chronic hemolytic
disorders who depend on active erythropoiesis to compensate for shortened red
cell survival. Patients with cellular immunodeficiency, including those infected with
HIV, are at risk for chronic viremia and associated hypoplastic anemia. Infection during pregnancy predisposes to spontaneous
abortion, fetal malformation, and hydrops
110
from severe anemia and circulatory failure.
The red cell P antigen is the cellular receptor for parvovirus B19, and people who
do not have the P antigen are naturally resistant to infection.111 About 30% to 60% of
normal blood donors have antibodies to parvovirus B19, which indicates immunity
rather than chronic persistent infection.109
Viremia occurs only in the early phases of
infection and there is no evidence for a carrier state; the incidence of viremia in blood
donors has been estimated to range from 1
in 3300 to 1 in 40,000.110 Parvovirus B19 lacks
a lipid envelope and is therefore not inactivated by solvent/detergent treatment or heat
inactivation using temperatures below 100
C.109 The virus has been found regularly in
clotting factor concentrates and has been
transmitted to persons with hemophilia. Rare
transmission through cellular blood components and plasma, but not intravenous immunoglobulin and albumin, has been reported.110
After the description of parvovirus B19
seroconversion (without clinical illness) in
volunteers during Phase IV clinical evaluation of solvent/detergent-treated plasma,
derivative manufacturers (with the concurrence of FDA) began development and implementation of NAT screening for high-titer parvovirus B19 viremic donations in

Chapter 28: Transfusion-Transmitted Diseases

minipools. The rationale, supported by observations from the Phase IV evaluation, is
that high-titer donations overwhelm neutralizing antibody in plasma pools, allowing
transmission of this highly resistant virus
by some derivatives. The FDA has classified
this MP-NAT as an in-process manufacturing control rather than a donor screening
test. Screening of whole blood donations has
not been a high priority because of the benign and/or transient nature of most parvovirus disease, the availability of effective
treatment (intravenous immunoglobulin)
for chronic hematologic sequelae, and the
extreme rarity of reports of parvovirus B19
transmission by individual components.112

Transmissible Spongiform
Encephalopathies
The transmissible spongiform encephalopathies (TSEs) are degenerative brain disorders caused by agents often called prions,
postulated to be infectious proteins. They
are characterized by long incubation periods, measured in years to decades, and by
the extreme resistance of the pathogens
to inactivation by physical and chemical
methods sufficient for classic pathogens.
Two TSEs, Creutzfeldt-Jakob disease (CJD)
and variant Creutzfeldt-Jakob disease
(vCJD), are of particular interest in transfusion medicine.

Classic CJD
CJD is a degenerative brain disorder that
is rapidly fatal once symptoms of progressive dementia and motor disturbances
develop. Approximately 85% of cases are
sporadic. Symptoms do not develop until
many years to several decades after the
initial infection. Ten to fifteen percent of
cases are familial, associated with inheritance of one of at least 20 described mu-

689

tations in the prion gene that, in its nonmutated form, encodes for a normal cellular protein. Worldwide, there is about
one case of CJD per million people per
year, nearly all in older individuals. In
sporadic CJD, the vast majority of cases,
the mode of acquisition is unknown. The
agent causing CJD is resistant to commonly
used disinfectants and sterilants. Iatrogenic CJD has been transmitted by administration of growth hormone and gonadotropic hormone derived from pooled
human pituitary tissue, through allografts
of dura mater, and through reuse of intracerebral electroencephalographic elec113
trodes from infected patients.
Early experimental studies in animals
raised the possibility that CJD could be
transmitted by blood transfusion. Additionally, iatrogenic transmission from peripheral injection of human pituitary-derived
hormones has been observed. Nevertheless, several population-based, case-controlled studies have shown no evidence that
blood transfusion is a risk factor for the development of CJD.114-116
Individuals at increased risk for CJD are
excluded from donating blood; this group
includes persons who have received tissue
or tissue derivatives known to be a source
of the CJD agent (eg, dura mater allografts,
pituitary growth hormone of human origin)
and persons with a family history of
CJD.117-119 For the purposes of donor exclusion, unit quarantine, and unit destruction,
family history has been defined as having
one blood relative who has had this diagnosis. However, a donor with CJD in a family
member may be accepted if gene sequences have been tested and found to be
normal.

Variant CJD
In 1996, the first cases of an unusual CJD
outbreak or cluster were described in the

690

AABB Technical Manual

United Kingdom (UK). These cases were
later termed variant CJD (vCJD) and appeared to be caused by the same prion
responsible for bovine spongiform encephalopathy (BSE). This prion is distinct
from the prion found in classical CJD. A
donor infected by dietary exposure to BSE
during the incubation period of vCJD might
theoretically infect a transfusion recipient. Consequently, UK health authorities
prohibited the use of UK plasma for further manufacture, restricted the use of UK
plasma for children (born on or after 1
January 1996), and implemented universal leukocyte reduction of cellular components (to reduce the prions known to be
present in white cells).
Experimental transfusion transmission
of BSE to two sheep (and four cases of
transmission of natural scrapie—a sheep
120
prion illness) have been reported. Positive
transmissions occurred with blood taken at
preclinical and clinical stages of infection.
Two cases of transfusion-transmitted BSE
in humans have been observed121,122 as a result of surveillance in the UK of 48 individuals identified as having received a labile
blood component from a total of 15 donors
who later developed vCJD. In the first possible case, the recipient developed symptoms
of vCJD 6.5 years after receiving a transfusion of red cells donated by an individual
3.5 years before the donor developed
symptoms of vCJD. Although the source of
the infection could have been caused by
past dietary exposure to the BSE agent, the
age of the patient was well beyond that of
most vCJD cases, and the chance of observing a case of vCJD in a recipient in the absence of transfusion-transmitted infection
was estimated to be about 1 in 15,000 to 1
in 30,000, making dietary transmission unlikely in this case.121 In the second possible
case, the person received a blood transfusion in 1999 from a donor who later developed vCJD. This patient died of causes

unrelated to vCJD, but a postmortem examination revealed the presence of the
abnormal prion protein in the patient’s
spleen and in a lymph node.122 Notably, unlike previous cases of vCJD (by any method
of transmission), in which all involved people were homozygous for methionine (MM)
at codon 129 of the prion protein gene
(PRNP), this individual was heterozygous
(methionine valine— MV). In the UK, the
population distribution of this gene is MM,
MV, or VV in 42%, 47%, and 11%, respectively.123
In the United States, blood donors who
were in the UK or Europe during the years
of potential exposure to the BSE agent are
deferred based on the duration of residence
there. Balancing the theoretical risk against
considerations of the adequacy of the blood
supply, the recommended deferral is for 3
months of cumulative residence in the UK
between 1980 and 1996 (and current and
former US military personnel, civilian military employees, and their dependents who
were stationed at European bases for 6
months or more during this period) or 5
years of cumulative residence in Europe.
Potential donors who may have injected
bovine insulin from the UK or received
transfusions in the UK during the BSE epidemic are also excluded.119

Bacterial Contamination
Bacterial contamination remains an important cause of transfusion morbidity
and mortality. Bacterial contamination of
blood components accounted for 29 (16%)
of the transfusion fatalities reported to
the FDA between 1986 and 1991. However, in 2002 alone, there were 17 deaths
reported to the FDA from bacterial contamination of blood components, most
commonly caused by contaminated
apheresis platelets and whole-blood-de-

Chapter 28: Transfusion-Transmitted Diseases

124,125

rived platelets.
Although the hepatitis
viruses, HIV, and WNV have been more
prominently featured in the media and
remain a primary concern of the public,
bacterial contamination is believed to be
the most common infectious source of
morbidity and mortality related to transfusion. To place the risk of bacterial contamination into some perspective, in 2002,
there were 23 transfusion-transmitted cases
of WNV identified in the United States.87
Of these 23 recipients, seven died, but
only five of these deaths were associated
with WNV meningoencephalitis. Thus, the
deaths from bacterial contamination were
more than three times more common than
those from WNV.
No matter how carefully blood is drawn,
processed, and stored, complete elimination of microbial agents is impossible. Bacteria are most often believed to originate
with the donor, either from the venipunc126
ture site or from unsuspected bacteremia.
Bacterial multiplication is more likely in
blood components stored at room temperature than in refrigerated components.126
Organisms that multiply in refrigerated
blood components are often psychrophilic
gram-negative organisms (such as Yersinia
enterocolitica, Serratia liquifaciens, and
Pseudomonas fluorescens). Gram-positive
organisms are more often seen in platelets
stored at 20 to 24 C.
For RBCs, the CDC estimates a symptomatic contamination rate of approximately
1 case per million units, primarily with Y.
127
enterocolitica, followed by S. liquifaciens.
In New Zealand, the incidence of symptomatic Yersinia contamination of RBC
units has been reported to be as high as
one in 65,000 units, with a fatality rate of
one in 104,000.128 Transfusion of an RBC
unit heavily contaminated with a gramnegative organism is often a rapid and catastrophic event, with a quick onset of sepsis
and a greater than 60% mortality rate.129,130

691

Because platelets are stored at 20 to 24 C
to retain their viability and function, they
serve as an excellent growth medium for
bacteria. Sepsis resulting from transfusion
of contaminated platelets is believed to be
both underrecognized and underreported.
Sepsis occurring after transfusion of contaminated platelets is usually not a catastrophic event, but it can occur several
hours or longer after transfusion, making it
more difficult to connect the transfusion to
the sepsis. Because many of the patients infected by bacteria from a platelet transfusion are immunocompromised by their
underlying condition and treatment (eg,
chemotherapy), the event is frequently attributed to other causes, such as an infected catheter, which often involves the
same organisms.
In the United States, 4 million platelet
units are transfused annually (1 million
apheresis platelets and 3 million wholeblood-derived platelet concentrates). 130
Given that approximately 1:1000 to 1:2000
platelet units are contaminated with bacteria (as measured by aerobic cultures done
in multiple studies before 2002), it would
be expected that 2000 to 4000 bacterially
contaminated units would be transfused.131
Estimates of the fraction of such units that
would result in signs or symptoms have
been as low as 1 in 10 cases. However, in
the only study that has prospectively cultured platelets that were transfused, symptoms occurred in 3 of 8 (35.8%) patients
who received culture-positive but Gram’s132
stain-negative platelet pools. Notably, six
Gram’s-stain-positive pools were interdicted and never transfused. Thus, of contaminated products, perhaps 1/10 to 2/5
would be expected to result in clinical sepsis (200 to 1600 cases) if transfused. Data
from national passive reporting studies in
the United States, Great Britain, and France
(Table 28-4) suggest that perhaps 1/5 to 1/3
would result in death (40 to 533 deaths per

692

AABB Technical Manual

Table 28-4. Summary of Organisms Identified in the BaCon, SHOT, and BACTHEM
Studies*
Organism

United
127
States

United
133
Kingdom

France

134

Total

Gram positive

Bacillus cereus

4 (1)

7 (1)

Coagulase-negative
Staphylococci

6 (1)

20 (1)

Streptococcus sp.

3 (1)

5 (1)

Staphylococcus aureus

2 (1)

6 (1)

Propionibacterium
acnes
Subtotal

3
17 (1 = 6%)†

14 (3 = 21%)

10 (0 = 0%)

3
41 (4 = 10%)

Gram negative

Klebsiella sp.
Serratia sp.

2 (2)

Escherichia coli

5 (1)

2 (1)

Acinetobacter

2 (1)

1 (1)

3 (3)

8 (2)

4 (2)

Enterobacter sp.

2 (1)

Providencia rettgeri

1 (1)

Yersinia enterocolitica

Subtotal
Total

1 (1)

11 (5 = 45%)

3 (2 = 67%)

6 (2 = 33%)

20 (9 = 45%)

28 (6 = 21%)

17 (5 = 29%)

16 (2 = 13%)

58 (13 = 14%)

*Number of cases (fatalities) and the percent of the subtotal and total cases are listed. This table illustrates that although
gram-positive organisms are associated with the majority of reported cases (41/58 = 71/%), gram-negative organisms
account for the majority of deaths (9/11 = 82%). Modified with permission from Brecher and Hay.131
†
There were 17 cases of gram-positive organisms identified in the US study; however, only one case (1/17 = 6%) resulted in a fatality.

127,133,134

year).
This translates to a risk of death
from a transfusion of a platelet unit contaminated with bacteria of between 1:7500
to 1:100,000. Clinical observations from
university hospitals with heightened awareness of platelet-related sepsis confirm such
estimates. A fatality rate of 1:17,000 has
been reported by Ness et al, from Johns
Hopkins, with pooled whole-blood-derived
platelets and 1:61,000 with apheresis platelets.135 University Hospitals of Cleveland

similarly observed a fatality rate of approximately 1:48,000 per whole-blood-derived
platelet concentrate.136 With the implementation of bacteria detection of platelets (see
below), it is anticipated that this rate will be
greatly reduced.

Clinical Considerations
Severe reactions are characterized by fever, shock, and disseminated intravascular

Chapter 28: Transfusion-Transmitted Diseases

coagulation (DIC). If bacterial contamination is suspected, the transfusion should
be stopped immediately and a Gram’s stain
and blood culture should be obtained
from the unit (not an attached segment of
tubing because the bag may be contaminated but an isolated segment of tubing
may be sterile) and recipient as promptly
as possible after the reaction is observed.
Bacterial multiplication may cause the
oxygen in an RBC unit to be consumed,
resulting in hemoglobin desaturation and
erythrocyte lysis, both of which contribute to a darkening of the unit compared
to the color of the blood in the attached
sealed segments. Color change (to dark purple or black), clots in the bag, or hemolysis suggest contamination, but the appearance of the blood in the bag is often
unremarkable. The presence of bacteria
on a Gram’s stain of the component is
confirmatory, but absence of visible organisms does not exclude the possibility.
Gram’s stain has a sensitivity of only 106 to
107 CFU/mL. The patient’s blood, the suspect component, and intravenous solutions in all the administration tubing used
should be cultured.
Treatment should not await the results of
these investigations and should include immediate intravenous administration of antibiotics combined with therapy for shock,
renal failure, and DIC, if present.

Preventive Measures
Prevention of septic reactions depends upon
reducing or preventing contamination of
components with bacteria. Careful selection of healthy blood donors is the first
and most important step.
The donor’s present appearance and recent medical history should indicate good
health; additional questioning may be
needed if there is a present or recent history of antibiotic use, of medical or surgical

693

interventions, or of any constitutional
symptoms. Questions to elicit the possibility of bacteremia are especially important
for autologous donors, who may have undergone recent hospitalization, antibiotic
therapy, or invasive diagnostic or therapeutic procedures; there have been several reports of Yersinia sepsis complications after
the infusion of stored autologous blood.
Scrupulous attention must be paid to selecting and cleansing the donor’s phlebotomy site. Skin preparation reduces but does
not completely abrogate the contamination
of components by bacteria. Scarred or dimpled areas associated with previous dermatitis or repeated phlebotomy can harbor
bacteria and should be avoided. Green
soap must not be used to prepare the
phlebotomy site.
Discarding the first aliquot of donor blood
removed (“diversion”) has been proposed
as a measure to reduce bacterial contamination of blood components. This measure
would remove the skin core that may enter
the collection from the hollow bore needle
used in the phlebotomy. Systems have been
developed to facilitate the application of
this approach and would be expected to reduce skin contaminants (mostly gram-positive organisms).
Phagocytosis of contaminating bacteria
by donor white cells in blood components
may be important for the minimization of
clinical bacterial contamination. Leukocyte
removal, with coincident removal of adherent or engulfed bacteria, has been advocated as an approach to reducing Yersinia
contamination of RBCs.137,138
Care in the preparation of components
and handling of materials used in blood administration is essential. If a waterbath is
used, components should be protected by
overwrapping, outlet ports should be inspected for absence of trapped fluid, and
the waterbath should be frequently emptied and disinfected.

694

AABB Technical Manual

Worldwide, screening of platelets for
bacteria is being implemented. Screening is
mandatory in several countries [eg, Belgium (Flemish Red Cross), the Netherlands,
Hong Kong (Red Cross), and Wales].139
In the United States, the College of
American Pathologists (CAP) Commission
on Laboratory Accreditation has added a
question to the Transfusion Medicine
Checklist to assess the presence of a laboratory system to detect bacteria in platelet
140
components (TRM.44955). Similarly, the
62(p11)
AABB requires bacteria detection.
Currently, two culture techniques are approved
by the FDA for quality control of leukocyte-reduced platelets and are available in
the United States. Because of expense and

logistics, whole-blood-derived platelets are
often tested in the United States with less
sensitive but more rapid detection strategies, such as staining or the use of surrogate
markers of bacterial metabolism (eg, pH
and glucose). Several other more rapid and
sensitive detection strategies are under development or are not readily available. One
possible investigative strategy after detecting a confirmed culture-positive platelet
unit is outlined in Fig 28-4.

Prospect for Extended Storage
The extent of bacterial growth in platelet
components correlates with the duration
of storage. In 1983, in recognition of tech-

Figure 28-4. Possible investigative strategy for a positive culture in a platelet unit. Modified with per141
mission.

Chapter 28: Transfusion-Transmitted Diseases

695

nically improved storage conditions, the
FDA increased storage limits of platelets
at room temperature from 3 to 7 days.
However, it reduced the limits to a maximum of 5 days in 1986, responding to reports of bacterial contamination after
142
more than 5 days of storage. The use of
bacteria detection systems has been given
as the rationale for an extension of platelet storage to 7 days in several European
countries and is being implemented in the
United States.139

gesting a low probability that the blood of
donors who have a confirmed positive
syphilis test result is infectious for syphilis.
Nevertheless, a study from the CDC showed
that from 1995-2000, 22 primary, 81 secondary, and 413 early latent syphilis cases
were identified through blood or plasma
144
donor screening in the United States.
Thus, screening of blood donors for syphilis
may have broader public health implications. Currently, performance of a STS is
62(pp33,34)
still required.

Syphilis

Tick-Borne Infections

Syphilis is caused by the spirochete Treponema pallidum and is characteristically
spread by sexual contact. The phase of
spirochetemia is brief and the organisms
survive only a few days at 4 C. Although
transmission by transfusion is possible,
its occurrence is exceedingly rare (the last
case reported in the United States occurred in 1965). Syphilis transmission by
transfusion may not be effectively prevented by subjecting the donor blood to
standard serologic tests for syphilis (STS)
because seroconversion often occurs after
the phase of spirochetemia. Most positive
STS results reflect immunologic abnormalities unrelated to syphilis (biologic
false-positives), inadequately treated noninfectious syphilis that is more of a threat
to the individual being tested than to a
transfusion recipient, or the serologic residual of an effectively treated infection.
A recent series described T. pallidum
DNA and RNA testing of 169 aliquots from
platelet concentrates that were reactive by
STS and confirmed positive by fluorescent
treponemal antibody absorption.143 This series included 48 donors who were positive
by rapid plasma reagin tests (compatible
with recent or active disease). No sample
contained T. pallidum DNA or RNA, sug-

Because many tick-borne infectious agents
circulate in the blood, it is theoretically
possible that they will be transmitted by
transfusion of blood components.

Babesia

Clinical Events
In the United States, the most frequently
recognized transfusion-associated tick-borne
infection is babesiosis. 145 Babesiosis is
usually transmitted by the bite of an infected deer (black-legged) tick and is reported most frequently in the coastal lands
and islands of northeastern United States,
including Martha’s Vineyard, Cape Cod, and
Long Island. Geographic areas of the hosts
and the vectors appear to be expanding.146
Transfusion-associated babesiosis has been
documented in more than 50 cases, caused
mostly by Babesia microti from the Northeast, but also by the recently recognized
WA1-type Babesia parasite, from asymp145-147
As hutomatic infected blood donors.
mans continue to encroach on the habitat
of vectors and natural reservoirs of infection [eg, deer (and other Cervidae) and
mice populations in the northeastern
United States], the incidence of transfusion-transmitted babesiosis may increase.

696

AABB Technical Manual

The vector and reservoir of the Babesia
more recently found in the northwestern
and western United States remain to be
defined. Babesia species survive blood
bank storage for up to at least 35 days and
can be transmitted by both RBCs and
platelet concentrates. Babesiosis classically causes a febrile illness with hemolytic anemia, but infection can also cause
chronic asymptomatic or mildly symptomatic parasitemia. Studies suggest that
untreated persons can harbor B. microti
DNA for long periods, despite mild or absent symptoms, and may transmit infec148
tion for months or possibly longer. Symptoms are often so mild that the infection
is not recognized, which likely explains
the low rate of reported transfusion-transmitted babesiosis. Symptomatic patients
develop fever 2 to 8 weeks after transfusion, sometimes associated with chills,
headache, hemolysis, or hemoglobinuria.
Rarely, life-threatening hemolytic anemia,
renal failure, and coagulopathy develop,
particularly in asplenic or severely immunocompromised patients.149

Preventive Measures
The Babesia carrier state may be asymptomatic and may exceed a year in duration. Persons with a history of babesiosis
are indefinitely deferred, because lifelong
parasitemia can follow recovery from
symptomatic illness. Restrictive policies,
such as not collecting blood in areas
where the disease vectors are endemic
during spring and summer months when
tick bites are more common, are in practice in some locations but probably are of
limited value. No test is available for mass
screening to detect asymptomatic carriers
of Babesia species.

Other Agents
One case of transfusion transmission of
Rocky Mountain spotted fever (Rickettsia
rickettsii) and no cases of human monocytic ehrlichiosis (caused by Erlichia chaffeensis) have been documented.150 A single
possible transfusion transmission of the
unnamed agent of human granulocytic
erlichiosis has been reported.151 In 1997,
Rocky Mountain spotted fever and/ or human monocytic erlichiosis developed in
National Guard trainees at Fort Chaffee,
AR. Ten components donated by infected
trainees had been transfused before a recall; however, none of the persons who received blood from infected donors became
152
clinically ill.
Lyme disease is the most common tickborne infection in the United States.
Borrelia burgdorferi, the causative spirochete, is transmitted through bites of the
deer (black-legged) tick. No transfusionrelated cases have been reported, but
chronic subclinical infections do occur
and experimentally inoculated organisms
can survive conditions of frozen, refrigerated, and room temperature storage.153 On
the other hand, the phase of spirochetemia seems to be associated with symptoms that would render a potential donor
ineligible, and in two reported cases where
the donor became ill shortly after donation, the recipient did not develop infec149
tion. Potential donors who give a history
of Lyme disease should be completely
asymptomatic and should have completed a full course of antibiotic therapy
before they are permitted to donate. Transfusion transmission of tick-borne agents is
biologically plausible and, for some agents,
has been demonstrated. Nevertheless,
modifications to current donor screening
are not likely to be useful because of their
low predictive value and the potential
for nonspecific questions to defer large

Chapter 28: Transfusion-Transmitted Diseases

numbers of donors for a small increment in
transfusion safety.153

Other Nonviral Infectious
Complications of Blood
Transfusion
Malaria
Malaria is caused by several species of
the intraerythrocytic protozoan genus
Plasmodium. Transmission usually results
from the bite of an anopheles mosquito, but
infection can follow transfusion of parasitemic blood. Although very rare in the
United States, malaria is probably the most
commonly recognized parasitic complication of transfusion; the risk in the United
States is estimated to be <0.3 case per mil154,155
lion transfusions.
From 1963 to 1999, 93
cases of transfusion-transmitted malaria (10
fatal) in the United States were reported to
CDC.155
The species involved in transfusiontransmitted malaria in the United States are
P. falciparum (35%), P. malariae (27%), P.
vivax (27%), and P. ovale (5%).155 Three percent were mixed infections, and 2% were
caused by unidentified species. Fever,
chills, headache, and hemolysis occur a
week to several months after the infected
transfusion; morbidity varies but can be severe, and deaths have occurred, especially
from P. falciparum. Adding to the risk of a
fatal outcome may be a lack of immunity in
the recipient, the patient’s underlying condition(s), and delay in the diagnosis because of lack of suspicion and unfamiliarity
with the disease in areas where the parasite
is not endemic.
Malaria parasites survive for at least a
week in components stored at room temperature or at 4 C. The parasites can also
survive cryopreservation with glycerol and
subsequent thawing. Any component that

697

contains red cells can transmit infection,
via the asexual form of the intraerythrocytic
parasite.
Asymptomatic carriers are generally the
source of transfusion-transmitted malaria,
although their parasite density is very low.
Asymptomatic infections rarely persist
more than 3 years, but asymptomatic P.
falciparum and P. vivax infections may persist for 5 years, P. ovale for 7 years, and P.
malariae can remain transmissible for the
lifetime of the asymptomatic individual. In
extreme cases, transmission of P. vivax, P.
ovale, P. falciparum, and P. malariae have
been reported at 27, 7, 13, and 53 years, respectively.156 There are no practical serologic tests to detect transmissible malaria in
asymptomatic donors. Malaria transmission is prevented by deferral of prospective
donors with increased risk of infectivity,
based on their medical and travel history.
The AABB requires that prospective donors
who have had a diagnosis of malaria, or
who have traveled or lived in a malaria-endemic area and have had unexplained
symptoms suggestive of malaria, be deferred for 3 years after becoming asymptomatic.62(p65) Individuals who have lived for
at least 5 consecutive years in areas in
which malaria is considered endemic by
the CDC Malarial Branch shall be deferred
for 3 years after departure from that area.
Individuals who have traveled to an area
where malaria is endemic shall be deferred
for 12 months after departing that area.
These deferral periods apply irrespective of
the receipt of antimalarial prophylaxis. Updated information on malaria risks worldwide is available from the CDC, including
an on-line resource (http://www.cdc.gov/
travel/yb/outline.htm#2).

Chagas’ Disease
American trypanosomiasis, or Chagas’
disease, is endemic in South and Central

698

AABB Technical Manual

America and is caused by the protozoan
parasite Trypanosoma cruzi. The human
host sustains infection after the bite of
reduviid bugs (called cone-nosed or “kissing” bugs), which usually exist in hollow
trees, palm trees, and in thatched-roofed
mud or wooden dwellings. Naturally acquired Chagas’ disease in the United States
is exceedingly rare. Five such cases have
been recognized in the United States since
1955, the most recent in 1998 in Tennessee.157

Clinical Events
T. cruzi infects humans whose skin or mucosa comes in contact with feces of infected reduviid bugs, usually as the result
of a bite. Recent infections are usually either asymptomatic or the very mild signs,
and symptoms go undetected. Rarely, the
site of entry evolves into an erythematous
nodule called a chagoma, which may be
accompanied by lymphadenopathy. Fever
and enlargement of the spleen and liver
may follow. Recently infected young children may experience acute myocarditis or
meningoencephalitis. Acute infection
usually resolves without treatment, but
persisting low-level parasitemia is usual
and up to one-third of infected individuals develop a chronic form associated with
cardiac or gastrointestinal symptoms
years or decades later.158

158

States : in New York, Los Angeles, Texas,
and Florida. All occurred in immunocompromised patients. Additionally, two
cases were reported in Manitoba, Canada.
In one interesting study, postoperative
blood specimens from 11,430 cardiac surgery patients were tested by EIA and, if
repeatedly reactive, were confirmed by
radioimmunoprecipitation. Six postoperative specimens (0.05%) were confirmed
positive. All six seropositive patients apparently were infected with T. cruzi before
surgery; however, a diagnosis of Chagas’
disease was not known or even considered in any of these patients. No evidence
for transfusion-transmitted T. cruzi was
found.159
Reasonably sensitive and specific EIA
screening tests for antibodies to T. cruzi, as
well as confirmatory Western blot and
radioimmunoprecipitation assays, have
been developed.158 Testing in several US
blood centers located in geographic areas
with a large immigrant population from
Central or South America found a seroprevalence of 0.1% to 0.2% among at-risk
donors, who were identified by questionnaire.160,161 However, look-back studies identified no infected recipients; it is also likely
that not all at-risk donors can be identified
by questionnaire.160 As a consequence, if blood
donor screening were to be implemented,
testing of all donors might be necessary.

Transfusion Considerations

Other Parasites

Blood transfusion has been a major source
of infection with T. cruzi in South American urban centers that receive large numbers of immigrants from rural areas where
the parasite is endemic. However, in many
countries, serologic screening has been
effective in reducing the risk of transfusion-transmitted Chagas’ disease. Four
cases of transfusion-transmitted Chagas’
disease have been reported in the United

Toxoplasmosis is caused by the ubiquitous
parasite Toxoplasma gondii, and infection
has been reported as an unusual transfusion complication in immunocompromi162
sed patients. The disease has not been
considered a problem in routine transfusion practice.
There have been occasional reports of
parasitic worm infections transmitted by
transfusion in countries other than the

Chapter 28: Transfusion-Transmitted Diseases

162

United States. Microfilariasis is a potential
transfusion risk in tropical zones, acquired
by donors through bites by insects carrying
Wuchereria bancrofti. Transfusion transmission of Leishmania species is a rare risk
in countries where such organisms are endemic. Currently, the AABB defers potential
donors who have been to Iraq in the previous 12 months as a result of possible Leishmania exposure.163

Reducing the Risk of
Infectious Disease
Transmission
Overall, the risk per unit of transfusiontransmitted disease is remarkably low
(Table 28-5). This low incidence is due to
both donor screening and specific disease
testing. Nevertheless, in pooled components, which may contain elements from
thousands of donors, the risk of disease
transmission is increased. Therefore, several strategies have been developed and
implemented to further reduce the risk of
disease transmission in pooled acellular
components and, in some cases, cellular
components.

Inactivation/Destruction of Agents in
Derivatives or Plasma Products
The first intervention specifically added
to reduce the risk of hepatitis transmission was heating (to 60 C for 10 hours),
which has been used for albumin products since at least 1948.167 In those rare instances when infections occurred with
plasma protein fractions prepared with
this step, the processing had been compromised.

Immunoglobulins
The plasma fractionation process used for
most immunoglobulin products employs

699

cold ethanol precipitation after removal
of cryoprecipitate. Historically, when antibodies to HCV were present in the plasma,
this process concentrated HCV in the Factor VIII-rich cryoprecipitate and other
fractions and left little in the immunoglobulin fraction. The immunoglobulin
fraction also has a high concentration of
virus-neutralizing antibodies and the resulting product for intramuscular application has a remarkably low risk of virus
transmission.168
Preparations of immunoglobulin intended
for intravenous administration (IGIV) were
expected to be similarly free of disease
transmission. However, NANB hepatitis
transmission did occur in the 1980s during
early clinical trials of IGIV products in the
United States and with routinely manufactured IGIV products in Europe.169 In late
1993 and early 1994, a worldwide outbreak
with more than 200 reported HCV infections was traced to a single IGIV preparation licensed in the United States.170,171 In
this case, transmission apparently occurred
because of lack of virus inactivation steps in
the specific manufacturing process for this
product172 and absence of complexing and
neutralizing anti-HCV subsequent to antiHCV screening of plasma donors, with resultant accumulation of virus particles in
the immunoglobulin fraction.170 Anti-HCVpositive source plasma has been excluded
from the manufacture of IGIV since 1992.
The importance of the manufacturing
method is underscored by outbreaks of HCV
infection from intravenous anti-D immunoglobulin in Germany in the late 1970s
and in Ireland from the late 1970s to the
early 1990s.173,174 Both products were prepared by anion exchange chromatography
rather than cold-ethanol (Cohn) fractionation.175 To prevent further HCV outbreaks,
the FDA has required, since 1994, virus
clearance steps in the manufacturing process of immunoglobulin or proof of ab-

700

AABB Technical Manual

Table 28-5. Infectious Risks of Blood Transfusion in the United States

Infectious Agent
or Outcome

Estimated Risk per
Unit Transfused

Estimated % of
Infected Units that
Transmit or Cause
Clinical Sequelae*

Reference

Viruses
HIV-1 and -2

1:1,400,0001:2,400,000

23, 29, 30

HTLV-I and -II

1:256,0001:2,000,000

HAV

1:1,000,000

164

HBV

1:58,000-1:147,000

HCV

1:872,000-1:
1,700,000

23, 29, 30

B19 parvovirus

1:3,300-1:40,000

Low

110

RBCs

1:1000

1:10,000,000 fatal

165

Platelets
(screened with
Gram’s stain, pH,
or glucose concentration)

1:2000-1:4000

>40% result in clinical sequelae

30, 132, 166

<1:10,000

Unknown

Babesia and malaria

<1:1,000,000†

Unknown

145, 156, 164

Trypanosoma cruzi

Unknown

<20

158

Bacteria

(screened with
early aerobic culture)

Parasites

*Units that were confirmed test positive for the infectious agent.
Note: West Nile virus is not included in this table because of regional, temporal, and testing (eg, minipool vs individual
donation testing) variation; decreasing rates of infection; and the fact that all testing in the United States is being conducted under an investigational new drug protocol.
†
Risk is higher in areas where Babesia is endemic.

Chapter 28: Transfusion-Transmitted Diseases

sence of HCV from the final product by
NAT. In addition, NAT technology is now
applied to screening of source plasma as an
additional layer of safety.

Coagulation Factors
Until the early 1980s, clotting factor concentrates frequently transmitted viral infections. As the significance of HIV transmission was recognized, virus inactivation
steps were applied more rigorously to
Factor VIII and other clotting factor concentrates, even though these steps were
initially introduced in the hope of reducing hepatitis transmission. Unfortunately,
a large proportion (over 50%) of the hemophilic population receiving concentrates
before processing was improved became
infected with HIV. Chronic hepatitis was
an additional complication in almost all
patients with hemophilia receiving older
clotting factor products.176
The thermal instability of Factor VIII
made it difficult to develop an effective heat
treatment, until a practical approach was
widely adopted in 1985. Since then, many
virus inactivation steps have been introduced,
and factor concentrates are now, in general,
very safe products. Each process has its
own set of advantages and disadvantages.
Application of organic solvents and detergents inactivates viruses with a lipid-containing envelope (eg, HIV, HBV, HCV, HTLV,
EBV, CMV, HHV-6, HHV-8) but is ineffective
against nonenveloped agents such as HAV
and parvovirus B19. Virus inactivation steps
have the potential drawback of reducing the
potency and biologic effectiveness of the
product. Another concern is whether virus
inactivation steps affect immunogenicity,
especially the induction of Factor VIII inhibitors in patients with hemophilia.
Current Risks of Human Plasma Derivatives. Many methods are highly effective
against enveloped viruses, but sporadic re-

701

ports of viral transmission continue to
occur, possibly resulting from accident or
error during the manufacturing process.
The combination of heat treatment, solvent/detergent treatment, and purification
steps with monoclonal antibodies provides
clotting factor concentrates with a risk of
transmitting hepatitis and HIV that is lower
than the risk associated with use of Cryoprecipitated Antihemophilic Factor derived
from individual voluntary whole blood donations. Documented transmission of HBV,
HCV, or HIV by US-licensed plasma derivatives is rare since the introduction of effective virus inactivation procedures and improved viral screening. Although absolute
safety of products derived from human
plasma cannot be guaranteed, starting with
the safest possible donated plasma reduces
the viral load and has contributed to the excellent safety record of the products subjected to virus inactivation/removal.177
Avoiding Human Plasma. Factor VIII
concentrates produced by recombinant
DNA technology are licensed for use and
have become the preparation of choice for
previously untreated patients with hemophilia.178 Batches are produced by culture of
mammalian cells engineered to secrete
Factor VIII into the supernatant medium,
which is purified by ion-exchange chromatography and immunoaffinity chromatography using a mouse monoclonal antibody.
Except for the fact that excipient human albumin is sometimes added to stabilize Factor VIII, the product is free of human proteins, HIV, hepatitis viruses, and other
unwanted agents, thus avoiding many of the
risks associated with using human plasma.
On the other hand, recombinant products
have a relatively short history of use and
there is no guarantee that they are risk-free.

Plasma
Virus reduction steps, originally developed
for purified plasma protein fractions, have

702

AABB Technical Manual

also been applied to plasma intended for
transfusion. Alternative approaches being
studied include organic solvents and detergents and use of photochemicals. Solvent/detergent treatment, which is effective against lipid-enveloped viruses, involves
addition of 1% Triton X-100 and 1% tri-nbutyl phosphate (TNBP) to pooled plasma,
followed by oil extraction of the TNBP and
chromatographic adsorption of the Triton
X-100. After several years of experience
with this method in Europe, solvent/detergent-treated plasma was transiently
available in the United States. However,
concern with the use of a pooled product,
expense, poor market penetration, and
possible thrombotic (or excessive bleeding) events led to the discontinuation of
this product. A psoralen (S59) activated by
ultraviolet A light is undergoing US clinical trials for pathogen inactivation in
platelets and plasma and is available in
some countries in Europe.

Processing Cellular Components
Photochemical and chemical pathogen
inactivation methods are theoretically applicable to cellular blood components;
another organic chemical (S-303) and a
nucleic acid targeting compound (PEN
110) have undergone initial clinical evaluation for pathogen inactivation in red cells.
These methods have the potential for reliable inactivation of bacteria, viruses, and
179,180
parasites, including intracellular forms.
However, both products have been shown
to result in the formation of antibodies in
recipients. Pathogen reduction trials in
platelets (eg, with S-59) have been associated with decreased cell recovery and survival and the need for increased platelet
transfusions. Such unintended consequences
of pathogen reduction have resulted in
some trials being halted and may ulti-

mately eliminate such molecules from
widespread clinical application.

Reporting Transfusion-Associated
Infections
Unexplained infectious disease reported
in a transfusion recipient must be investigated for the possibility of transfusiontransmitted illness.62(pp83,85) Hepatitis is expected to become apparent within 2 weeks
to 6 months if it resulted from transfusion, but, even within this interval, the
cause need not necessarily have been
blood-borne infection. Blood centers and
transfusion services must have a mechanism to encourage recognition and reporting of possible transfusion-associated
infections. HIV infection thought to be a
result of transfusion should also be reported to the blood supplier, although the
interval between transfusion and the recognition of infection or symptoms may be
years.
Infection in a recipient should be reported to the collecting agency so that donors shown or suspected of being infectious can be evaluated and recipients of
other components from the implicated or
other donations can be contacted and, if
necessary, tested. A donor who proves to
have positive results on tests during the investigation must be placed on an appropriate deferral list.

Reporting Fatalities
The Code of Federal Regulations [21 CFR
606.170(b)] requires that fatalities attributed to transfusion complications (eg,
hepatitis, AIDS, and hemolytic reactions)
be reported to the Director, Center for
Bi o l o g i c s Eva l u a t i o n a n d Re s e a rc h
( C B E R ) , O f f i c e o f Co m p l i a n c e a n d
Biologics Quality, Attn: Fatality Program
Manager (HFM-650), 1401 Rockville Pike,
Suite 200N, HFM-650, Rockville, MD

Chapter 28: Transfusion-Transmitted Diseases

20852-1448. A report should be made as
s o o n a s p o s s i b l e by t e l e p h o n e
(301-827-6220), fax (301-827-6748), or
email to fatalities2@cber.fda.gov and a
written report should be submitted within
7 days. Current information can be found
on the Internet at http://www.fda.gov/
cber/gdlns/bldfatal.pdf.

Management of Posttransfusion Infections

Implicated Donors
If documented transfusion-associated
hepatitis, HIV, or HTLV-I/II occurs in a patient who received only a single unit, that
donor must be permanently excluded
from future donations, and the name
placed in a file of permanently deferred
individuals. If posttransfusion viral infection occurs after exposure to blood from
several donors, it is not necessary to exclude all of the potentially implicated donors. If only a few donors are involved, it
may be desirable to recall them to obtain
an interim medical history and to perform additional tests. Donors found to
have been implicated in more than one
case of transfusion-associated viral infection should be appropriately investigated
and possibly deferred permanently according to procedures established by the
collecting agency.

703

physician, a blood bank physician or
other trained staff member should provide initial counseling and appropriate
medical referral. The notification process
and counseling must be done with tact
and understanding, and the concerns of
the donor should be addressed. The donor should be told clearly why he or she is
deferred and, when appropriate, about
the possibility of being infectious to others. Notification should occur promptly
because a delay in notification can delay
initiation of treatment or institution of
measures to prevent the spread of infection to others.

Use of Immunoglobulins
It is not recommended practice to give intramuscular or intravenous immune serum
globulin or HBIG prophylactically to prevent posttransfusion hepatitis 181 ; these
agents have not been shown to prevent
posttransfusion hepatitis B, and the available evidence is conflicting about their effect on posttransfusion hepatitis C.182,183 If
there has been inadvertent transfusion of
known marker-positive blood or needlestick exposure to infectious material, HBIG
may prevent or attenuate HBV infection.184
Prophylaxis with immunoglobulin is ineffective in preventing HCV transmission
following occupational exposures and is
not recommended for this indication.185

Notification
A donor who will be permanently excluded as a future blood donor because of a
positive test implication in posttransfusion viral infection must be notified of
this fact. Follow-up testing should, ideally,
be done by the donor’s own physician,
and the collecting agency should obtain
the donor’s consent to release available
information to a designated health-care
provider. If the donor does not have a

References
1. Alter MJ. Epidemiology and prevention of
hepatitis B. Semin Liver Dis 2003;23:39-46.
2. Centers for Disease Control and Prevention.
Recommendations for prevention and control of hepatitis C virus (HCV ) infection and
HCV-related chronic disease. MMWR Morb
Mortal Wkly Rep 1998;47(No. RR019):1-39.
3. Conry-Cantilena C, Menitove J. Hepatitis. In:
Anderson K, Ness P, eds. Scientific basis of
transfusion medicine. Implications for clini-

704

10.
11.
12.
13.

14.

15.

16.

17.

AABB Technical Manual

cal practice. Philadelphia: WB Saunders,
2000:472-87.
Dodd R. Hepatitis. In: Petz LD, Swisher SN,
Kleinman S, et al, eds. Clinical practice of
transfusion medicine. 3rd ed. New York:
Churchill Livingstone, 1996:847-73.
Dienstag JL. Transfusion-transmitted hepatitis B, A, and D. In: Simon TL, Dzik WH,
Snyder EL, et al, eds. Rossi’s principles of
transfusion medicine. 3rd ed. Philadelphia:
Lippincott Williams and Wilkins, 2002:73341.
Alter HJ, Nakatsuji Y, Melpolder J, et al. The
incidence of transfusion-associated hepatitis
G virus infection and its relation to liver disease. N Engl J Med 1997;336:747-54.
Sayers M. Cytomegalovirus and other herpesviruses. In: Petz LD, Swisher SN, Kleinman S,
et al, eds. Clinical practice of transfusion
medicine. 3rd ed. New York: Churchill Livingstone, 1996:875-89.
Unemura T, Yeo AET, Sottini A, et al. SEN virus infection and its relationship to transfusion-associated hepatitis. Hepatology 2001;
33:1303-11.
Alter HJ. Transfusion-transmitted hepatitis C
and non-A, non-B, non-C virus infections. In:
Simon TL, Dzik WH, Snyder EL, et al, eds.
Rossi’s principles of transfusion medicine.
3rd ed. Philadelphia: Lippincott Williams and
Wilkins, 2002:718-32.
Kumar A, Beniwal B, Kar P, et al. Hepatitis E in
pregnancy. Obstet Gynecol Surv 2005;60: 7-8.
Lee WM. Hepatitis B virus infection. N Engl J
Med 1997;337:1733-45.
Alter HJ. To C or not to C: These are the questions. Blood 1995;85:1681-95.
Seeff LB, Buskell-Bales Z, Wright EC, et al.
Long-term mortality after transfusion-associated non-A, non-B hepatitis. The National
Hea r t, Lu ng, a nd Blood Institu te Stu d y
Group. N Engl J Med 1992;327:1906-11.
Vogt M, Lang T, Frosner G, et al. Prevalence
and clinical outcome of hepatitis C infection
in children who underwent cardiac surgery
before the implementation of blood-donor
screening. N Engl J Med 1999;341:866-70.
National Institutes of Health. National Institutes of Health consensus development conference panel statement: Management of
hepatitis C. Hepatol 1997;26:2S-10S.
Sherman M, Bain V, Villeneuve JP, et al. The
management of chronic viral hepatitis: A Canadian consensus conference 2004. Can J
Gastroenterol 2004;18:715-28.
Busch MP. Closing the windows on viral transmission by blood transfusion. In: Stramer S,
ed. Blood safety in the new millennium.
Bethesda MD: AABB, 2000:33-54.

18. Westhoff TH, Jochimsen F, Schmittel A, et al.
Fatal hepatitis B virus reactivation by an escape mutant following rituximab therapy
(letter). Blood 2003;102:1930.
19. Busch MP, Kleinman SH, Jackson B, et al. Nucleic acid amplification testing of blood donors for transfusion-transmitted diseases:
Report of the Interorganizational Task Force
on Nucleic Acid Amplification Testing of
Blood Donors. Transfusion 2000;40:143-59.
20. Alter HJ, Purcell RH, Shih JW, et al. Detection
of antibody to hepatitis C virus in prospectively followed transfusion recipients with
acute and chronic non-A, non-B hepatitis. N
Engl J Med 1989;321:1494-500.
21. Brecher ME, ed. Collected questions and answers. 6th ed. Bethesda, MD: AABB, 2000:21.
22. Van der Poel CL, Cuypers HT, Reesink HW, et
al. Confirmation of hepatitis C virus infection
by new four-antigen recombinant immunoblot assay. Lancet 1991;337:317-9.
23. Stramer SL, Glynn SA, Kleinman SH, et al for
the National Heart, Lung, and Blood Institute
Nucleic Acid Test Study Group. Detection of
HIV-1 and HCV infections among antibodynegative blood donors by nucleic acid-amplification testing. N Engl J Med 2004;351:760-8.
24. Variance request to use NAT results and be
exempted from HIV and HCV supplemental
testing in specific circumstances (Association Bulletin #05-03). Bethesda, MD: AABB,
2005.
25. Busch MP, Korelitz JJ, Kleinman SH, et al. Declining value of alanine aminotransferase in
screening of blood donors to prevent posttransfusion hepatitis B and C virus infection.
The Retrovirus Epidemiology Donor Study.
Transfusion 1995;35:903-10.
26. Jongerius JM, Webster M, Cuypers HT, et al.
New hepatitis B virus mutant form in a blood
donor that is undetectable in several hepatitis B surface antigen screening assays. Transfusion 1998;38:56-9.
27. Alter H. Transfusion-transmitted non-A,
non-B and hepatitis C infections. In: Rossi
EC, Simon TL, Moss GL, Gould S, eds. Principles of transfusion medicine. 2nd ed. Baltimore, MD: Williams and Wilkins, 1996:687-98.
28. Armstrong GL, Alter MJ, McQuillan GM,
Margolis HS. The past incidence of hepatitis
C virus infection: Implications for the future
burden of chronic liver disease in the United
States. Hepatology 2000;31:777-82.
29. Goodnough LT, Shander A, Brecher ME.
Transfusion medicine: Looking to the future.
Lancet 2003;361:161-9.
30. Busch MP, Glynn SA, Stramer S, et al. A new
strategy for estimating risks of transfusiontransmitted viral infections based on rates of

Chapter 28: Transfusion-Transmitted Diseases

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

detection of recently infected donors. Transfusion 2005;45:254-64.
65th Blood Product Advisory Committee
Meeting (transcripts). March 16, 2000. [Available at http://www.fda.gov/ohrms/dockets/
ac/00/transcripts/3603t1.rtp.]
Food and Drug Administration. Draft guidance for industry: Current good manufacturing practice for blood and blood components: (1) quarantine and disposition of
units from prior collections from donors
with repeatedly reactive screening tests for
antibody to hepatitis C virus (anti-HCV ); (2)
supplemental testing and the notification of
consignees and blood recipients of donor
test results for anti-HCV. ( June 17, 1999)
Rockville, MD: CBER Office of Communication, Training, and Manufacturers Assistance, 1999.
Goldman M, Juodvalkis S, Gill P, Spurli G.
Hepatitis C lookback. Transfus Med Rev
1998; 12:84-93.
Culver DH, Alter MJ, Mullan RJ, Margolis HS.
Evaluation of the effectiveness of targeted
lookback for HCV infection in the United
States—interim results. Transfusion 2000;40:
1176-81.
Bowker SL, Smith LJ, Rosychuk RJ, Preiksaitis
JK. A review of general hepatitis C virus lookbacks in Canada. Vox Sang 2004;86:21-7.
Centers for Disease Control. Pneumocystis
carinii pneumonia among persons with hemophilia A. MMWR Morb Mortal Wkly Rep
1982;31:365-7.
Centers for Disease Control. Possible transfusion-associated acquired immune deficiency syndrome (AIDS)—California. MMWR
Morb Mortal Wkly Rep1982;31:652-4.
Ragni MV, Winkelstein A, Kingsley L, et al.
1986 update of HIV seroprevalence, seroconversion, AIDS incidence, and immunologic correlates of HIV infection in patients
with hemophilia A and B. Blood 1987;70:
786-90.
Busch MP, Young MJ, Samson SM, et al. Risk
of human immunodeficiency virus (HIV )
transmission by blood transfusions before
t h e i m p l e m e n t a t i o n o f HI V- 1 a n t i b o d y
screening. The Transfusion Safety Study
Group. Transfusion 1991;31:4-11.
Levine A, Liebman H. The acquired immunodeficiency syndrome (AIDS). In: Beutler E,
Lichtman M, Coller B, Kipps T, eds. Williams’
hematology. New York: McGraw-Hill, 1995:
975-97.
Mayer A, Busch M. Transfusion-transmitted
HIV infection. In: Anderson K, Ness P, eds.
Scientific basis of transfusion medicine. Im-

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

705

plications for clinical practice. Philadelphia:
WB Saunders, 1994:659-68.
Centers for Disease Control. 1993 revised
classification system for HIV infection and
expanded surveillance case definition for
AIDS among adolescents and adults. MMWR
Morb Mortal Wkly Rep 1992;41:1-19.
Brookmeyer R. Reconstruction and future
trends of the AIDS epidemic in the United
States. Science 1991;253:37-42.
Centers for Disease Control and Prevention.
US HIV and AIDS cases reported through December 2000. HIV/AIDS Surveillance Report
2000;12(2):1-44. [Available at http://www.
cdc.gov/hiv/stats/hasr1202.pdf.]
Petersen LR, Doll LS, White CR, et al. Heterosexually acquired HIV infection and the US
blood supply: Consideration for screening of
potential donors. Transfusion 1993;33:552-7.
Simon F, Loussert-Ajaka I, Damond F, et al.
HIV type 1 diversity in northern Paris, France.
AIDS Res Hum Retroviruses 1996;12:1427-33.
Parry JV, Murphy G, Barlow KL, et al. National surveillance of HIV-1 subtypes for
England and Wales: Design, methods, and
initial findings. J Acquir Immune Defic Syndr
2001;26:381-8.
Leads from the MMWR. AIDS due to HIV-2
infection—New Jersey. JAMA 1988;259:969,
972.
Sullivan MT, Guido EA, Metler RP, et al. Identification and characterization of an HIV-2
antibody-positive blood donor in the United
States. Transfusion 1998;38:189-93.
de Oliveira CF, Diaz RS, Machado DM, et al.
Surveillance of HIV-1 genetic subtypes and
diversity in the US blood supply. Transfusion
2000;40:1399-406.
Delwart EL, Orton S, Parekh B, et al. Two percent of HIV-positive US blood donors are infected with non-subtype-B strains. AIDS Res
Hum Retroviruses 2003;19:1065-70.
Peeters M, Gueye A, Mboup S, et al. Geographical distribution of HIV-1 group O viruses in Africa. AIDS 1997;11:493-8.
Food and Drug Administration. Memorandum: Interim recommendations for deferral
of donors at increased risk for HIV-1 group O
infection. (December 11, 1996) Rockville,
MD: CBER Office of Communication, Training, and Manufacturers Assistance, 1996.
Kaiser Family Foundation. Cumulative adult/
adolescent HIV infection cases reported
through 2003 among states with confidential
name-based reporting. [Available at http://
www.statehealthfacts.kff.org.]
Delwart EL, Kalmin ND, Jones TS, et al. First
case of HIV transmission by an RNA-

706

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

AABB Technical Manual

screened blood donation. Vox Sang 2004;86:
771-7.
Phelps R, Robbins K, Liberti T, et al. Windowperiod human immunodeficiency virus
transmission to two recipients by an adolescent blood donor. Transfusion 2004;44:92933.
Ling AE, Robbins KE, Brown TM, et al. Failure
of routine HIV-1 tests in a case involving
transmission with preseroconversion blood
components during the infectious window
period. JAMA 2000;284:210-4 [comment in
JAMA 2000;284:238-40].
Fiebig E, Busch MP. Retroviral infections. In:
Simon TL, Dzik WH, Snyder EL, et al, eds.
Rossi’s principles of transfusion medicine.
3rd ed. Philadelphia: Lippincott Williams and
Wilkins, 2002:742-56.
Tabor E. The epidemiology of virus transmission by plasma derivatives: Clinical studies
verifying the lack of transmission of hepatitis
B and C viruses and HIV type 1. Transfusion
1999;39:1160-8.
Dufoort G, Courouce AM, Ancelle-Park R,
Bletry O. No clinical signs 14 years after HIV-2
transmission via blood transfusion (letter).
Lancet 1988;ii:510.
Kleinman S, Busch MP, Korelitz JJ, Schreiber
GB. The incidence/window period model and
its use to assess the risk of transfusion-transmitted human immunodeficiency virus and
hepatitis C virus infection. Transfus Med Rev
1997;11:155-72.
Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.
Code of federal regulations. Title 21 CFR
610.40. Washington, DC: US Government
Printing Office, 2004 (revised annually).
Busch MP. HIV and blood transfusions: Focus
on seroconversion. Vox Sang 1994;67 (Suppl
3):13-8.
Fiebig EW, Wright DJ, Rawal BD, et al. Dynamics of HIV viremia and antibody seroconversion in plasma donors: Implications for
diagnosis and staging of primary HIV infection. AIDS 2003;17:1871-9.
Food and Drug Administration. Talk paper:
FDA approves first nucleic acid test (NAT)
system to screen whole blood donors for infections with human immunodeficiency virus (HIV ) and hepatitis C virus (HCV ). (Februar y 28, 2002) [Available at http://fda.
gov/cber/products/hivhcvgen022702.htm.]
Food and Drug Administration. Guidance for
industry: Use of nucleic acid tests on pooled
and individual samples from donations of
whole blood and blood components (including Source Plasma and Source Leukocytes) to

68.

69.

70.

71.

72.

73.
74.

75.

76.

77.

78.

79.

adequately and appropriately reduce the risk
of transmission of HIV-1 and HCV. (October
21, 2004) Rockville, MD: CBER Office of Communication, Training, and Manufacturers Assistance, 2004. [Available at http://www.fda.
gov/cber/gdlns/hivhcvnatbld.htm.]
Centers for Disease Control. Interpretive criteria used to report Western blot results for
HIV-1-antibody testing: United States. MMWR
Morb Mortal Wkly Rep 1991;40:692-5.
Kleinman S, Busch MP, Hall L, et al. Falsepositive HIV-1 test results in a low-risk
screening setting of voluntary blood donation. Retrovirus Epidemiology Donor Study.
JAMA 1998;280:1080-5.
Food and Drug Administration. Memorandum: Revised recommendations for the prevention of human immunodeficiency (HIV )
transmission by blood and blood products.
(April 23, 1992) Rockville, MD: CBER Office of
Communication, Training, and Manufacturers Assistance, 1992.
Food and Drug Administration. Memorandum:
Use of fluorognost HIV-1 immunofluorescent
assay (IFA). (April 23, 1992) Rockville, MD:
CBER Office of Communication, Training,
and Manufacturers Assistance, 1992.
Mintz P. Participation of HIV-infected patients in autologous blood programs. JAMA
1993;269:2892-4.
Pub. Law. 101-336, 104 Stat. 327 (1990). Codified at 42 U.S.C. §12101-12213.
Food and Drug Administration. Current good
manufacturing practice for blood and blood
components: Notification of consignees receiving blood and blood components at increased risk for transmitting HIV infection.
(September 9, 1996) Fed Regist 1996;61:
47413-23.
Health Care Financing Administration. Medicare and Medicaid programs; hospital standard for potentially HIV infectious blood and
blood products. (September 9, 1996) Fed Regist
1996;61:47423-34.
Code of federal regulations. 42 CFR 610.46-47.
Washington, DC: US Government Printing
Office, 2004 (revised annually).
Code of federal regulations. 21 CFR 482.27(c).
Washington, DC: US Government Printing
Office, 2004 (revised annually).
Murphy EL, Glynn SA, Fridey J, et al. Increased incidence of infectious diseases during prospective follow-up of human T-lymphotropic virus type II- and I-infected blood
donors. Retrovirus Epidemiology Donor
Study. Arch Intern Med 1999;159:1485-91.
Orland JR, Engstrom J, Fridey J, et al. Prevalence and clinical features of HTLV neuro-

Chapter 28: Transfusion-Transmitted Diseases

80.

81.

82.

83.

84.

85.

86.

87.

88.

89.

90.

logic disease in the HTLV Outcomes Study.
Neurology 2003;61:1588-94.
Murphy EL, Wang B, Sacher RA, et al. Respiratory and urinary tract infections, arthritis,
and asthma associated with HTLV-II and
HTLV-I infection. Emerg Infect Dis 2004;10:
109-16.
Orland JR, Wang B, Wright DJ, et al. Increased mortality associated with HTLV-II
infection in blood donors: A prospective cohort study. Retrovirology 2004;1:4.
Levine PH, Manns A. Transfusion transmission of human T-lymphotropic virus types I
and II: Lessons to be learned from look-back
investigations and implications for patient
counseling (editorial). Transfusion 1993;33:
4-6.
Kleinman S, Swanson P, Allain JP, Lee H.
Transfusion transmission of human T-lymphotropic virus types I and II: Serologic and
polymerase chain reaction results in recipients identified through look-back investigations. Transfusion 1993;33:14-8.
Schreiber GB, Busch MP, Kleinman SH,
Korelitz JJ. The risk of transfusion-transmitted viral infections. The Retrovirus Epidemiology Donor Study. N Engl J Med 1996;334:
1685-90.
Food and Drug Administration. Guidance for
industry: Donor screening for antibodies to
HTLV-II. (August 15, 1997) Rockville, MD:
CBER Office of Communication, Training,
and Manufacturers Assistance, 1997.
Dual enzyme immunoassay (EIA) approach
for deferral and notification of anti-HTLVI/II EIA reactive donors. Association Bulletin
99-9. Bethesda, MD: AABB, 1999.
Pealer LN, Marfin AA, Petersen LR, et al for
the West Nile Virus Transmission Investigation Team. Transmission of West Nile virus
through blood transfusion in the United
States in 2002. N Engl J Med 2003;349:123645.
Biggerstaff BJ, Petersen LR. Estimated risk of
transmission of the West Nile virus through
blood transfusion in the US, 2002. Transfusion 2003;43:1007-17.
Food and Drug Administration. Guidance for
industry: Revised recommendations for the
assessment of donor suitability and blood
and blood product safety in cases of known
or suspected West Nile virus infection. (May
1, 2003) Rockville, MD: CBER Office of Communication, Training, and Manufacturers
Assistance, 2003.
Kleinman S, Glynn SA, Busch M, et al for the
N H L B I Re t r ov i r u s Ep i d e m i o l o g y St u d y
(REDS). The 2003 West Nile virus United

91.

92.

93.

94.

95.

96.

97.
98.

99.

100.

101.

102.

103.

707

States epidemic: The America's Blood Centers experience. Transfusion 2005;45:469-79.
Centers for Disease Control and Prevention.
Update: West Nile virus screening of blood
donations and transfusion-associated transmission—United States, 2003. MMWR Morb
Mortal Wkly Rep 2004;53:281-4.
Busch MP, Caglioti S, Robertson GF, et al.
Screening the blood supply for West Nile virus RNA by nucleic acid amplification testing. N Engl J Med 2005 (in press).
Update on WNV-related activities and considerations, 2004; a summary of the WNV
Task Force meeting. Association Bulletin
#04-03. Bethesda, MD: AABB, 2004.
Centers for Disease Control and Prevention.
2004 West Nile virus activity in the United
States (reported as of January 11, 2005).
[Available at http://www.cdc.gov/ncidod/
dvbid/westnile/surv&controlCaseCount04_
detailed.htm.]
Centers for Disease Control and Prevention.
Transfusion-associated transmission of West
Nile virus—Arizona, 2004. MMWR Morb
Mortal Wkly Rep 2004;53:842-4.
Roback JD. Human herpes infections. In:
Hillyer CD, Silberstein LE, Ness PM, Anderson KC, eds. Blood banking and transfusion
medicine. New York: Churchill Livingstone,
2003:465-85.
Roback JD. CMV and blood transfusions. Rev
Med Virol 2002;12:211-19.
Leukocyte reduction for the prevention of
transfusion-transmitted cytomegalovirus.
Association Bulletin #97-2. Bethesda, MD:
AABB, 1997.
Bowden RA, Slichter SJ, Sayers M, et al. A
comparison of filtered leukocyte-reduced
and cytomegalovirus (CMV ) seronegative
blood products for the prevention of transfusion-associated CMV infection after marrow
transplant. Blood 1995;86:3598-603.
Preiksaitis JK. The cytomegalovirus-“safe”
blood product: Is leukoreduction equivalent
to antibody screening? Transfus Med Rev
2000;14:112-36.
McMonigal K, Horwitz CA, Henle W. Postperfusion syndrome due to Epstein-Barr virus. Report of two cases and review of the literature. Transfusion 1983;23:331-5.
Campadelli-Fiume G, Mirandola P, Menotti
L. Human herpesvirus 6: An emerging pathogen. Emerg Infect Dis 1999;5:353-66.
Chatlynne LG, Lapps W, Handy M, et al. Detection and titration of human herpesvirus8-specific antibodies in sera from blood donors, acquired immunodeficiency syndrome
patients, and Kaposi’s sarcoma patients us-

708

104.

105.

106.

107.

108.

109.

110.

111.

112.

113.
114.

115.

116.

AABB Technical Manual

ing a whole virus enzyme-linked immunosorbent assay. Blood 1998;92:53-8.
Cannon MJ, Dollard SC, Smith DK, et al.
Blood-borne and sexual transmission of human herpesvirus 8 in women with or at risk
for human immunodeficiency virus infection. N Engl J Med 2001;344:637-43.
Luppi M, Barozzi P, Santagostino G, et al. Molecular evidence of organ-related transmission
of Kaposi sarcoma-associated herpesvirus or
human herpesvirus-8 in transplant patients.
Blood 2000;96:3279-81.
Regamey N, Tamm M, Wernli M, et al. Transmission of human herpesvirus 8 infection
from renal-transplant donors to recipients. N
Engl J Med 1998;339:1358-63.
Parravicini C, Olsen SJ, Capra M, et al. Risk of
Kaposi’s sarcoma-associated herpes virus
transmission from donor allografts among
Italian posttransplant Kaposi’s sarcoma patients. Blood 1997;90:2826-9.
Mbulaiteye SM, Biggar RJ, Bakaki PM, et al.
Human herpesvirus 8 infection and transfusion history in children with sickle-cell disease in Uganda. J Natl Cancer Inst 2003;95:
1330-5.
Luban NL. Human parvoviruses: Implications for transfusion medicine. Transfusion
1994;34:821-7.
Prowse C, Ludlam CA, Yap PL. Human parvovirus B19 and blood products. Vox Sang
1997;72:1-10.
Brown KE, Hibbs JR, Gallinella G, et al. Resistance to parvovirus B19 infection due to lack
of virus receptor (erythrocyte P antigen). N
Engl J Med 1994;330:1192-6.
Nucleic acid testing of blood donors for human parvovirus B19 (presentations, public
hearing, discussion, and recommendations).
64th meeting of the Blood Products Advisory
Committee, Bethesda, MD, September 16,
1999. [Available at http://www.fda.gov/cber/
advisory/bp/bpmain.htm.]
Manuelidis L. The dimensions of CreutzfeldtJakob disease. Transfusion 1994;34:915-28.
Ricketts MN, Cashman NR, Stratton EE, El
Saadany S. Is Creutzfeldt-Jakob disease
transmitted in blood? Emerg Infect Dis 1997;
3:155-63.
Evatt B, Austin H, Barnhart E, et al. Surveillance for Creutzfeldt-Jakob disease among
persons with hemophilia. Transfusion 1998;
38:817-20.
van Duijn CM, Delasner ie-Laupretre N,
Masullo C, et al. Case-control study of risk
factors of Creutzfeldt-Jakob disease in Europe during 1993-95. European Union (EU)
Collaborative Study Group of CreutzfeldtJakob Disease (CJD). Lancet 1998;351:1081-5.

117. Food and Drug Administration. Memorandum: Revised precautionary measures to reduce the possible risk of transmission of
Creutzfeldt-Jakob disease (CJD) by blood and
blood products. (August 5, 1995) Rockville,
MD: CBER Office of Communication, Training, and Manufacturers Assistance, 1995.
118. Food and Drug Administration. Memorandum: Revised precautionary measures to reduce the possible risk of transmission of
Creutzfeldt-Jakob disease (CJD) by blood and
blood products. (December 11, 1996) Rockville, MD: CBER Office of Communication,
Training, and Manufacturers Assistance, 1996.
119. Food and Drug Administration. Draft guidance for industry: Preventive measures to reduce the possible risk of transmission of
Creutzfeldt-Jakob disease (CJD) and variant
Creutzfeldt-Jakob disease (vCJD) by human
cells, tissues, and cellular and tissue-based
products (HCT/Ps). ( June 25, 2002) Fed
Regist 2002;67:42789-90.
120. Hunter N, Foster J, Chong A, et al. Transmission of prion diseases by blood transfusion. J
Gen Virol 2002;83:2897-905.
121. Llewelyn CA, Hewitt PE, Knight RS, et al. Possible transmission of variant CreutzfeldtJakob disease by blood transfusion. Lancet
2004;363:417-21.
122. Peden AH, Head MW, Ritchie DL, et al. Preclinical vCJD after blood transfusion in a
PRNP codon 129 heterozygous patient. Lancet 2004;364:521-9.
123. Nurmi MH, Bishop M, Strain L, et al. The normal population distribution of PRNP codon
129 polymorphism. Acta Neurol Scand 2003;
108:374-8.
124. Sazama K. Bacteria in blood for transfusion.
A review. Arch Pathol Lab Med 1994;118:350-65.
125. Food and Drug Administration. CBER annual
report FY 2002 (October 1, 2001 through September 30, 2002). Rockville, MD: CBER Office
of Communication, Training, and Manufacturers Assistance, 2002. [Available at http://
www. fda.gov/cber/inside/annrptpart3.htm.]
126. Morrow JF, Braine HG, Kickler TS, et al. Septic
reactions to platelet transfusions. A persistent
problem. JAMA 1991;266:555-8.
127. Kuehnert MJ, Roth VR, Haley NR, et al. Transfusion-transmitted bacterial infection in the
United States, 1998 through 2000. Transfusion 2001;41:1493-9.
128. Theakston EP, Morris AJ, Streat SJ, et al.
Transfusion transmitted Yersinia enterocolitica infection in New Zealand. Aust N Z J
Med 1997;27:62-7.
129. Cookson ST, Arduino MJ, Aguero SM, Jarvis
WR, and the Yersinia Study Group. Yersinia
enterocolitica-contaminated Red Blood Cells

Chapter 28: Transfusion-Transmitted Diseases

130.

131.

132.

133.

134.

135.

136.

137.

138.

139.

140.

141.

142.

(RBCs)—an emerging threat to blood safety
(abstract). In: Program and abstracts of the
36th Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, September 15-18, 1996. Washington,
DC: American Society for Microbiology,
1996:237.
National Blood Data Resource Center. Comprehensive report on blood collection and
transfusion in the United States in 2001.
Bethesda, MD: AABB, 2003.
Brecher ME, Hay SN. Improving platelet
safety: Bacterial contamination of platelets.
Curr Hematol Rep 2004;3:121-7.
Dykstra A, Hoeltge G, Jacobs M, et al. Platelet
bacterial contamination (PBC) rate is surveillance method (SM) dependent (abstract).
Transfusion 1998;38(Suppl):104S.
Serious Hazards of Transfusion (SHOT). Annual report 2000-2001. Manchester, UK:
SHOT Office, 2002. [Available at http://www.
shot.demon.co.uk/toc.htm.]
Perez P, Salmi LR, Follea G, et al. Determinants of transfusion-associated bacterial
c o n t a m i n a t i o n : Re s u l t s o f t h e Fre n c h
BACTHEM case-control study. Transfusion
2001;41:862-71.
Ness PM, Braine HG, King K, et al. Single donor platelets reduce the risk of septic transfusion reactions. Transfusion 2001;41:857-61.
Engelfriet CP, Reesink HW, Blajchman MA, et
al. Bacterial contamination of blood components. Vox Sang 2000;78:59-67.
Kim DM, Brecher ME, Bland LA, et al. Prestorage removal of Yersinia enterocolitica
from red cells with white cell-reduction filters. Transfusion 1992;32:658-62.
Buchholz DH, AuBuchon JP, Snyder EL, et al.
Removal of Yersinia enterocolitica from AS-1
red cells. Transfusion 1992;32:667-72.
Pietersz RNI, Engelfriet CP, Reesink HW, et
al. Detection of bacterial contamination of
platelet concentrates. International Forum.
Vox Sang 2003;85:224-39.
Commission on Laboratory Accreditation.
Transfusion medicine checklist. TRM.44955
Phase I. Northfield, IL: College of American
Pathologists, January 2003. [Available at
http://www.cap.org/html/checklist_html/
transfusionmedicine_1202.html.]
Brumit MC, Hay SN, Brecher ME. Bacteria
detection. In: Brecher ME, ed. Bacterial and
parasitic contamination of blood components. Bethesda, MD: AABB Press, 2003:5782.
Anderson KC, Lew MA, Gorgone BC, et al.
Transfusion-related sepsis after prolonged
platelet storage. Am J Med 1986;81:405-11.

709

143. Orton SL, Liu H, Dodd RY, Williams AE. Prevalence of circulating Treponema pallidum
DNA and RNA in blood donors with confirmed positive syphilis tests. Transfusion
2002;42:94-9.
144. Gardella C, Marfin AA, Kahn RH, et al. Persons with early syphilis identified through
blood or plasma donation screening in the
United States. J Infect Dis 2000;185:545-9.
145. Leiby DA. Babesia and other parasites. In:
Brecher ME, ed. Bacterial and parasitic contamination of blood components. Bethesda,
MD: AABB Press, 2003:179-200.
146. Herwaldt BL, Springs FE, Roberts PP, et al.
Babesiosis in Wisconsin: A potentially fatal
disease. Am J Trop Med Hyg 1995;53:146-51.
147. Herwaldt BL, Kjemtrup AM, Conrad PA, et al.
Transfusion-transmitted babesiosis in Washington State: First reported case caused by a
WA1-type parasite. J Infect Dis 1997;175:
1259-62.
148. Krause PJ, Spielman A, Telford SR 3rd, et al.
Persistent parasitemia after acute babesiosis.
N Engl J Med 1998;339:160-5.
149. Cable R, Trouern-Trend J. Tick-borne infections. In: Linden JV, Bianco C, eds. Blood
safety and surveillance. New York: Marcel
Dekker, 2001:399-422.
150. Wells GM, Woodward TE, Fiset P, Hornick RB.
Rocky Mountain spotted fever caused by
blood transfusion. JAMA 1978;239:2763-5.
151. Eastlund T, Persing D, Mathiesen D, et al.
Human granulocytic ehrlichiosis after red
cell transfusion (abstract). Transfusion 1999;
39(Suppl):117S.
152. Arguin PM, Singleton J, Rotz LD, et al. An investigation into the possibility of transmission of tick-borne pathogens via blood
transfusion. Transfusion-Associated TickBorne Illness Task Force. Transfusion 1999;
39:828-33.
153. McQuiston JH, Childs JE, Chamberland ME,
Tabor E for the Working Group on Transfusion Transmission of Tick-Borne Diseases.
Transmission of tick-borne agents of disease
by blood transfusion: A review of known and
potential risks in the United States. Transfusion 2000;40:274-84.
154. Mungai M, Tegtmeier G, Chamberland M,
Parise M. Transfusion-transmitted malaria
in the United States from 1963 through 1999.
N Engl J Med 2001;344:1973-8.
155. Centers for Disease Control and Prevention.
Probable transfusion-transmitted malaria—
Houston, Texas, 2003. MMWR Morb Mortal
Wkly Rep 2003;52:1075-6.
156. Katz LM. Transfusion-induced malaria. In:
Brecher ME, ed. Bacterial and parasitic con-

710

157.

158.

159.

160.

161.

162.

163.

164.

165.

166.

167.

168.

169.

AABB Technical Manual

tamination of blood components. Bethesda,
MD: AABB Press, 2003:127-55.
Herwaldt BL, Grijalva MJ, Newsome AL, et al.
Use of polymerase chain reaction to diagnose
the fifth reported US case of autochthonous
transmission of Trypanosoma cruzi, in Tennessee, 1998. J Infect Dis 2000; 181:395-9.
Shulman IA. Transfusion of T. cruzi infection
by blood transfusion. In: Brecher ME, ed.
Bacterial and parasitic contamination of
blood components. Bethesda, MD: AABB
Press, 2003:157-78.
Leiby DA, Rentas FJ, Nelson KE, et al. Evidence of Trypanosoma cruzi infection (Chagas’ disease) among patients undergoing cardiac surgery. Circulation 2000;102:2978-82.
Leiby DA, Read EJ, Lenes BA, et al. Seroepidemiology of Trypanosoma cruzi, etiologic
agent of Chagas’ disease, in US blood donors.
J Infect Dis 1997;176:1047-52.
Shulman IA, Appleman MD, Saxena S, et al.
Specific antibodies to Trypanosoma cruzi
among blood donors in Los Angeles, California. Transfusion 1997;37:727-31.
Shulman I, Haimowitz MD. Transmission of
parasitic infections by blood transfusion. In:
Simon TL, Dzik WH, Snyder EL, et al, eds.
Rossi’s principles of transfusion medicine.
3rd ed. Philadelphia: Lippincott Williams and
Wilkins, 2002:774-83.
Deferral for risk of Leishmaniasis exposure.
Association Bulletin #03-14. Bethesda, MD:
AABB, 2003.
US General Accounting Office. Blood supply:
Transfusion-associated risks. GAO/PEMD-97-1.
Washington, DC: US Government Printing
Office, 1997.
Brecher ME. Bacterial contamination of
blood products. In: Simon TL, Dzik WH,
Snyder EL, et al, eds. Rossi’s principles of
transfusion medicine. 3rd ed. Philadelphia:
L i ppi nco t t Wi lli a m s a nd Wi lk i ns, 2002:
789-801.
Rock G, Neurath D, Toye B, et al. The use of a
bacteria detection system to evaluate bacterial contamination in PLT concentrates.
Transfusion 2004;44:337-42.
Suomela H. Inactivation of viruses in blood
and plasma products. Transfus Med Rev 1993;
7:42-57.
Centers for Disease Control. Safety of therapeutic immune globulin preparations with
respect to transmission of human T-lymphotropic virus type III/lymphadenopathy-associated virus infection. MMWR Morb Mortal
Wkly Rep 1986;35:231-3. [Erratum in MMWR
Morb Mortal Wkly Rep 1986;35:607.]
Williams PE, Yap PL, Gillon J, et al. Non-A,
non-B hepatitis transmission by intravenous

170.

171.

172.

173.

174.

175.

176.
177.

178.

179.

180.

181.

182.

183.

immunoglobulin (letter). Lancet 1988;ii:501.
[Erratum in Lancet 1988;ii:584.]
Yu MW, Mason BL, Guo ZP, et al. Hepatitis C
transmission associated with intravenous
immunoglobulins (letter). Lancet 1995;345:
1173-4.
Centers for Disease Control. Outbreak of
hepatitis C associated with intravenous immunoglobulin administration: United States,
October 1993-June 1994. MMWR Morb Mortal Wkly Rep 1994;43:505-9.
Farrugia A, Walker E. Hepatitis C virus transmission by intravenous immunoglobulin
(letter). Lancet 1995;346:373-5.
Meisel H, Reip A, Faltus B, et al. Transmission
of hepatitis C virus to children and husbands
by women infected with contaminated anti-D
immunoglobulin. Lancet 1995;345:1209-11.
Power JP, Lawlor E, Davidson F, et al. Hepatitis C viraemia in recipients of Irish intravenous anti-D immunoglobulin (letter). Lancet
1994;344:1166-7.
Foster PR, McIntosh RV, Welch AG. Hepatitis
C infection from anti-D immunoglobulin
(letter). Lancet 1995;346:372.
Makris M, Preston FE. Chronic hepatitis in
haemophilia. Blood Rev 1993;7:243-50.
Prowse C. Kill and cure. The hope and reality
of virus inactivation. Vox Sang 1994;67(Suppl
3):191-6.
Lusher JM, Arkin S, Abildgaard CF, Schwartz
RS. Recombinant factor VIII for the treatment
of previously untreated patients with hemophilia A. Safety, efficacy, and development of
inhibitors. Kogenate Previously Untreated
Patient Study Group. N Engl J Med 1993;328:
453-9.
Ben-Hur E, Moor AC, Margolis-Nunno H, et
al. The photodecontamination of cellular
blood components: Mechanisms and use of
photosensitization in transfusion medicine.
Transfus Med Rev 1996;10:15-22.
Zhang Q-X, Edson C, Budowsky E, Purmal A.
InactineTM—a method for viral inactivation
in red blood cell concentrates (abstract).
Transfusion 1998;38(Suppl):75S.
Seeff L. The efficacy of and place for HBIG in
t h e p re v e n t i o n o f t y p e B h e p a t i t i s. In :
Szmuness W, Alter H, Maynard J, eds. Viral
hepatitis: 1981 International Symposium.
Philadelphia: The Franklin Institute Press,
1982:585-95.
Sanchez-Quijano A, Pineda JA, Lissen E, et al.
Prevention of post-transfusion non-A, non-B
hepatitis by non-specific immunoglobulin in
heart surgery patients. Lancet 1988;i:1245-9.
Conrad ME. Prevention of post-transfusion
hepatitis (letter). Lancet 1988;ii:217.

Chapter 28: Transfusion-Transmitted Diseases

184. Kobayashi R, Stiehm E. Immunoglobulin
therapy. In: Petz LD, Swisher SN, Kleinman S,
et al, eds. Clinical practice of transfusion medicine. 3rd ed. New York: Churchill Livingstone, 1996:985-1010.
185. Centers for Disease Control. Recommendations for follow-up of health-care workers after occupational exposure to hepatitis C virus. MMWR Morb Mortal Wkly Rep 1997;46:
603-6.

711

Suggested Reading
Actions following an initial positive test for possible bacterial contamination of a platelet unit (Association Bulletin #04-07). Bethesda, MD: AABB,
2004.
Criteria for donor deferral in known or suspected
common source outbreaks of hepatitis A virus infection (Association Bulletin #04-08). Bethesda,
MD: AABB, 2004.
Guidance on management of blood and platelet
donors with positive or abnormal results on bacterial contamination tests (Association Bulletin
#05-02). Bethesda, MD: AABB, 2005.

The inclusion of methods in this edition
of the Technical Manual is a subjective
decision of the Technical Manual Program Unit. Readers are encouraged to refer to previous editions of the manual for
methods not appearing in this edition because exclusion from the current edition
does not necessarily indicate that their
use is prohibited. However, some procedures, such as xylene and chloroform elution techniques, were removed because
the chemicals used in the procedures
could present a safety risk. Thus, readers
are cautioned when referring to procedures in previous editions because they
have not been reviewed for content and
safety.

There are often many different ways to
perform the same test procedure. Although
some workers may prefer other methods,
those given here are reliable, straightforward, and of proven value. Although the investigation of unusual serologic problems
often requires flexibility in thought and
methodology, the adoption of uniform
methods for routine procedures in the laboratory is imperative. In order for laboratory personnel to have reproducible and
comparable results in a test procedure, it is
essential that everyone in the laboratory
perform the same procedure in the same
manner.

Methods

Methods Section 1: General Laboratory Methods

Methods Section 1

General Laboratory Methods
Reagent Preparation

The methods outlined in the following
sections are examples of acceptable procedures. Other acceptable procedures
may be used by facilities if desired. To the
greatest extent possible, the written procedures conform to the Guidelines for
Clinical Laboratory Technical Procedure
Manuals developed by the National Committee for Clinical Laboratory Standards.
As indicated in Title 21 of the Code of
Federal Regulations (CFR) Part 606.65(e),
the manufacturer’s instructions (eg, product insert) for reagents and supplies licensed by the Food and Drug Administration (FDA) should be followed. Any deviation
should be validated using appropriate
controls and incorporated into a standard
operating procedure before approval by
the medical director. (Note: Deviations
may also require concurrence from the
FDA.) It is important to use Standard Precautions when appropriate (see Chapter
2).

Many procedures include formulas for reagent preparation. Labels for reagents prepared in-house must contain the following:
■
Name of solution.
■
Date of preparation.
■
Expiration date (if known).
■
Storage temperature and/or conditions.
■
Mechanism to identify the person preparing the solution.
■
Universal hazardous substance label.

Temperatures
Whenever specific incubation or storage
temperatures are given, the following ranges
are considered satisfactory:

Stated Temperature
4C
Room temperature
37 C
56 C

Acceptable Range
2-8 C
20-24 C
36-38 C
54-58 C

Section 1

Introduction

716

AABB Technical Manual

Centrifugation Variables
Centrifugation speeds (relative centrifugal
force) and times should be standardized
for each piece of equipment. (See Methods Section 8.)

Reference
Guidelines for clinical laboratory technical procedure manuals. 3rd ed. (NCCLS Document GP2-A3,
Vol. 12, No. 10.) Wayne, PA: National Committee
for Clinical Laboratory Standards, 1996.

Method 1.1. Transportation
and Shipment of Dangerous
Goods
Several agencies specify packaging and
shipping requirements for dangerous or
hazardous materials, depending on how
the material is shipped (mail, ground, or
air). For transport by mail of infectious
materials, clinical specimens, or biologic
products, the United States Postal Service
(USPS) Dangerous Goods Regulations
must be followed.1,2 For interstate transport of infectious materials by ground or
air, the United States Department of
Transportation (DOT) regulations apply.3
Most air carriers apply the International
Air Transport Association (IATA)4 regulations and the technical instructions of the
International Civil Aviation Organization
(ICAO).5 These agencies adopt the recommendations of the United Nations (UN)
Committee of Experts on the Transport of
Dangerous Goods for the international
transport of infectious substances and
clinical specimens.
The Centers for Disease Control and Pre6
7
vention (CDC) and the IATA provide packing and labeling requirements for shipments of infectious materials in order to
protect the public health by minimizing the
potential for direct contact with such materials, contamination of the environment,

and the spread of disease. The CDC also
serves as a Center for Applied Biosafety and
Training for the World Health Organization
(WHO)8 and for the UN. The Occupational
9
Safety and Health Administration (OSHA)
regulates worker safety related to handling,
packing, and transport. Both the DOT and
the IATA require active training for anyone
who packages infectious or toxic materials
for shipment.3,4 DOT and IATA documents
offer general shipping advice for all hazardous materials because the regulations are
similar and most carriers follow the shipping guidelines set forth in the IATA Dangerous Goods Regulations4 and the Infec7
tious Substances Shipping Guidelines.
Facilities should also consult their local carriers for additional requirements. In general, all dangerous goods regulations are
based on public risk from the materials and
therefore have specific packaging, labeling,
and documentation requirements. It is the
responsibility of the shipper of biologic or
infectious material to properly classify,
package, label, and document the substance being shipped.

Dangerous Goods Classifications
IATA classifies hazards into nine categories:
1.
Explosives.
2.
Compressed gases.
3.
Flammable liquids.
4.
Other flammable hazards.
5.
Oxygen-rich material, oxidizers, and
organic peroxides.
6.
Material affecting health, poisons,
and infectious substances.
7.
Radioactive materials.
8.
Corrosive material.
9.
Miscellaneous hazards.4
The infectious category includes:
■

Infectious substances: microbiologic
agents or their toxins that cause, or

Methods Section 1: General Laboratory Methods

■

■
■

may cause, disease; also called etiologic agents.
Biologic products: products that are
prepared and shipped in compliance with the provisions of 9 CFR
part 102 (Licensed Veterinary Biological Products), 9 CFR part 103 (Biological Products for Experimental
Treatment of Animals), 9 CFR part
104 (Imported Biological Products),
21 CFR part 312 (Investigational
New Drug Application), 21 CFR parts
600-680 (Biologics), 29 CFR Part
1910.1030 (Occupational Exposure
to Bloodborne Pathogens), 42 CFR
Part 72 (Interstate Shipment of Etiological Agents), or 49 CFR Parts 171178 (Hazardous Materials Regulations).
Clinical or diagnostic specimens: human or animal material (including
excreta, secreta, blood and its components, body fluids, tissue) being
shipped for the purpose of diagnosis.
Genetically modified organisms.
Clinical and medical waste.

Blood Bank Applications
Although the USPS regulates some biologic products, such as live poliovirus vaccine, the DOT and IATA take the position
that biologic products and diagnostic
specimens may be shipped with less
stringent packaging and labeling requirements, unless they contain, or are reasonably believed to contain, an infectious
substance. The CDC has proposed regulations to harmonize these requirements
with other federal and international re10
quirements. Units of blood that meet
disease testing requirements and unscreened blood from healthy donors
meeting all FDA donor eligibility criteria
including specimens for screening may

717

be shipped without hazard restrictions.
Specimens being shipped for initial diagnosis purposes and with a low probability
that infectious agents are present are
packaged as diagnostic samples (IATA
packing instruction 650); specimens from
individuals known, or thought likely, to
have an infectious disease are shipped as
infectious substances (IATA packing instruction 602). The IATA Dangerous
Goods Regulations should be consulted
for the most recent amendments to ship
biologic specimens.

Method 1.1.1. Shipping Diagnostic
Specimens and Infectious Substances

Principle
The safe transport of hazardous materials
requires that they be packaged in a way to
protect the materials and those who handle them. Prevention from leakage and
from being crushed in unexpected accidents are required. Primary containers
should be leakproof and securely closed.
The primary container should be placed
in a watertight secondary container. Absorbent material capable of absorbing the
entire liquid contents in the package is
placed between the primary and secondary containers. The outer packaging should
have adequate strength for its intended
use and capacity and be labeled in accordance with applicable regulations. Packaging requirements for clinical specimens
are similar to those for infectious substances except that performance standards of the packaging materials are less
rigorous.

Materials
1.

Leakproof, watertight primary container (eg, test tube), heat sealed,
crimped, or otherwise reinforced to
prevent cap slippage.

718

7.
8.

10.

AABB Technical Manual

Leakproof, watertight secondary package (eg, sealable plastic bag or container with screw cap).
Nonparticulate absorbent material (eg,
paper towels, gauze, disposable diaper).
For infectious substances and for total shipment volumes greater than
50 mL (but less than 4000 mL or 4
kg), shock-absorbent material equal
in volume to the nonparticulate-absorbent material.
Outer packaging.
a.
For infectious substances with a
shipping volume greater than 50
mL (but less than 4000 mL or 4
kg), an outer package of corrugated cardboard, fiberboard,
wood, metal, or rigid plastic meeting UN strength requirements.
b.
For biologic and diagnostic
specimens with a shipping volume less than 50 mL, a noncertified outer container.
Coolant material (if necessary).
a.
Wet ice, enclosed in sealed bags
to prevent leakage.
b.
Dry ice, not to exceed 5 lb (for
air shipments).
Itemized listing of package contents.
Address label that includes the names,
addresses, and contact names and
telephone numbers of both the sender
and intended recipient.
Special labels, as applicable to circumstances (see Table 1.1.1-1).
a.
Diagnostic specimens.
b.
Infectious substances.
c.
Dry ice.
d.
Liquid nitrogen.
e.
Cargo aircraft only.
Carrier-specific documents, such as
airbills or dangerous goods declarations.

Procedure
1.
2.

3.
4.
5.

Place the sealed primary container
into the secondary container.
Add sufficient absorbent material
around the primary container (between the primary and secondary
containers) to cover all sides of the
primary container and to absorb the
entire contents of the primary container should breakage occur.
Seal the secondary container securely.
Place the secondary container into
the applicable outer package.
For infectious substances with a
shipping volume greater than 50 mL,
place shock-absorbent materials at
the top, bottom, and sides between
the secondary container and the
outer packaging. See Fig 1.1.1-1.
Place any necessary coolant material
(eg, wet ice or dry ice) between the
secondary container and the applicable outer packaging. Provide interior support to secure the secondary
packaging in the original position
because the ice or dry ice melts or
dissipates.

Figure 1.1.1-1. Appropriate packaging of clinical
specimen material.

Methods Section 1: General Laboratory Methods

719

Table 1.1.1-1. Special Labels for Shipping
Clinical (Diagnostic) Specimens
Specimens collected for diagnosis, research, or other purposes must be labeled as biohazardous. Both the
primary container and the outer packaging must contain a biohazard label. The outer packaging label must
appear as follows:
1. The color of the material on which the label is printed must be bright orange, with the biohazard symbol
and printing in black.
2. The label must be a rectangle measuring 2 inches high by 4 inches long.
3. The biohazard symbol, measuring 1.56 inches in diameter, must be centered on a square measuring 2
inches on each side.
4. The size of the letters, printed in Helvetica, must be:
Biohazard
16 pt.
Clinical specimens
14 pt.
Packaged in compliance with 42 CFR part 72
6 pt.
In case of damage or leakage, notify
10 pt.
Shipper and receiver
10 pt.

Infectious Substances (Etiologic Agents)
For substances known to contain infectious agents or reasonably anticipated to contain such agents,
biohazardous labels must be placed on the primary container and outer packaging. The outer packaging
label must appear as follows:
1. The color of the material on which the label is printed must be white, with the biohazard symbol and
printing in black.
2. The label must be a diamond-on-point measuring, at a minimum, 4 inches on each side.
3. The biohazard symbol, measuring 0.81 inches in diameter, must be centered on a square measuring 2
inches on each side.
4. The Class 6 symbol for infectious substances must be centered at the bottom of the label.
5. The size of the letters, printed in Helvetica, must be:
Infectious substance
16 pt.
Packaged in compliance with 42 CFR part 72
5 pt.
In case of damage or leakage, notify
7 pt.
Immediately notify
7 pt.
Public Health Authority
7 pt.
In the United States
5 pt.
Centers for Disease Control and Prevention
5 pt.
Atlanta, GA
5 pt.
1-800-232-0124
5 pt.
6
24 pt.

Dry Ice
Packages with dry ice must be labeled with the following information:
1. The diamond-shaped Class 9 symbol for miscellaneous hazardous materials.
2. The words “Dry Ice” or “Carbon Dioxide, Solid.”
3. “UN 1845,” the United Nations hazardous material category for dry ice (if shipped out of the country).
4. The name of the contents being cooled.
5. The weight of the dry ice in kg (not to exceed 5 lb if transported by air).

Liquid Nitrogen
Packages with liquid nitrogen must be labeled with a green IATA label stating “Contains Cryogenic Liquid.”

Cargo Aircraft Only
If the mode of transport is air and more than 50 mL of infectious substance is enclosed, the package must
have a warning label “Cargo Aircraft Only” to preclude the carrier from transporting the package on a
passenger plane.

720

AABB Technical Manual

Enclose the itemized listing of container contents between the secondary and outer packaging.
Seal the outer packaging and label
with address label and applicable
special labels.
Complete applicable shipping forms
and send them with the package.

8.
9.
10.

Notes
1.

11.

Package specimens within a container such that they will remain in
an upright position to help prevent
leakage.
Closures on primary containers can
be reinforced with adhesive tape or
paraffin. It is not necessary to reinforce unopened evacuated specimen
collecting tubes.
For infectious substances, the name,
address, and telephone number of
the shipper must appear on both the
secondary and outer shipping containers.
Shipments of infectious substances
may contain multiple secondary containers, but the total volume of the
shipment may not exceed 4 L or 4
kg, excluding the packaging and
coolant weights.
Primary containers or secondary
packaging must be capable of withstanding an internal pressure differential of 95 kPA (0.95 bar, 13.8 lb/in2)
between the temperatures of –40 C
and 55 C.
Styrofoam, plastic bags, and paper
envelopes are unacceptable for outer
packaging.
UN-certified containers must be able
to withstand a 30-foot “drop test,” as
specified in 49 CFR 178.609, without
breaking enclosed tubes. The UN
certification number must appear
on the container. Noncertified con-

12.

tainers must be able to withstand at
least a 1.2-meter drop on a hard unyielding surface without release of
the container’s contents.
Ensure that all state and local regulations are followed.
Some exceptions may apply for
ground transportation.
If unsure about how to package and
ship materials, contact the CDC.
If breakage occurs during shipment,
the package should be handled with
extreme caution (wear personal protective equipment) and the entire
package (including containers, contents, and packaging materials) should
be autoclaved before discard. Supervisory personnel should be notified
if the contents are lost in transit or if
the damage appears related to inadequate packaging by the sender.
The carrier, the receiver, or anyone
handling damaged or leaking packages must isolate the package and
notify the shipper and intended recipient immediately. In addition, for
infectious substances, notify the CDC
as soon as possible (1-800-232-0124).
When notifying the CDC, the caller
should provide a description of the
condition of the package, the name,
address, and telephone number of
the shipper, and any other pertinent
information, so that decontamination and disposal procedures can be
provided.

Additional Considerations with the Use of
Dry Ice
1.

Solid carbon dioxide or “dry ice” is
classified as a hazardous material
because it can cause burns on contact and gives off carbon dioxide gas
as it volatilizes.

Methods Section 1: General Laboratory Methods

Insulated gloves must be worn when
handling dry ice; eye protection must
be worn when breaking up chunks
of solid ice or when breaking apart
ice pellets.
Dry ice should be handled in a wellventilated area; its gas can cause lightheadedness and, in extreme cases, asphyxiation.
When kept in a tightly sealed shipping container, dry ice could rupture
the packaging. If dry ice is used, it
must be placed outside the secondary packaging or in an overpack with
one or more complete packages. The
outer packaging must be leakproof.
Procedures for packing with dry ice
must include instructions for sealing
the outer container in a manner that
allows the gas to escape and prevents loosening of secondary containers as the dry ice dissipates.
Consult the Domestic Mail Manual
(section C023),2 49 CFR 173.217 and
175.10 (a)(13),3 IATA Dangerous Goods
4
Regulations, and IATA packing instruction 904.
HAZMAT training requirements for
dry ice are found in 49 CFR 172.704.3
Training for staff who come in contact with dry ice should include information on:
■
Potential hazards.
■
Personal protective equipment
to use when handling or chipping.
■
Procedures for packing, sealing, labeling, and handling
boxes containing dry ice.
■
Special considerations in operating a vehicle used to transport boxes containing dry ice.
■
Procedures for storing or disposing of dry ice (eg, allowing
the gas to dissipate by natural
means in a well-ventilated

721

area) after a shipment is received.

Additional Considerations with the Use of
Liquid Nitrogen
1.
2.

Liquid nitrogen is classified as a cryogenic liquid.
Insulated gloves, eye protection, and
a protective laboratory coat must be
worn when working with liquid nitrogen.
A watertight material capable of withstanding cryogenic temperatures must
be used for the primary container.
This container must maintain its containment integrity at the temperature of the refrigerant as well as at
the temperature and pressure of air
transport if refrigeration is lost.
The secondary packaging must be
designed to withstand cryogenic temperatures.
The design of the outer packaging
must allow for relief of pressure when
the liquid nitrogen evaporates. A
loose-fitting, but secured, container
cover or pressure relief valve may be
used.

References
1.

Code of Federal Regulations. Title 39 CFR.
Washington, DC: US Government Printing
Office, 2004 (revised annually).
Etiologic agent preparations, clinical specimens, and biological products. Domestic
Mail Manual (section C023), issue 57, July 10,
2003.
Code of Federal Regulations. Title 49 CFR Part
171-180. Washington, DC: US Government
Printing Office, 2004 (revised annually).
Dangerous goods regulations. 45th ed. Montreal, Canada: International Air Transport Association, 2004 (revised annually).
Technical instructions for the safe transport
of dangerous goods by air. 2002-2004 ed.
Montreal, Canada: International Civil Aviation Organization, Documents 9284 and
9284SU, 2002.

722

AABB Technical Manual

Code of Federal Regulations. Title 42 CFR Part
72. Washington, DC: US Government Printing
Office, 2004 (revised annually).

Infectious substances and diagnostic specimens shipping guidelines. 4th ed. Montreal,
Canada: International Air Transport Association, 2003.

World Health Organization. Guidelines for
the safe transport of infectious substances
and diagnostic specimens. Geneva, Switzerland: World Health Organization, 1997.

Code of Federal Regulations. Title 29 CFR
1910.1030. Washington, DC: US Government
Printing Office, 2004 (revised annually).

10.

Centers for Disease Control and Prevention.
Packaging and handling of infectious substances and select agents, notice of proposed
rulemaking. Fed Regist 1999;64(208):5802231.

Method 1.1.2. Monitoring Temperature
During Shipment of Blood

Principle
Some form of temperature indication or
monitoring is desirable when shipping
blood. The temperature of the contents of
a shipping container used for whole blood
or liquid-stored red cell components can
be ascertained when the shipment is received, as follows:

Procedure
1.

2.
3.
4.

Open the shipping container and
promptly place the sensing end of a
calibrated liquid-in-glass or electronic thermometer between two
bags of blood or components (labels
facing out) and secure the “sandwich” with two rubber bands.
Close the shipping container.
After approximately 3 to 5 minutes,
read the temperature.
If the temperature of red-cell-containing components exceeds 10 C,
quarantine the units until their appropriate disposition can be determined.

Notes
Other suitable methods for monitoring
shipments are:
1.
Use time/temperature indicators,
one such indicator per shipping carton. These indicators will change
color or show another visible indication if the temperature has exceeded
10 C.
2.
Place a “high-low” thermometer in
the shipping container. This simple,
reusable thermometer measures and
records the highest and lowest temperatures during any period.

Method 1.2. Treatment of
Incompletely Clotted Specimens
Principle
Fibrin generation may continue in serum
separated from incompletely clotted blood,
especially during incubation at 37 C. This
produces strands of protein that entrap
red cells and make it difficult to evaluate
agglutination. Blood from patients who
have recently received heparin may not
clot at all, and blood from patients with
excessive fibrinolytic activity may reliquefy or may contain protein fragments that
interfere with examination for agglutination.

Materials
1.

2.
3.
4.

Thrombin: dry human/bovine thrombin
or thrombin solution (50 units/mL
in saline).
Glass beads.
Protamine sulfate: 10 mg/mL in saline.
Epsilon aminocaproic acid (EACA):
0.25 g/mL in saline.

Methods Section 1: General Laboratory Methods

Procedure
1.

To accelerate clotting: Either of the
following techniques may be used:
a.
Add to the specimen the amount
of dry thrombin that adheres to
the tip of an applicator stick or
1 drop of thrombin solution
per mL of whole blood or serum. Allow 10 to 15 minutes for
the clot to form. Use standard
centrifugation to separate the
clot and serum.
b.
Gently agitate the separated serum with small glass beads, at
37 C, for several minutes. Then,
use low speed centrifugation to
pellet the glass beads. Transfer
the serum to another tube.
To neutralize heparin: Protamine
sulfate can be added to the specimen to neutralize heparin; however,
excess protamine promotes rouleaux
formation and, in great excess, will
inhibit clotting. Add 1 drop of protamine sulfate solution to 4 mL of
whole blood and wait 30 minutes to
evaluate the effect on clotting. If
clotting does not occur, add additional protamine sparingly. Note:
protamine sulfate may work more
rapidly when briefly incubated (5-10
minutes) at 37 C.
To inhibit fibrinolytic activity: Add 0.1
mL of EACA to 4 mL of whole blood.

Notes
1.

The use of anticoagulated (eg, ACD
or EDTA) collection tubes may help
to avoid the problem of incompletely clotted specimens. The use of
anticoagulated specimens must be
validated in accordance with each
standard operating procedure.
Because preparations of human
thrombin may contain red cell anti-

723

bodies, test results must be carefully
observed for false-positive reactions.
Quality control should be performed
on thrombin reagents before or concurrent with their use to identify
those with contaminating antibodies.

Method 1.3. Solution
Preparation—Instructions
Principle
The basic definitions, calculations, and
instructions given below serve as a review
of simple principles necessary for solution preparation.
1.
Mole, gram-molecular weight: Weight,
expressed in grams equal to the
atomic or molecular weight of the
substance.
2.
Molar solution: A one molar (1 M)
solution contains one mole of solute
in a liter of solvent. The solvent is assumed to be distilled or deionized
water unless otherwise indicated.
3.
Gram-equivalent weight: Weight, in
grams, of a substance that will produce or react with 1 mole of hydrogen ion.
4.
Normal solution: A one normal (1 N)
solution contains one gram-equivalent weight of solute in a liter of
solution.
5.
Percentage solutions: The percent
designation of a solution gives the
weight or volume of solute present
in 100 units of total solution. Percent
can be expressed as:
a.
Weight/weight (w/w), indicating
grams of solute in 100 g of solution.
b.
Volume/volume (v/v), indicating
milliliters of solute present in
100 mL of solution.

724

AABB Technical Manual

7.
8.

Weight/volume (w/v), indicating
grams of solute in 100 mL of solution. Unless otherwise specified, a solution expressed in
percentage can be assumed to
be w/v.
Water of crystallization, water of hydration: Molecules of water that form
an integral part of the crystalline
structure of a substance. A given
substance may have several crystalline forms, with different numbers of
water molecules intrinsic to the entire molecule. The weight of this water must be included in calculating
molecular weight of the hydrated
substance.
Anhydrous: The salt form of a substance with no water of crystallization.
Atomic weights (rounded to whole
numbers): H, 1; O, 16; Na, 23; P, 31; S,
32; Cl, 35; K, 39.
Molecular weights:
HCl: 1 + 35 = 36; NaCl: 23 + 35 = 58
KCl: 39 + 35 = 74
H2O: (2 × 1) + 16 = 18
NaH2PO4: 23 + (2 × 1) + 31 + (4 × 16) =
120
NaH2PO4 • H2O: 23 + (2 × 1) + 31 +
(4 × 16) + (2 × 1) + 16 = 138
KH2PO4: 39 + (2 × 1) + 31 + (4 × 16) =
136
H2SO4: (2 × 1) + 32 + (4 × 16) = 98

Examples
1.

Molar solutions:
1 M KH2PO4 = 136 g of solute made up
to 1 L.
0.15 M KH2PO4 = (136 × 0.15) = 20.4 g
of solute made up to 1 L.
0.5 M NaH2PO4 = (120 × 0.5) = 60 g of
solute made up to 1 L.
Molar solution with hydrated salt:
0.5 M NaH2PO4 • H2O = (138 × 0.5) =

69 g of the monohydrate crystals
made up to 1 L.
Normal solutions:
1 N HCl = 36 g of solute made up to 1
L. One mole HCl dissociates into one
+
mole H , so gram-equivalent weight
and gram-molecular weight are the
same.
12 N HCl = (36 × 12) = 432 g of solute
made up to 1 L.
1 N H2SO4 = (98 ÷ 2) = 49 g of solute
made up to 1 L. One mole H2SO4 dis+
sociates to give two moles of H , so
the gram-equivalent weight is half
the gram-molecular weight.
Percent solution:
0.9% NaCl (w/v) = 0.9 g of solute
made up to 100 mL of solution.

Notes
Accurate results require accurate preparation of reagents. It is important to carefully read and follow all instructions and
labels.
1.
Weigh only quantities appropriate
for the accuracy of the equipment.
The operator’s manual should give
these specifications.
2.
Prepare the largest volume that is
practical. There is greater accuracy
in measuring larger volumes than
smaller volumes. If a reagent balance is accurate to ±0.01 g, the potential error in weighing 0.05 g (50
mg) will be 20%, whereas the potential error in weighing 0.25 g (250 mg)
will be only 4%. If the solution retains its activity when stored appropriately, it is usually preferable to
prepare a large volume. If the solution deteriorates rapidly, smaller volumes may be preferred to reduce
waste.
3.
Note whether a substance is in the
hydrated or anhydrous form. If the

Methods Section 1: General Laboratory Methods

instructions give solute weight for
one form, and the available reagent
is in another form, be sure to adjust
the measurements appropriately.
For example, if instructions for 0.5 M
NaH2PO4 call for 60 g, and the reagent is NaH2PO4
H2O, find the ratio between the weights of the
two forms. The molecular weight of
NaH2PO4
H2O is 138 and the molecular weight of NaH 2 PO 4 is 120.
Therefore, the ratio is 138 ÷ 120 =
1.15. Multiply the designated weight
by the ratio (60 g × 1.15 = 69 g) to obtain the final weight needed.
Dissolve the solute completely before making the solution to the final
volume. This is especially important
for substances, such as phosphates,
that dissolve slowly. For example, to
make 500 mL of 0.15 M KH2PO4:
a.
Weigh 10.2 g of solute in a weighing boat or glass [(0.15 × 136) ÷
2] because only 500 mL will be
made.
b.
Place 350 mL of water in a
500-mL volumetric flask on a
magnetic stirrer. Add the stirring bar and adjust it to a slow,
steady stirring speed.
c.
Add 10.2 g of salt, then rinse the
boat with several aliquots of
water until no salt remains. Numerous small-volume rinses
remove adherent material more
effectively than a few larger
volumes. Add the rinse water to
the material in the flask and stir
until the salt has completely
dissolved.
d.
If pH measurement is unnecessary, add water to the 500-mL
mark, adjusting the volume for
the stirring bar, and mix thoroughly. For solutions needing pH
adjustment, see the next step.

725

Adjust the pH of the solution before
bringing it to its final volume so that
the addition of water (or other solvent) does not markedly change the
molarity. For example, to bring 500
mL of 0.1 M glycine to a pH of 3:
a.
Ad d 3 . 7 5 g o f g l y c i n e
(H2 NCH2 COOH:
molecular
weight, 75) to 400-475 mL of
water in a beaker. Dissolve
completely, using a magnetic
stirrer.
b.
Add a few drops of concentrated
(12 N) HCl and measure pH after acid is thoroughly mixed.
Continue adding HCl until pH
is 3.0.
c.
Transfer the solution to a 500-mL
volumetric flask. Rinse beaker
and stirring bar with aliquots of
water, adding the rinse water to
the flask. Use the rinses to contribute to the total 500-mL volume.
d.
Measure the pH of the solution
at final volume.

References
1.

Remson ST, Ackerman PG. Calculations for
the medical laboratory. Boston, MA: Little,
Brown & Co., 1977.
Henry JB, ed. Clinical diagnosis and management by laboratory methods. 18th ed. Philadelphia: WB Saunders, 1991.

Method 1.4. Serum Dilution
Principle
Serum is sometimes diluted in saline or
other diluents to determine its relative
antibody concentration. It is customary to
express the dilution as 1 part of serum
contained in the total number of parts of
the dilution. For example, to test the serum at one-tenth its original concentration, a dilution of 1 part in 10 may be

726

AABB Technical Manual

quantity of a new higher final
dilution is:

made by mixing 1 mL of serum with 9 mL
of saline. The final volume is 10, and the
dilution is expressed as a 1 in 10 dilution.
The diluted material contains one-tenth
(1/10 or 0.1) of the unmodified serum. It
is often customary to report the titer of an
antibody as the reciprocal of the highest
dilution that retains a 1+ agglutination.
Therefore, serum that reacts at a dilution
of 1/32 is considered to have a titer of 32.
Note: A 1 in 10 dilution is 1 part in 9 parts,
whereas a 1 to 10 or 1:10 is 1 part in 10 parts.

reciprocal of present dilution
volume of present dilution needed
= reciprocal of final dilution
total final volume required
b.

Procedure
1.

Diluting an existing dilution:
a.
A new higher dilution can be
prepared from diluted material
by adding more diluent. The
formula for calculating either
the new higher final dilution or
the amount of diluent to add to
obtain a higher final dilution is:

Example: Present serum dilution is one in two, total final
volume is 100 mL, and new final serum dilution is 1 in 10.
How much serum (diluted one
in two) will have to be added to
make up a final volume of 100
mL of a 1 in 10 dilution?
2
X

10
100

X = 20 or 20 mL of serum (dilution of one in two) must be added
to 80 mL of diluent to obtain
100 mL of a 1 in 10 dilution.

reciprocal of present serum dilution
volume of serum dilution used
= reciprocal of new final dilution
total final volume
b.

Example: Serum dilution is one
in two and volume of serum dilution is 1.0 mL. If 4.0 mL of saline is added, what will be the
new final dilution?
2
1

X
5

X = 10 or 1 in 10 dilution
2.

Diluting a dilution to a specified volume:
a.
The formula for calculating the
volume of diluent to add to a
dilution to achieve a certain

Method 1.5. Dilution of %
Solutions
Procedure
1.

Dilutions can be prepared from more
concentrated solutions by use of the
following formula:
(Volume1 × Concentration1) =
(Volume2 × Concentration2)
V1 × C1 = V2 × C2
where V1 and C1 represent original
volume and concentration, and V2
and C2 represent final desired volume and concentration.

Methods Section 1: General Laboratory Methods

Example: 30% albumin is available,
but 2 mL of 6% albumin is needed.
How should the albumin be diluted?
V1 × 30 = 2 × 6
30V1 = 12
V1 = 12 ÷ 30 = 0.4
Therefore, mix 0.4 mL of 30% albumin with 1.6 mL saline to obtain 2.0
mL of 6% albumin, or for small-volume use, mix 4 drops 30% albumin
with 16 drops saline to obtain 20
drops of 6% albumin.

1.
2.

Method 1.6. Preparation of a
3% Red Cell Suspension
Principle

A 3% red cell suspension is a common reagent in many serologic procedures. The
suspension need not be exactly 3%; an
approximation achieves the appropriate
serum-to-cell ratio for most test procedures and an adequate number of red
cells to read and grade the reactions. The
following steps are intended to help an individual gain confidence in approximating a 3% red cell suspension visually, both
as a suspension of cells and in the appropriate size of the cell pellet achieved after
centrifugation.

Materials
1.
2.
3.
4.
5.
6.

Whole blood sample.
Test tubes.
Disposable pipettes (1 mL and 10 mL
serologic).
Saline.
Centrifuge (3000 rpm or equivalent).
Commercially prepared 3% reagent
red cell suspension.

Procedure
To prepare 10 mL of a 3% red cell suspension:

727

Transfer at least 1 mL of whole blood
to a 10-mL tube.
Wash the red cells in saline or phosphate-buffered saline (PBS), centrifuging for 5 minutes to pellet the
cells. Repeat two or three times. The
final supernate should be clear and
should be completely removed by
aspiration.
Transfer 0.3 mL of the washed red
cells to a tube with 9.7 mL of saline,
PBS, or Alsever’s solution.
Cap or cover the tube with parafilm.
Thoroughly mix the red cells and saline by gently inverting the tube several times.
To compare the color and density of
the suspension by eye, transfer a volume of the prepared suspension to a
10 × 75 mm tube. Also transfer a similar volume of a known 3% red cell
suspension (eg, commercial reagent
red cell suspension) to another 10 ×
75 mm tube. Hold the two tubes in
front of a light source to compare them.
To compare the size of the cell pellet
expected from a 3% red cell suspension, transfer one drop of the prepared suspension to a 10 × 75 mm
tube. Similarly, transfer one drop of
a known 3% commercial reagent red
cell suspension to another 10 × 75 mm
tube. Centrifuge the tubes in a serologic centrifuge, using the spin time
designated for “saline.” The size of
the two cell pellets should be similar.

Note
For best results use red cell suspensions
on the day of preparation only, unless stability for a longer time has been validated.

728

AABB Technical Manual

Method 1.7. Preparation and
Use of Phosphate Buffer
Principle

References
1.

Mixtures of acids and bases can be prepared at specific pH values and used to
buffer (render) other solutions to that pH.
The following procedure includes a method
for preparing phosphate-buffered saline
(PBS), which can be used as a diluent in
serologic tests.

Hendry EB. Osmolarity of human serum and
of chemical solutions of biologic importance.
Clin Chem 1961;7:156-64.
Dacie JV, Lewis SM. Practical haematology. 4th
ed. London, England: J and A Churchill, 1968:
540-1.

Method 1.8. Reading and
Grading Tube Agglutination
Principle

Reagents
1.

Prepare acidic stock solution (solution A) by dissolving 22.16 g of
NaH2PO4 • H2O in 1 L of distilled water. This 0.16 M solution of the monobasic phosphate salt (monohydrate)
has a pH of 5.0.
Prepare alkaline stock solution (solution B) by dissolving 22.7 g of
Na2 HPO4 in 1 L of distilled water.
This 0.16 M solution of the dibasic
phosphate salt (anhydrous) has a pH
of 9.0.

Procedure
1.

Prepare working buffer solutions of
the desired pH by mixing appropriate volumes of the two solutions. A
few examples are:
pH
Solution A
Solution B
5.5
94 mL
6 mL
7.3
16 mL
84 mL
7.7
7 mL
93 mL
Check the pH of the working solution before using it. If necessary, add
small volumes of acid solution A or
alkaline solution B to achieve the desired pH.
To prepare PBS of a desired pH, add
one volume of phosphate buffer at
that pH to nine volumes of normal
saline.

The purpose of grading reactions is to allow comparison of reaction strengths.
This is beneficial in detecting multiple
antibody specificities or antibodies exhibiting dosage. The grading of agglutination
reactions should be standardized among
all members of the laboratory staff, in the
interest of uniformity and reproducibility
of test results. Most laboratories define
their own version of a grading system,
which is described in a written procedure
available to all staff. Some systems use assigned numeric values (scores) for the
observed reactions.

Materials
1.
2.

Centrifuged serologic tests for agglutination.
Agglutination viewer.

Procedure
1.

2.
3.

Gently shake or tilt the tube and resuspend the red cell button in the
tube. The tilt technique uses the meniscus to gently dislodge the red cell
button from the wall of the tube.
Observe the way that cells are dispersed from the red cell button.
Record reactivity by comparing the
agglutinates to the descriptions in
Table 1.8-1. The reactivity should be
assessed when the red cells have been

Methods Section 1: General Laboratory Methods

729

Table 1.8-1. Interpretation of Agglutination Reactions
Macroscopically Observed Findings

Designation

Score

4+
3+
2+
1+
1w
w+ or +/–
0
mf

12
10
8
5
4
2
0

One solid agglutinate
Several large agglutinates
Medium-size agglutinates, clear background
Small agglutinates, turbid background
Very small agglutinates, turbid background
Barely visible agglutination, turbid background
No agglutination
Mixtures of agglutinated and unagglutinated red cells
(mixed field)
Complete hemolysis
Partial hemolysis, some red cells remain

completely resuspended from the
button.
3.

Interpretation
Refer to Table 1.8-1.

Notes
1.

An agglutination viewer may facilitate
the reading of tube tests. However,
the manufacturer’s test recommendation must be followed for interpreting the test results.
Serum overlying the centrifuged cell
button must be inspected for
hemolysis, which is a positive sign of
an antigen-antibody reaction, provided the pretest serum was not

H
PH

hemolyzed and no hemolytic agent
was added to the test.
The character of the agglutination
should be noted and recorded. This
information provides valuable clues
in the investigation such as the characteristic refractile agglutination of
anti-Sda.
Mixed-field agglutination is expected when using pooled cells for
donor antibody detection and adding check cells to negative antiglobulin tests.

Reference
Race RR, Sanger R. Blood groups in man. 6th ed.
Oxford: Blackwell Scientific Publications, 1975.

Methods Section 2: Red Cell Typing

Section 2

Methods Section 2

Red Cell Typing

Method 2.1. Slide Test for
Determination of ABO Type
of Red Cells

All reagents must be used in accordance
with the manufacturer’s instructions.

Procedure
1.

Principle
See Chapter 13 for a discussion of the
principles of testing for ABO groups.

2.
3.

Specimen
The reagent manufacturer’s instructions
must be consulted before slide tests are
performed; some manufacturers recommend performing slide tests with whole
blood, whereas others specify the use of
red cell suspensions of lighter concentrations prepared in saline, serum, or plasma.

Reagents
1.
2.
3.

Anti-A.
Anti-B.
Anti-A,B (optional).

Place 1 drop of anti-A on a clean, labeled glass slide.
Place 1 drop of anti-B on a separate
clean, labeled glass slide.
Place 1 drop of anti-A,B on a third
slide, if parallel tests are to be performed with this reagent, or on a
single clean, labeled slide if this is
the only test performed.
Add to each drop of reagent on the
slides 1 drop of well-mixed suspension (in saline, serum, or plasma) of
the red cells to be tested. (Consult
the reagent manufacturer’s instructions to determine the correct cell
concentration to be used.)
Mix the reagents and red cells thoroughly, using a clean applicator stick
for each reagent. Spread the mixture
731

732

AABB Technical Manual

over an area approximately 20 mm ×
40 mm.
Gently tilt the slide continuously for
up to 2 minutes. Do not place the slide
over a heated surface, such as an Rh
viewbox, during this period.
Read, interpret, and record the results of the reactions on all slides.

Interpretation
1.

Strong agglutination of red cells in
the presence of any ABO typing reagent constitutes a positive result.
A smooth suspension of red cells at
the end of 2 minutes is a negative result.
Samples that give weak or doubtful
reactions should be retested using
Method 2.2.

Notes
1.

Slide testing imposes a greater risk
of exposure to infectious samples.
Personnel should follow safety measures detailed in the facility’s procedures manual.
Slide testing is not suitable for detection of ABO antibodies in serum/
plasma.

Specimen
The reagent manufacturer’s package insert must be consulted to determine specific specimen requirements. Generally,
clotted or anticoagulated blood samples
may be used for ABO testing. The red cells
may be suspended in autologous serum,
plasma, or saline, or may be washed and
resuspended in saline.

Reagents
1.
2.
3.

Anti-A.
Anti-B.
Anti-A,B. Note: Use of this reagent is
optional.
4.
A1, A2, and B red cells. They can be
obtained commercially or the testing
laboratory can prepare a 2% to 5%
suspension on each day of use. (Note:
The use of A2 cells is optional.)
All reagents must be used in accordance
with the manufacturer’s instructions.

Procedures

Testing Red Cells
1.
2.
3.

Method 2.2. Tube Tests for
Determination of ABO Group
of Red Cells and Serum

Principle
See Chapter 13 for a discussion of the
principles of testing for ABO groups. The
following procedure is an acceptable representative method, but the manufacturer’s
instructions for the specific reagents must
be consulted.

Place 1 drop of anti-A in a clean, labeled test tube.
Place 1 drop of anti-B in a clean, labeled tube.
Place 1 drop of anti-A,B in a clean,
labeled tube, if tests are to be performed with this reagent.
Add to each tube 1 drop of a 2% to
5% suspension (in saline, serum, or
plasma) of the red cells to be tested.
Alternatively, the equivalent amount
of red cells can be transferred to each
tube with clean applicator sticks.
Mix the contents of the tubes gently
and centrifuge them for the calibrated spin time.
Gently resuspend the cell buttons
and examine them for agglutination.

Methods Section 2: Red Cell Typing

733

interpretation is recorded for the
patient’s or donor’s ABO group (see
Chapter 13).

Read, interpret, and record the test results. Compare the red cell test results with those obtained in the serum/plasma tests (see below).

Note

Testing Serum/Plasma
1.

2.
3.
4.
5.

Label two clean test tubes as A1 and
B. (Note: Label an additional tube if
an optional test with A2 red cells is to
be performed.)
Add 2 or 3 drops of serum/plasma to
each tube.
Add 1 drop of A1 reagent cells to the
tube labeled A1.
Add 1 drop of B reagent cells to the
tube labeled B.
Add A2 cells to the appropriate tube,
if this optional test is being performed.
Mix the contents of the tubes gently
and centrifuge them for the calibrated spin time.
Examine the serum overlying the cell
buttons for evidence of hemolysis.
Gently resuspend the cell buttons
and examine them for agglutination.
Read, interpret, and record test results.
Compare serum test results with
those obtained in testing red cells
(see above).

Interpretation
1.

Agglutination of tested red cells and
either hemolysis or agglutination in
tests with serum constitute positive
test results.
A smooth cell suspension after resuspension of the cell button is a
negative test result.
Interpretation of serum/plasma and
cell tests for ABO is given in Table
13-1.
Any discrepancy between the results
of the tests with serum/plasma and
cells should be resolved before an

Positive reactions characteristically show
3+ to 4+ agglutination by reagent ABO antibodies; reactions between test serum
and reagent red cells are often weaker.
The serum tests may be incubated at
room temperature for 5 to 15 minutes to
enhance weak reactions. See Chapter 13
for a discussion of weakly reactive samples.

Method 2.3. Microplate Test
for Determination of ABO
Group of Red Cells and
Serum
Principle
See Chapter 13 for a discussion of the
principles of testing for ABO blood group.
Microplate techniques can be used to test
for antigens on red cells and for antibodies in serum.
A microplate can be considered as a matrix of 96 “short” test tubes; the principles
that apply to hemagglutination in tube tests
also apply to tests in microplates.
Microplates may be rigid or flexible, with
either U-shaped or V-shaped bottoms.
U-shaped bottom plates are more widely
used because results can be read either after centrifuging the plate and observing the
characteristics of resuspended red cells or
by observing the streaming pattern of the
cells when the plate is placed at an angle.
Either reading technique permits estimation of the strength of agglutination.

Specimen
Refer to Method 2.2.

734

AABB Technical Manual

Equipment
1.

Dispensers (optional): Semiautomated devices are available for dispensing equal volumes to a row of wells.
Special plate carriers can be purchased to fit common table-top centrifuges.
Microplate readers (optional): Automated photometric devices are available that read microplate results by
the light absorbance in U-shaped
bottom wells to differentiate between positive and negative tests.
The microprocessor component of
the reader interprets the reactions
and prints the blood testing results.
The manufacturer’s instructions for
the collection and preparation of serum/plasma and cell specimens
must be followed.
Centrifuges: Appropriate conditions
must be established for each centrifuge. The following times and relative centrifugal forces, expressed as
g, are suggested. Consult the manufacturer’s directions for specific information.
For a flexible U-shaped bottom
microplate: 700 × g for 5 seconds for
red cell testing and serum/plasma
testing.
For a rigid U-shaped bottom microplate: 400 × g for 30 seconds for red
cell testing and serum/plasma testing.

Procedure

Testing Red Cells
1.

3.
4.

Place 1 drop of anti-A and anti-B in
separate clean wells of a U-bottom
microplate. If tests with anti-A,B are
to be performed, add this reagent to
a third well.
Add 1 drop of a 2% to 5% saline suspension of red cells to each well containing blood typing reagent.
Mix the contents of the wells by
gently tapping the sides of the plate.
Centrifuge the plate at the appropriate conditions established for the
centrifuge.
Resuspend the cell buttons by manually tapping the plate or with the
aid of a mechanical shaker, or place
the plate at an angle for the tilt and
stream method.
Read, interpret, and record results.
Compare red cell test results with those
obtained in testing serum/plasma.

Testing Serum/Plasma
1.

Reagents
Many manufacturers supply ABO or Rh
typing reagents that are licensed by the
Food and Drug Administration (FDA) for
use as undiluted reagents in microplate
tests.
1.
Anti-A.
2.
Anti-B.
3.
Anti-A,B. Note: Use of this reagent is
optional.

Group A1, A2, and B red cells. They
can be obtained commercially or the
testing laboratory can prepare a 2%
to 5% suspension on each day of use.
(Note: The use of A2 cells is optional.)

2.
3.
4.

Add 1 drop of a 2% to 5% suspension
of reagent A1 and B red cells to separate clean wells of a U-bottom microplate. (Note: If an optional test on A2
cells will be performed, add A2 cells
to a third well.)
Add 1 drop of serum or plasma under test to each well.
Mix the contents of the wells by gently
tapping the sides of the plate.
Centrifuge the plate at the appropriate conditions established for the
centrifuge.

Methods Section 2: Red Cell Typing

Resuspend the cell buttons by manually tapping the plate or with aid of
a mechanical shaker, or place the plate
at an angle for the tilt and stream
method.
Read, interpret, and record results.
Compare test results on serum/plasma
with those obtained in testing red
cells.

Specimen
Refer to Method 2.2.

Reagents
1.

Note
To enhance weak serum/plasma reactions, the plates may be incubated at
room temperature for 5 to 10 minutes,
then the centrifugation, reading, and recording steps may be repeated.

3.
4.

Agglutination in any well of red cell
tests and hemolysis or agglutination
in any well of a serum test constitute
positive results.
A smooth suspension of red cells after resuspension of the cell button is
a negative test.
The interpretation of ABO tests is
given in Table 13-1.
Any discrepancy between results on
cell and serum/plasma tests should
be resolved before an interpretation
is recorded for the patient’s or donor’s
ABO group (see Chapter 13).

Method 2.4. Confirmation of
Weak A or B Subgroup by
Adsorption and Elution
Principle

5.
6.

See Chapter 13 for a discussion of the
principles of testing for ABO groups.

Human anti-A and/or anti-B. Because some monoclonal ABO typing
reagents are sensitive to changes in
pH and osmolarity, they may not be
suitable for use in adsorption/elution
tests.
Eluting agent: See Methods Section
4.

Procedure

Interpretation
1.

735

Wash 1 mL of the red cells to be
tested at least three times with saline.
Remove and discard the supernatant
saline after the last wash.
Add 1 mL of reagent anti-A (if a weak
variant of A is suspected) or 1 mL of
anti-B (if a weak variant of B is suspected) to the washed cells.
Mix the red cells with the reagent
antibody and incubate them at 4 C
for 1 hour, mixing occasionally.
Centrifuge the mixture to pack the
red cells. Remove all supernatant reagent.
Transfer the red cells to a clean test
tube.
Wash the cells at least eight times
with large volumes (10 mL or more)
of cold (4 C) saline. Save an aliquot
of the final wash supernatant fluid
and test it in parallel with the eluate.
Use an elution method suitable for
recovery of ABO antibodies, eg, heat
or Lui freeze-thaw elution techniques can be used to remove antibody from the cells (see Methods
Section 4).
Centrifuge to pack the cells and transfer the supernatant eluate to a clean
test tube.

736

AABB Technical Manual

Test the eluate and the final wash
solution (from step 6), in parallel,
against two examples of group O
cells and two examples of cells expressing the relevant antigen (A 1
cells for suspected anti-A, B cells for
anti-B). Add 2 drops of eluate or
wash to 1 drop of cells and examine
them for agglutination after immediate centrifugation; if negative, incubate 15 to 30 minutes at room temperature. If these phases are both
negative, a 15-minute incubation at
37 C and the indirect antiglobulin
test may also be performed.

beginning the elution, if the cells
were not adequately washed, or if
there was dissociation of bound antibody during the wash process.
A and B cells can be used in the adsorption/elution procedure as positive/negative controls and tested in
parallel. Group O cells can also be
used as a negative control.

Note
The eluate may be stained by hemoglobin
and be difficult to read except at the indirect antiglobulin phase.

Reference
Interpretation
1.

The presence of anti-A or anti-B in
the eluate, hence the presence of A
or B antigen on the test cells, is confirmed if: a) the eluate reacts with
both antigen-positive cells, at any
phase; b) the eluate is nonreactive at
all phases with all group O cells; and
c) the final wash solution is nonreactive with all four cells.
If the eluate does not react with
the A or B cells, it may indicate that
the test cells do not express the antigen and cannot adsorb the relevant
antibody; alternatively, it could reflect failure to prepare the eluate
correctly.
If the eluate reacts with one or
both of the A or B cells and also with
one or both or all of the O cells, it indicates recovery of some other or
additional antibody in the adsorption/elution process.
If the final wash solution reacts with
the A or B cells, tests on the eluate
cannot be considered valid. This can
occur if unbound reagent antibody
was not adequately removed before

Beattie KM. Identifying the causes of weak or
“missing” antigens in ABO grouping tests. In: The
investigation of typing and compatibility problems caused by red blood cells. Washington, DC:
AABB, 1975:15-37.

Method 2.5. Saliva Testing
a
b
for A, B, H, Le , and Le
Principle
Approximately 78% of all individuals possess the Se gene that governs the secretion
of water-soluble ABH antigens into all
body fluids with the exception of cerebrospinal fluid. These secreted antigens
can be demonstrated in saliva by inhibition tests with ABH and Lewis antisera
(see Chapter 13).

Specimen
1.

Collect 5 to 10 mL of saliva in a small
beaker or wide-mouthed test tube.
Most people can accumulate this
amount in several minutes. To encourage salivation, the subject may
be asked to chew wax, paraffin, or a
clean rubber band, but not gum or

Methods Section 2: Red Cell Typing

2.
3.

anything else that contains sugar or
protein.
Centrifuge saliva at 900 to 1000 × g
for 8 to 10 minutes.
Transfer supernatant to a clean test
tube and place it in a boiling waterbath for 8 to 10 minutes to inactivate
salivary enzymes.
Recentrifuge at 900 to 1000 × g for 8
to 10 minutes, remove clear or slightly
opalescent supernatant fluid, and
discard the opaque or semisolid material. Dilute the supernatant fluid
with an equal volume of saline.
Refrigerate, if testing is to be done
within several hours. If testing will
not be done on the day of collection,
freeze the sample and store it at –20
C. Frozen samples retain activity for
several years.

737

or nonsecretors, to use as positive
and negative controls.

Procedures

Selection of Blood Grouping Reagent
Dilution
1.
2.

3.
4.

Prepare doubling dilutions of the appropriate blood typing reagent.
To 1 drop of each reagent dilution,
add 1 drop of 2% to 5% saline suspension of red cells. Use A, B, or O
cells to determine, respectively, A, B,
or H secretor status. Use Le(a+b–)
red cells to determine Lewis secretor
status.
Centrifuge each tube and examine
macroscopically for agglutination.
Select the highest reagent dilution
that gives 2+ agglutination.

Inhibition Test for Secretor Status
1.

Reagents
1.

4.
5.

Human (polyclonal) anti-A and anti-B.
Note: Some monoclonal reagents
may not be appropriate for use;
therefore, appropriate controls are
essential.
Anti-H lectin from Ulex europaeus
obtained commercially or prepared
by saline extraction of Ulex europaeus seeds.
Polyclonal (rabbit/goat/human)
a
anti-Le . There are no published data
on the suitability of monoclonal
Lewis antibodies.
A1 and B red cells, as used in Method
2.2.
Group O, Le(a+b–) red cells, as used
for antibody detection or identification (see Chapter 19).
Specimens, frozen or fresh saliva,
from persons known to be secretors

Add 1 drop of appropriately diluted
blood grouping reagent to each of
four tubes. For ABH studies, the
tubes should be labeled “Secretor,”
“Nonsecretor,” “Saline,” and “Unknown.” For Lewis studies, they will
be “Lewis-positive,” “Lewis-negative,” “Saline,” and “Unknown.”
Add 1 drop of the appropriate saliva
to the “Secretor,” “Nonsecretor,” and
“Unknown” tubes, and 1 drop of saline to the tube marked “Saline.”
Mix the contents of the tubes. Incubate the tubes for 8 to 10 minutes at
room temperature.
Add 1 drop of 2% to 5% saline suspension of washed indicator cells to
each tube, group A, B, or O for ABH
secretor status, as appropriate, or
Le(a+) for Lewis testing.
Mix the contents of the tubes. Incubate the tubes for 30 to 60 minutes
at room temperature.

738

AABB Technical Manual

Centrifuge each tube and inspect
each cell button macroscopically for
agglutination.

For further interpretation, see Table
2.5-1.

Notes
Interpretation
1.

Agglutination of indicator cells by
antibody in tubes containing saliva
indicates that the saliva does not
contain the corresponding antigen.
The failure of known antibody to agglutinate indicator cells after incubation with saliva indicates that the
saliva contains the corresponding
antigen.
The failure of antibody in the saline
control tube to agglutinate indicator
cells invalidates the results of saliva
tests; this usually reflects use of reagents that are too dilute. Redetermine the appropriate reagent dilution, as described above, and repeat
the testing.

Include, as controls, saliva from a
known secretor and nonsecretor. For
ABH status, use saliva from previously tested Se and sese persons. For
Lewis testing, use saliva from a person whose red cells are Le(a+b–) or
Le(a–b+) as the positive control; use
saliva from a Le(a–b–) person as the
negative control. Aliquots of saliva
from persons of known secretor status may be frozen for later use.
This screening procedure can be
adapted for the semiquantitation of
blood group activity by testing serial
saline dilutions of saliva. The higher
the dilution needed to remove inhibitory activity, the more blood group
substance is present in the saliva.

Table 2.5-1. Interpretation of Saliva Testing
Testing with Anti-H

Unknown
Saliva

Se Saliva
(H Substance
Present)

Non- Se Saliva
(H Substance Not
Saline
Present)
(Dilution Control)

Nonsecretor of H

Secretor of H

Testing With Anti-Le

Interpretation

Unknown
Saliva

Le-positive Saliva

Le-negative
Saliva

Saline
(Dilution Control)

Interpretation

Lewis-negative

Lewis-positive*

*A Lewis-positive person shown to be a secretor of ABH can be assumed to have Le as well as Le in saliva. A Le(a+)
a
person who is sese and does not secrete ABH substance will have only Le in saliva.

Methods Section 2: Red Cell Typing

Saliva should be diluted before it is
incubated with antibody. To detect
or to measure salivary A or B substance in addition to H substance,
the same procedure can be used
with diluted anti-A and anti-B reagents. The appropriate dilution of
anti-A or anti-B is obtained by titrating the reagent against A1 or B red
cells, respectively.
A Lewis-positive person shown to be
a secretor of A, B, and H can be assumed to have Leb as well as Lea in
the saliva. A Le(a+) person who does
not secrete A, B, or H substances
lacks the Se gene and will have only
Lea in the saliva.
Specimens with a high concentration of soluble antigen may give a
false-negative result and require dilution before testing.

Rh control reagent: The manufacturer’s instructions will indicate the
type of reagent to use, if needed.

Procedure
1.
2.

Method 2.6. Slide Test for
Determination of Rh Type
Principle
See Chapter 14 for a discussion of the
principles of Rh typing.
6.

Specimen

739

Place 1 drop of anti-D onto a clean,
labeled slide.
Place 1 drop of the appropriate control reagent, if needed, onto a second labeled slide.
To each slide, add 2 drops of a wellmixed 40% to 50% suspension (in autologous or group-compatible serum
or plasma) of the red cells to be tested.
Thoroughly mix the cell suspension
and reagent. Using a clean applicator stick for each test, spread (mix)
the reaction mixture over an area approximately 20 mm × 40 mm.
Place both slides on the viewbox and
tilt the slides gently and continuously to observe them for agglutination (see note 1). Most manufacturers stipulate that the test must be
read within 2 minutes because drying of the reaction mixture may
cause the formation of rouleaux,
which may be mistaken for agglutination.
Interpret and record the results of
the reactions on both slides.

Refer to Method 2.2.

Interpretation
Equipment

View box.

Reagents
1.

Reagent anti-D: Suitable reagents include polyclonal high-protein or lowprotein (eg, monoclonal) reagents.
Follow the instructions from the
manufacturer of the anti-D in use
before performing slide tests; the
method presented here is a representative procedure.

Agglutination with anti-D and a
smooth suspension on the control
slide constitute a positive test result
and indicate that the cells being
tested are D+.
No agglutination with either anti-D
or the Rh control suggests that the
cells are D–. Testing by the antiglobulin procedure (see Method 2.9)
will show weak expression of D on
cells that are not agglutinated on
slide testing.

740

AABB Technical Manual

If there is agglutination on the control slide, results of the anti-D test
must not be interpreted as positive
without further testing.
Drying around the edges of the reaction mixture must not be confused
with agglutination.

Procedure
1.
2.

Notes
1.

Slide testing imposes a much greater
risk of biohazardous exposure. Personnel should follow safety measures detailed in the facility’s procedures manual.
For slide tests using low-protein
anti-D, a negative result on slide
testing with either anti-A or anti-B
serves as the control reaction.

Method 2.7. Tube Test for
Determination of Rh Type

Place 1 drop of anti-D in a clean, labeled test tube.
Place 1 drop of the appropriate control reagent, if needed, in a second
labeled tube.
Add to each tube 1 drop of a 2% to
5% suspension (in saline, serum or
plasma) of the red cells to be tested;
alternatively, the equivalent amount
of red cells can be transferred to each
tube with clean applicator sticks.
Mix gently and centrifuge for the
time and at the speed specified by
the manufacturer.
Gently resuspend the cell button and
examine it for agglutination. If a
stick was used to transfer the red
cells, adding 1 drop of saline to each
tube will make it easier to resuspend
the cell button.
Grade reactions and record test and
control results.

Principle
See Chapter 14 for a discussion of the
principles of Rh typing.

Interpretation
1.

Specimen
Refer to Method 2.2.
2.

Reagents
1.

Reagent anti-D: Suitable reagents include polyclonal high-protein or lowprotein (eg, monoclonal) reagents.
Follow the instructions from the
manufacturer of the anti-D in use
before performing tube tests. The
method presented here is a representative procedure.
Rh control reagent: The manufacturer’s instructions will indicate the
type of control to use, if needed.

Agglutination in the anti-D tube,
combined with a smooth suspension
in the control tube, indicates that
the red cells under investigation are
D+.
A smooth suspension of red cells in
both the anti-D and the control
tubes is a negative test result. Specimens from patients may be designated as D– at this point. Donor
blood must be further tested for the
presence of weak D antigen. The serum-and-cell mixture used in steps 1
through 5, above, may be used to test
for weak D, providing the manufacturer’s directions state that the reagent is suitable for the test for weak
D.

Methods Section 2: Red Cell Typing

Notes
1.

Most commercially prepared antisera
provide a 2+ or greater agglutination
with D+ cells. A facility may choose
to do additional testing on results
with an agglutination of less than 2+.
Required testing must be defined in
the facility’s procedures manual.
A negative tube test with anti-A and/
or anti-B serves as a valid control
when a low-protein anti-D reagent
has been used.

3.
4.

6.
7.

Method 2.8. Microplate Test
for Determination of Rh Type

Principle

See Chapter 14 for a discussion of the
principles of Rh typing

741

Add 1 drop of a 2% to 5% saline suspension of red cells to each well.
Mix the contents of the wells by
gently tapping the sides of the plate.
Centrifuge the plate at the appropriate conditions established for the
centrifuge.
Resuspend the cell buttons by manually tapping the plate or with the
aid of a mechanical shaker, or place
the plate at an angle for the tilt and
stream method.
Read, interpret, and record the results.
Incubate negative tests at 37 C for 15
minutes.
Centrifuge the plate at the appropriate conditions established for the
centrifuge.
Resuspend the cell buttons by manually tapping the plate or with the
aid of a mechanical shaker, or place
the plate at an angle for the tilt and
stream method.
Read, interpret, and record the results.

Specimen

10.

Refer to Method 2.2. Clotted or anticoagulated samples may be used for Rh testing.
Follow the manufacturer’s instructions for
specimen preparation when using semiautomated microplate readers.

Interpretation

Use only anti-D approved for use in microplate tests (see the discussion in Method
2.3).

Agglutination with anti-D reagent after
the immediate-spin or 37 C incubation
phase indicates a positive test provided
there is no agglutination with the control
reagent. See Table 14-3 for determining
Rh phenotypes from reactions obtained
with Rh blood typing reagents.

Procedure

Note

The following is a representative method;
the manufacturer’s instructions should be
followed for specific reagents and equipment.
1. Place 1 drop of the Rh reagent in a
clean well of the microplate. If the
reagent requires the use of an Rh
control, add 1 drop of the control to
a second well.

Refer to the manufacturer’s instructions
for the necessity for weak D testing.

Reagents

Method 2.9. Test for Weak D
Principle
Some red cells express the D antigen so
weakly that most anti-D reagents do not

742

AABB Technical Manual

directly agglutinate the cells. Weak D expression can be recognized most reliably
by an indirect antiglobulin procedure after incubation of the test red cells with
anti-D.

Specimen
Refer to Method 2.2.

Reagents
1.

2.
3.

Reagent anti-D: Suitable reagents include polyclonal high-protein or lowprotein (eg, monoclonal blend) reagents, but the manufacturer’s package insert should be consulted before any anti-D reagent is used for
this purpose.
Antihuman globulin reagent, either
anti-IgG or polyspecific.
IgG-coated red cells.

8.
9.

Procedure

10.

If the original, direct test with anti-D was
performed by tube testing, the same tube
may be used for the weak D test, providing the manufacturer’s directions so state.
In this case, proceed directly to step 4, after recording the original anti-D tube test
as negative.
1. Place 1 drop of anti-D in a clean, labeled test tube.
2. Place 1 drop of the appropriate control reagent in a second labeled test
tube.
3. To each tube, add 1 drop of a 2% to
5% suspension in saline of the red
cells to be tested. It is permissible to
use a direct antiglobulin test (DAT)
on the test cells as a control, but an
indirect antiglobulin procedure with
an Rh control reagent is preferable
because this ensures that all reagent
components that might cause a
false-positive result are represented.

11.

Mix and incubate both tubes according to the reagent manufacturer’s directions. Typically, this is 15 to 30
minutes at 37 C.
If a reading is desired after the 37 C
incubation phase, centrifuge the tubes
according to the reagent manufacturer’s directions.
Gently resuspend the cell buttons
and examine the tubes for agglutination. If the test red cells are strongly
agglutinated in the anti-D tube but
not in the control tube, record the test
sample as D+ and do not proceed with
the antiglobulin phase of the test.
If the test cells are not agglutinated
or the results are doubtful, wash the
cells three or four times with large
volumes of saline.
Add antiglobulin reagent, according
to the manufacturer’s directions.
Mix gently and centrifuge according
to the calibrated spin times.
Gently resuspend each cell button,
examine the tubes for agglutination,
and grade and record the test result.
If the test result is negative, add IgGcoated red cells.

Interpretation
1.

Either a diluent control or a direct
antiglobulin test (DAT) must accompany the test for weak D. Agglutination in the anti-D tube and none in
the control tube constitutes a positive test result. If the facility chooses
to perform the test for weak D, and
the result is clearly positive, the
blood should be classified as D+. It is
incorrect to report such red cells as
u ”
being “D–, weak D” or “D–, D .
Absence of agglutination in the tube
with anti-D is a negative result, indicating that the cells do not express D
and should be classified as D–.

Methods Section 2: Red Cell Typing

If there is agglutination at any phase
in the control tube, no valid interpretation of the weak D test can be
made. If the specimen is from a potential transfusion recipient, Rhnegative blood should be given until
the D type can be resolved. If the
specimen is from a donor, the unit
should not be used for transfusion.

polyagglutinable red cells are shown in
Table 2.10-1. If commercially made lectins
are used, follow the manufacturer’s instructions.

Reagents
Seeds may be obtained from health-food
stores, pharmacies, or commercial seed
companies. The seeds should be raw.

Note

Procedure

Some facilities may elect to do an additional reading after the 37 C incubation
and before completing the antiglobulin
phase of testing. Refer to the manufacturer’s instructions. If this optional reading is performed, the facility’s procedures
manual should indicate its policy on the
interpretation of this result and on the additional testing requirements.

Method 2.10. Preparation
and Use of Lectins
Principle
Saline extracts of seeds react with specific
carbohydrates on red cell membranes and
make useful typing reagents that are
highly specific at appropriate dilutions.
Diluted extract of Dolichos biflorus agglutinates A 1 red cells but not A 2 . Ulex
europaeus extract reacts with the H determinant; it agglutinates in a manner proportional to the amount of H present
(O>A2>B>A1>A1B red cells). Other lectins
useful for special purposes include
Arachis hypogaea (anti-T), Glycine max
(anti-T, -Tn), Vicia graminea (anti-N), and
the Salvia lectins (S. horminum, antiTn/Cad; S. sclarea, anti-Tn). To investigate red cell polyagglutination, prepare
and test the cells with Arachis, Glycine,
Salvia, and Dolichos lectins. The anticipated reactions with various types of

743

Grind the seeds in a food processor
or blender until the particles look
like coarse sand. A mortar and pestle
may be used, or seeds can be used
whole.
In a large test tube or small beaker,
place ground seeds and three to four
times their volume of saline. (Seeds
vary in the quantity of saline they
absorb.)
Incubate at room temperature for 4
to 12 hours, stirring or inverting occasionally.
Transfer supernatant fluid to a centrifuge tube and centrifuge it for 5
minutes, to obtain clear superna-

Table 2.10-1. Reactions Between Lectins
and Polyagglutinable Red Cells

Arachis hypogaea*
Dolichos biflorus†
Glycine max (soja)
Salvia sclarea
Salvia horminum

Tn Cad

+
0
+
0
0

+
0
0
0
0

0
+
+
+
+

0
+
+
0
+

*T and Th cells give weaker reactions with Arachis after
protease treatment; Tk reactivity is enhanced after protease treatment.
†
A and AB cells may react due to anti-A reactivity of Dolichos lectin.

744

AABB Technical Manual

tant. Collect and filter the supernatant fluid and discard seed residue.
5.
Test dilutions of the extract to find
dilution for the desired activity. Determine the activity of the extract
with the appropriate red cells, as below.
For Dolichos biflorus:
a.
Add 1 drop of 2% to 5% saline
suspension of known A 1 , A 2 ,
A1B, A2B, B, and O red cells to
appropriately labeled tubes.
b.
Add 1 drop of the extract to each
tube.
c.
Centrifuge for calibrated time.
d.
Inspect for agglutination and
record results.
e.
The lectin should agglutinate
A1 and A1B cells but not A2, A2B,
B, or O cells. The native extract
often agglutinates all the cells
tested. To make the product
useful for reagent purposes, add
enough saline to the extract so
that there is 3+ or 4+ agglutination of A1 and A1B cells, but not
of A2, A2B, B, or O cells.
For Ulex europaeus:
a.
Add 1 drop of 2% to 5% saline
suspension of known A 1 , A 2 ,
A1B, B, and O cells to appropriately labeled tubes.
b.
Add 1 drop of extract to each tube.
c.
Centrifuge for the calibrated
time.
d.
Inspect for agglutination and
record results.
e.
The strength of the agglutination should be in the order of
O>A2>B>A1>A1B.
f.
Dilute extract with saline, if
necessary, to a point that O
cells show 3+ or 4+ agglutination, A2 and B cells show 1+ to
2+ agglutination, and A1 or A1B
cells are not agglutinated.

Notes
1.

To facilitate grinding hard seeds, the
seeds can be covered with saline and
soaked for several hours before
grinding. The container used for
soaking should not be tightly closed
because some beans release gas during the soaking process, which could
cause the container to explode.
The saline extracts may be stored in
the refrigerator for several days; they
may be stored indefinitely if frozen.
Tests should include a positive and
negative control.

Method 2.11. Use of
Sulfhydryl Reagents to
Disperse Autoagglutination
Principle
See Chapter 20 for a discussion of autoagglutination dispersion.

Specimen
Immunoglobulin-coated red cells to be
evaluated.

Reagents
1.

0.01 M dithiothreitol (DTT): 0.154 g
of DTT dissolved in 100 mL of phosphate-buffered saline (PBS) at pH
7.3, or 0.1 M 2-mercaptoethanol
(2-ME), 0.7 mL of stock solution of
14 M 2-ME diluted in 100 mL of PBS
at pH 7.3.
PBS at pH 7.3.

Procedure
1.
2.

Dilute red cells to a 50% concentration in PBS.
Add an equal quantity of 0.01 M DTT
in PBS, or 0.1 M 2-ME in PBS, to the
cells.

Methods Section 2: Red Cell Typing

3.
4.
5.

Incubate at 37 C for 10 minutes (2-ME)
or 15 minutes (DTT).
Wash cells three times in saline and
resuspend them.
Dilute the treated red cells to a 2% to
5% concentration in saline for use in
blood grouping tests. Verify that red
cells do not spontaneously agglutinate before typing or use.

Specimen
Test cells with a positive direct antiglobulin test (DAT) result.

Procedure
1.

Note
This procedure is normally used only for
ABO forward cell typing, Rh testing, and
the direct antiglobulin test. At this concentration of DTT, some antigens, in particular Jsa and Jsb, may be weakened or destroyed by 0.01M DTT.

Reference
Reid ME. Autoagglutination dispersal utilizing
sulfhydryl compounds. Transfusion 1978;18:353-5.

Method 2.12. Gentle Heat
Elution for Testing Red Cells
with a Positive DAT

3.
4.

Principle
Red cells heavily coated with IgG may
spontaneously agglutinate in high-protein reagents and will cause false-positive
AHG test results. To perform red cell antigen typing, it may be necessary to dissociate antibody from the cells by elution
without damaging membrane integrity or
altering antigen expression. The gentle
heat elution procedure employed to prepare immunoglobulin-free red cells differs from procedures intended to recover
active antibody.

Reagent
Antihuman globulin.

745

Place one volume of washed antibody-coated red cells and three volumes of normal saline in a test tube
of appropriate size. In another tube,
place the same volumes of saline
and washed red cells positive for the
antigen under test. This will provide
a check that the elution technique
does not destroy the antigen reactivity.
Incubate the contents of both tubes
at approximately 45 C for 10 to 15
minutes. The tubes should be agitated frequently. The time of incubation should be roughly proportional
to the degree of antibody coating, as
indicated by strength of antiglobulin
reactivity.
Centrifuge the tubes and discard the
supernatant saline.
Test the person’s cells for degree of
antibody removal by comparing a
DAT on the treated cells with the
antiglobulin results on untreated red
cells. If the antibody coating is reduced but still present, steps 1
through 3 can be repeated; the control cells should be subjected to a
similar second treatment.
Test the treated cells for the desired
antigen.

Notes
1.

This procedure may be unnecessary
if IgM monoclonal reagents are
available; these reagents cause direct agglutination and are not usually affected by bound immunoglobulin.

746

AABB Technical Manual

As with untreated patient cells, results of antigen testing in recently
transfused patients should be interpreted with caution because of the
potential presence of donor cells.

Method 2.13. Dissociation of
IgG by Chloroquine for Red
Cell Antigen Testing of Red
Cells with a Positive DAT

2.
3.

4.
5.

Principle

phate solution. Similarly treat the
control sample.
Mix and incubate at room temperature for 30 minutes.
Remove a small aliquot (eg, 1 drop)
of the treated test cells and wash
them four times with saline.
Test the washed cells with anti-IgG.
If this treatment has rendered the
cells nonreactive with anti-IgG, wash
the total volumes of treated test cells
and control cells three times in saline and make a 2% to 5% suspension in saline to use in subsequent
blood typing tests.
If the treated red cells react with
anti-IgG after 30 minutes of incubation with chloroquine diphosphate,
steps 3 and 4 should be repeated at
30-minute intervals (for a maximum
incubation period of 2 hours), until
the sample tested is nonreactive
with anti-IgG. Then proceed as described in step 5.

Red cells with a positive direct antiglobulin
test (DAT) cannot be tested accurately
with blood typing reagents that require an
indirect antiglobulin technique. Under
controlled conditions, chloroquine diphosphate dissociates IgG from the red cell
membrane with little or no damage to its
integrity. Use of this procedure permits
complete phenotyping of red cells coated
with warm-reactive autoantibody, including tests with reagents solely reactive by
indirect antiglobulin techniques.

Notes

Specimen

Red cells with a positive DAT due to IgG
coating.

Reagents
1.

Chloroquine diphosphate solution
prepared by dissolving 20 g of chloroquine diphosphate in 100 mL of saline. Adjust to pH 5.1 with 1 N NaOH,
and store at 2 to 6 C.
Control red cells carrying a single-dose
expression of antigens for which the
test samples are to be phenotyped.
Anti-IgG antiglobulin reagent.

3.
4.

Procedure
1.

To 0.2 mL of washed IgG-coated cells,
add 0.8 mL of chloroquine diphos-

Chloroquine diphosphate does not
dissociate complement proteins
from the cell membrane. If red cells
are coated with both IgG and C3,
only anti-IgG should be used in tests
performed after chloroquine treatment.
Incubation with chloroquine diphosphate should not be extended beyond 2 hours. Prolonged incubation
at room temperature or incubation
at 37 C may cause hemolysis and
loss of red cell antigens.
Some denaturation of Rh antigens
may occur.
Many serologists run chloroquinetreated control cells for each antigen
tested. Select control cells that are
positive for the antigen correspond-

Methods Section 2: Red Cell Typing

ing to the antisera that will be used
to type the patient’s cells.
Chloroquine diphosphate may not
completely remove antibody from
sensitized red cells. DAT results on
red cells from some persons, particularly those with a strongly positive
initial test, may only be diminished
in strength.
In addition to its use for removal of
autoantibodies, this method can be
used for removal of Bg (HLA)-related
antigens from red cells. Appropriate
Bg controls should be used.
If a commercial kit is used, manufacturer’s instructions should be followed
for testing and controls.

Specimen
Red cells with a positive direct antiglobulin
test (DAT).

Reagents
1.

References
1.

Edwards JM, Moulds JJ, Judd WJ. Chloroquine diphosphate dissociation of antigenantibody complexes: A new technique for
phenotyping rbcs with a positive direct
antiglobulin test. Transfusion 1982;22:59-61.
Swanson JL, Sastamoinen R. Chloroquine
stripping of the HLA-A,B antigens from red
cells (letter). Transfusion 1985;25:439-40.

1.
2.

4.
5.
6.

Principle
Acid glycine/EDTA can be used to dissociate antibody molecules from red cell
membranes. This procedure is routinely
used for blood typing tests or adsorption
procedures. All common red cell antigens
can be detected after treatment with acid
glycine/EDTA except antigens of the Kell
system, Bg antigens, and Er antigens.
Thus, cells treated in this manner cannot
be used to determine these phenotypes.

10% EDTA prepared by dissolving 2 g
of disodium ethylenediamine tetraacetic acid (Na2EDTA) in 20 mL of
distilled or deionized water.
0.1 M glycine-HCl buffer (pH 1.5)
prepared by diluting 0.75 g of glycine
to 100 mL with isotonic (unbuffered)
saline. Adjust the pH to 1.5 using
concentrated HCl.
1.0 M TRIS-NaCl prepared by dissolving 12.1 g of tris (hydroxymethyl)
aminomethane (TRIS) and 5.25 g of
sodium chloride (NaCl) to 100 mL
with distilled or deionized water.

Procedure

Method 2.14. Acid Glycine/
EDTA Method to Remove
Antibodies from Red Cells

747

7.
8.

9.
10.

Wash the red cells to be treated six
times with isotonic saline.
In a test tube, mix together 20 volumes of 0.1 M acid glycine-HCl (pH
1.5) with five volumes of 10% EDTA.
This is the acid glycine/EDTA reagent.
Place 10 volumes of washed red cells
in a clean tube.
Add 20 volumes of acid glycine/EDTA.
Mix the contents of the tube thoroughly.
Incubate the mixture at room temperature for no more than 2 to 3
minutes.
Add one volume of 1.0 M TRIS-NaCl
and mix the contents of the tube.
Centrifuge at 900 to 1000 × g for 1 to
2 minutes, then aspirate, and discard the supernatant fluid.
Wash the red cells four times with
saline.
Test the washed cells with anti-IgG;
if nonreactive with anti-IgG, the cells

748

AABB Technical Manual

are ready for use in blood typing or
adsorption procedures. If the DAT is
still positive, one additional treatment
can be performed.

Notes
1.

3.
4.

Overincubation of red cells with acid
glycine/EDTA causes irreversible
damage to cell membranes.
Include a parallel control reagent,
such as 6% bovine albumin or inert
plasma, when typing treated red cells.
Use anti-IgG, not a polyspecific antiglobulin reagent, in step 10.
Many serologists run acid glycine/
EDTA treated control cells for each
antigen tested. Select control cells that
are positive for the antigen corresponding to the antisera that will be
used to type the patient’s cells.
If a commercial kit is used, manufacturer’s instructions should be followed for testing and controls.

References
1.

transfused red cells and may be separated
from the transfused population by simple
centrifugation. Newly formed autologous
cells concentrate at the top of the column
of red cells when blood is centrifuged in a
microhematocrit tube, providing a simple
method for recovering autologous cells in
a blood sample from recently transfused
patients. Note: Red cells from patients
with hemoglobin S or spherocytic disorders are not effectively separated by this
method (see Method 2.16 for an alternative procedure).

Specimen
Red cells from whole blood collected into
EDTA.

Materials
1.
2.
3.

Louie JE, Jiang AF, Zaroulis CG. Preparation of
intact antibody-free red cells in autoimmune
hemolytic anemia (abstract). Transfusion
1986;26:550.
Champagne K, Spruell P, Chen J, et al. EDTA/
glycine-acid vs. chloroquine diphosphate
treatment for stripping Bg antigens from red
blood cells (abstract). Transfusion 1996;36
(Suppl):21S.
Reid ME, Lomas-Francis C. The blood group
antigen factsbook. New York: Academic Press,
2004.

Procedure
1.

Method 2.15. Separation of
Transfused from Autologous
Red Cells by Simple
Centrifugation

Principle

Newly formed autologous red cells generally have a lower specific gravity than

Microhematocrit centrifuge.
Plain (not heparinized) glass or plastic hematocrit tubes.
Sealant.

Wash the red cells three times in saline. For the last wash, centrifuge
them at 900 to 1000 g for 5 to 15
minutes. Remove as much of the
supernatant fluid as possible without disturbing the buffy coat. Mix
thoroughly.
Fill 10 microhematocrit tubes to the
60-mm mark with well-mixed washed
red cells.
Seal the ends of the tubes by heat or
with sealant.
Centrifuge all tubes in a microhematocrit centrifuge for 15 minutes.
Cut the microhematocrit tubes 5
mm below the top of the column of
red cells. This 5-mm segment con-

Methods Section 2: Red Cell Typing

tains the least dense, hence youngest, circulating red cells.
Place the cut microhematocrit tubes
into larger test tubes (10 or 12 × 75
mm), add saline, and mix well to
flush the red cells from the microhematocrit tubes. Then, either a)
centrifuge them at 1000 × g for 1
minute and remove the empty hematocrit tubes or b) transfer the salinesuspended red cells to a clean test
tube.
Wash the separated red cells three
times in saline before resuspending
them to 2% to 5% in saline for testing.

Notes
1.

Separation is better if 3 or more days
have elapsed since transfusion than
if the sample has been obtained
shortly after transfusion.
The red cells should be mixed continuously while the microhematocrit
tubes are being filled.
Separation techniques are only effective if the patient is producing
normal or above-normal numbers of
reticulocytes. This method will be
ineffective in patients with inadequate reticulocyte production.
Some red cell antigens may not be as
strongly expressed on reticulocytes
as on older cells. Particular attention
should be given to determinations of
the E, e, c, Fya, Jka, and Ge antigens.

References
1.

Method 2.16. Separation of
Transfused Red Cells from
Autologous Red Cells in
Patients with Hemoglobin S
Disease
Principle
Red cells from patients with sickle cell
disease, either hemoglobin SS or SC, are
resistant to lysis by hypotonic saline, in
contrast to red cells from normal persons
and those with hemoglobin S trait. This
procedure permits isolation of autologous
red cells from patients with hemoglobin
SS or SC disease who have recently been
transfused.

Specimen
Red cells to be evaluated.

Reagents
1.
2.

Hypotonic saline (0.3% w/v NaCl):
NaCl, 3 g; distilled water to 1 L.
Normal saline (0.9% w/v NaCl):
NaCl, 9 g; distilled water to 1 L.

Procedure
1.
2.

Reid ME, Toy P. Simplified method for recovery of autologous red blood cells from transfused patients. Am J Clin Pathol 1983;79:364-6.
Vengelen-Tyler V, Gonzales B. Reticulocyte
rich RBCs will give weak reactions with many
blood typing antisera (abstract). Transfusion
1985;25:476.

749

Place 4 or 5 drops of red cells into a
10 or 12 × 75-mm test tube.
Wash the cells six times with 0.3%
NaCl, or until the supernatant fluid
no longer contains grossly visible
hemoglobin. For each wash, centrifuge at 1000 × g for 1 minute.
Wash the cells twice with 0.9% NaCl
to restore tonicity. For each wash,
centrifuge at 200 × g for 2 minutes to
facilitate removal of residual stroma.
Resuspend the remaining intact red
cells to a 2% to 5% concentration for
phenotyping.

750

AABB Technical Manual

Note

Reference

Larger volumes, for use in adsorption studies, can be processed in a 16 × 100-mm
test tube.

Brown D. A rapid method for harvesting autologous red cells from patients with hemoglobin S
disease. Transfusion 1988;28:21-3.

Methods Section 3: Antibody Detection/Identification and Compatibility Testing

Antibody Detection,
Antibody Identification, and
Serologic Compatibility
Testing

the donor red cells in EDTA saline because high-titered anti-A or -B can
initiate complement coating, which can
cause steric hindrance of agglutination.2 The use of a patient’s sample
collected in EDTA is an alternative
approach to prevent this phenomenon.

Method 3.1. Immediate-Spin
Compatibility Testing to
Demonstrate ABO
Incompatibility
Principle
See Chapter 18 for a discussion of the principles of compatibility testing.

Specimen

Procedure

Patient’s serum or plasma may be used.
The age of the specimen must comply with
the pretransfusion specimen requirements
in AABB Standards for Blood Banks and
Transfusion Services.1(p38)

Reagents
1.
2.

Normal saline.
Donor red cells, 2% to 5% suspension in normal saline or EDTA saline.
Some serologists prefer to suspend

Label a tube for each donor red cell
suspension being tested with the patient’s serum.
Add 2 drops of the patient’s serum or
plasma to each tube.
Add 1 drop of the suspension of donor red cells to the appropriate test
tube.
Mix the contents of the tube(s) and
centrifuge according to the calibration of the centrifuge.
751

Section 3

Methods Section 3

752

AABB Technical Manual

Examine the tube(s) for hemolysis,
gently resuspend the red cell button(s),
and examine for agglutination.
Read, interpret, and record test results.

2.
3.

Interpretation
1.
2.

Agglutination or hemolysis constitutes
a positive (incompatible) test result.
A smooth suspension of red cells after resuspension of the red cell button constitutes a negative result and
indicates a compatible immediatespin crossmatch.

References
1.

Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.
Judd WJ, Steiner EA, O’Donnell DB, Oberman
HA. Discrepancies in ABO typing due to
prozone; how safe is the immediate-spin
crossmatch? Transfusion 1988;28:334-8.

Method 3.2. Indirect
Antiglobulin Test (IAT) for
the Detection of Antibodies
to Red Cell Antigens

Principle
For a discussion of the principles of saline, albumin, low-ionic-strength saline
(LISS), and polyethylene glycol (PEG) indirect antiglobulin testing, see Chapters 12,
18, and 19.

Bovine albumin (22% or 30%).
LISS made as follows:
a.
Add 1.75 g of NaCl and 18 g of
glycine to a 1-liter volumetric flask.
b.
Add 20 mL of phosphate buffer
prepared by combining 11.3 mL
of 0.15 M KH2PO4 and 8.7 mL of
0.15 M Na2HPO4.
c.
Add distilled water to the 1-liter
mark.
d.
Adjust the pH to 6.7 ± 0.1 with
NaOH.
e.
Add 0.5 g of sodium azide as a
preservative.
Note: LISS may be used as an additive (Method 3.2.2) or for the suspension of test red cells (Method 3.2.3).
LISS preparations are also available
commercially.
PEG, 20% w/v: To 20 g of 3350 MW
PEG, add phosphate-buffered saline
(PBS) pH 7.3 (see Method 1.7) to 100 mL.
PEG is also available commercially.
Antihuman globulin (AHG) reagent.
Polyspecific or anti-IgG may be used
unless otherwise indicated.
Commercially available group O antibody detection cells. Pooled group
O antibody detection cells may be
used only for donor testing. Testing
of patients’ samples must be performed with unpooled cells.
IgG-coated red cells.

Method 3.2.1. Saline Indirect Antiglobulin
Test

Specimen

Procedure

Serum or plasma may be used. The age of
the specimen must comply with pretransfusion specimen requirements in AABB
Standards for Blood Banks and Transfusion
Services.

Reagents
1.

Normal saline.

Add 2 drops of serum or plasma to
properly labeled tubes.
Add 1 drop of 2% to 5% saline-suspended reagent group O cells or donor red cells to each tube and mix.
Centrifuge and observe for hemolysis and agglutination. Grade and record the results.

Methods Section 3: Antibody Detection/Identification and Compatibility Testing

4.
5.

8.
9.

Incubate at 37 C for 30 to 60 minutes.
Centrifuge and observe for hemolysis and agglutination. Grade and record the results.
Wash the cells three or four times with
saline and completely decant the final wash.
Add AHG to the dry cell button according to the manufacturer’s directions. Mix well.
Centrifuge and observe for agglutination. Grade and record the results.
Confirm the validity of negative tests
by adding IgG-coated red cells.

Method 3.2.2. Albumin or LISS-Additive
Indirect Antiglobulin Test

3.
4.

Add 2 drops of serum or plasma to
properly labeled tubes.
Add an equivalent volume of 22% or
30% bovine albumin or LISS additive
(unless the manufacturer’s directions
state otherwise.)
Add 1 drop of a 2% to 5% saline-suspended reagent or donor red cells to
each tube and mix.
For albumin, incubate at 37 C for 15
to 30 minutes. For LISS, incubate for
10 to 15 minutes or follow the manufacturer’s directions.
Centrifuge and observe for hemolysis and agglutination. Grade and record the results.
Perform the test described in Method
3.2.1, steps 6 through 9.

Procedure
1.

2.
3.
4.
5.

Method 3.2.3. LISS Indirect Antiglobulin Test

Wash reagent or donor red cells three
times in normal saline and completely decant saline.
Resuspend the cells to a 2% to 3%
suspension in LISS.

For each cell sample to be tested, mix
2 drops of test serum, 4 drops of 20%
PEG in PBS, and 1 drop of a 2% to
5% suspension of red cells.
Incubate at 37 C for 15 minutes.
DO NOT CENTRIFUGE.
Wash the cells four times with saline
and completely decant the final wash.
Perform the AHG test, using anti-IgG,
described in Method 3.2.1, steps 7
through 9.
Note: The manufacturer’s instructions
should be followed for the proper use
of commercial PEG solutions.

Interpretation (for Antiglobulin Tests,
Methods 3.2.1 through 3.2.4)

Procedure

Add 2 drops of serum to a properly
labeled tube.
Add 2 drops of LISS-suspended red
cells, mix, and incubate at 37 C 10 to
15 minutes or follow the manufacturer’s directions.
Centrifuge and observe for hemolysis and agglutination by gently resuspending the cell button. Grade and
record results.
Perform the test described in Method
3.2.1, steps 6 through 9.

Method 3.2.4. PEG Indirect Antiglobulin
Test

Procedure
1.

753

The presence of agglutination/hemolysis after incubation at 37 C constitutes a positive test.
The presence of agglutination after
addition of AHG constitutes a positive test.
Antiglobulin tests are negative when
no agglutination is observed after
initial centrifugation and the IgGcoated red cells added afterward are
agglutinated. If the IgG-coated red
cells are not agglutinated, the nega-

754

AABB Technical Manual

tive result is invalid and the test must
be repeated.

Method 3.3. Prewarming
Technique

Controls
The procedure used for the detection of
unexpected antibodies in pretransfusion
testing should be checked daily with weak
examples of antibody. Control sera can be
prepared from reagent grade typing sera
diluted with 6% bovine albumin to give 2+
reactions by an IAT. Human sources of IgG
antibodies are also acceptable.

Notes
1.

The incubation times and the volume
and concentration of red cells indicated are those given in the literature.
Individual laboratories may choose to
standardize techniques with somewhat
different values. See Chapter 12 for
other limitations when modifying
procedures. In all cases, the manufacturer’s package insert should be
consulted before modifying a procedure.
For the saline procedure, step 3 may
be omitted to avoid the detection of
antibodies reactive at room temperature.
For the PEG procedure:
a.
Omit centrifugation after 37 C
incubation because red cells will
not resuspend readily.
b.
Use anti-IgG rather than polyspecific AHG to avoid unwanted
positive reactions due to C3binding autoantibodies.
Steps 6 through 9 of the IAT (Method
3.2.1) must be performed without interruption.

Principle
Prewarming may be useful in the detection and identification of red cell antibodies that bind to antigen only at 37 C. This
test is particularly useful for testing sera
of patients with cold-reactive autoantibody
activity that may mask the presence of
clinically significant antibodies. However,
use of the prewarming technique for this
application has become controversial.1-2 It
has been shown to result in decreased
reactivity of some potentially significant
antibodies and weak antibodies can be
missed.3 The technique should be used
with caution and not used to eliminate
unidentified reactivity.
Strong cold-reactive autoantibodies may
react in prewarmed tests; other techniques
such as cold allo- or autoadsorption or
dithiothreitol treatment of plasma may be
required to detect underlying clinically significant antibodies.

Specimen
Serum or plasma may be used. The age of
the specimen must comply with pretransfusion specimen requirements in AABB
Standards for Blood Banks and Transfusion
Services.4(p38)

Reagents
1.
2.
3.

Reference
Silva MA, ed. Standards for blood banks and transfusion services. 23rd ed. Bethesda, MD: AABB, 2005:
38.

Normal saline.
Anti-IgG.
Commercially available group O antibody detection cells. Pooled group
O antibody detection cells may be used
only for donor testing. Testing of patients’ samples must be done with
unpooled cells.
IgG-coated red cells.

Methods Section 3: Antibody Detection/Identification and Compatibility Testing

saline instead of 37 C saline is used
in the wash step.2 The use of roomtemperature saline may avoid the
elution of clinically significant antibody(ies) from reagent red cells that
can occur with the use of 37 C saline.
Some strong cold-reactive autoantibodies, however, may still react and
therefore require the use of 37 C saline to avoid their detection.

Procedure
1.
2.
3.
4.

6.
7.

8.
9.
10.

Prewarm a bottle of saline to 37 C.
Label one tube for each reagent or
donor sample to be tested.
Add 1 drop of 2% to 5% saline-suspended red cells to each tube.
Place the tubes containing red cells
and a tube containing a small volume
of the patient’s serum and a pipette
at 37 C; incubate for 5 to 10 minutes.
Using the prewarmed pipette, transfer 2 drops of prewarmed serum to
each tube containing prewarmed red
cells. Mix without removing tubes
from the incubator.
Incubate at 37 C for 30 to 60 minutes.
Without removing the tubes from the
incubator, fill each tube with prewarmed (37 C) saline. Centrifuge
and wash three or four times with 37
C saline.
Add anti-IgG, according to the manufacturer’s directions.
Centrifuge and observe for reaction.
Grade and record the results.
Confirm the validity of negative tests
by adding IgG-coated red cells.

755

References
1.

Judd WJ. Controversies in transfusion medicine. Prewarmed tests: Con. Transfusion 1995;
35:271-7.
Mallory D. Controversies in transfusion medicine. Prewarmed tests: Pro—why, when, and
how—not if. Transfusion 1995;35:268-70.
Leger RM, Garratty G. Weakening or loss of
antibody reactivity after prewarm technique.
Transfusion 2003;43:1611-14.
Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.

Method 3.4. Saline
Replacement to Demonstrate
Alloantibody in the Presence
of Rouleaux

Notes

Principle

Rouleaux are aggregates of red cells that,
characteristically, adhere to one another
on their flat surface, giving a “stack of coins”
appearance when viewed microscopically.
Rouleaux formation is an in-vitro phenomenon resulting from abnormalities of
serum protein concentrations. The patient is often found to have liver disease,
multiple myeloma, or another condition
associated with abnormal globulin levels.
It may be difficult to detect antibody-associated agglutination in a test system
containing rouleaux-promoting serum. In
the saline replacement technique, serum
and cells are incubated to allow antibody

The prewarming procedure described
above will not detect alloantibodies
that agglutinate at 37 C or lower and
are not reactive in the antiglobulin
phase. If detection of these antibodies is desired, testing and centrifugation at 37 C are required. If time permits, a tube containing a prewarmed
mixture of serum and cells can be
incubated at 37 C for 60 to 120 minutes, and the settled red cells examined for agglutination by resuspending the button without centrifugation.
Cold-reactive antibodies may not be
detectable when room-temperature

756

AABB Technical Manual

attachment, but the serum is removed
and saline is added as the resuspending
medium.

Reagents

1.
2.

Dry enzyme powder, 1 g.
Phosphate-buffered saline (PBS), pH
7.3: see Method 1.7.
Phosphate buffer, pH 5.4.

Saline.

Procedure
Procedure

After routine incubation and resuspension,
proceed with the following steps if the appearance of the resuspended cells suggests
rouleaux formation:
1.
Recentrifuge the serum/cell mixture.
2.
Remove the serum, leaving the cell
button undisturbed.
3.
Replace the serum with an equal
volume of saline (2 drops).
4.
Resuspend the cell button gently and
observe for agglutination. Rouleaux
will disperse when suspended in saline,
whereas true agglutination will remain.

Reference
Issitt PD, Anstee DJ. Applied blood group serology.
4th ed. Durham, NC: Montgomery Scientific Publications, 1998:1135.

Method 3.5. Enzyme
Techniques
For a discussion of the principles of enzyme testing, see Chapter 19.

Method 3.5.1. Preparation of Ficin
Enzyme Stock, 1% w/v

Place 1 g of powdered ficin in a 100mL volumetric flask. Handle the
ficin carefully; it is harmful if it gets
in the eyes or is inhaled. It is desirable to wear gloves, mask, and apron,
or to work under a hood.
Add PBS, pH 7.3 to 100 mL, to dissolve the ficin. Agitate vigorously by
inversion, rotate for 15 minutes, or
mix with a magnetic stirrer until
mostly dissolved. The powder will not
dissolve completely.
Collect clear fluid, either by filtration
or centrifugation, and prepare small
aliquots. Store the aliquots at –20 C
or colder. Do not refreeze a thawed
solution.

Method 3.5.2. Preparation of Papain
Enzyme Stock, 1% w/v

Principle
The enzyme preparations used in blood
banking differ from lot to lot; each time a
stock enzyme solution is prepared, its reactivity should be tested and incubation
periods standardized for optimal effectiveness. See Method 3.5.3.

Principle

Reagents

The enzyme preparations used in blood
banking differ from lot to lot; each time a
stock enzyme solution is prepared, its reactivity should be tested and incubation
periods standardized for optimal effectiveness. See Method 3.5.3.

1.
2.
3.

L-cysteine hydrochloride 0.5 M, 0.88
g in 10 mL distilled water.
Dry enzyme powder, 2 g.
Phosphate buffer 0.067 M at pH 5.4,
prepared by combining 3.5 mL of
Na2HPO4 and 96.5 mL of KH2PO4.

Methods Section 3: Antibody Detection/Identification and Compatibility Testing

Procedure
1.

2.
3.
4.
5.

Procedure

Add 2 g of powdered papain to 100
mL of phosphate buffer (pH 5.4).
Handle papain carefully; it is harmful to mucous membranes. Use appropriate protective equipment.
Agitate enzyme solution for 15 minutes at room temperature.
Collect clear fluid by filtration or
centrifugation.
Add L-cysteine hydrochloride and
incubate solution at 37 C for 1 hour.
Add phosphate buffer (pH 5.4) to final volume of 200 mL. Store aliquots
at –20 C or colder. Do not refreeze
aliquots.

2.
3.
4.

5.
6.

Method 3.5.3. Standardization of Enzyme
Procedures

Principle
For a two-stage enzyme procedure, the
optimal treatment time must be determined for each new lot of stock solution.
The technique given below for ficin can
be modified for use with other enzymes.

8.
9.

10.
11.

Reagents
1.
2.
3.

4.
5.
6.

757

1% stock solution of ficin in PBS, pH
7.3.
Several sera known to lack unexpected antibodies.
Anti-D that agglutinates only enzyme-treated D+ red cells and does
not agglutinate untreated D+ cells.
Anti-Fya of moderate or strong reactivity.
D+ and Fy(a+b–) red cell samples.
Antihuman globulin (AHG) reagent.
Polyspecific or anti-IgG may be used
unless otherwise indicated.
IgG-coated red cells.

12.

Prepare 0.1% ficin by diluting one
volume of stock ficin solution with
nine volumes of PBS, pH 7.3.
Label three tubes: 5 minutes, 10
minutes, and 15 minutes.
Add equal volumes of washed red cells
and 0.1% ficin to each tube.
Mix and incubate at 37 C for the time
designated. Incubation times are
easily controlled if the 15-minute
tube is prepared first, followed by
the 10- and 5 -minute tubes at
5-minute intervals. Incubation will
be complete for all three tubes at the
same time.
Immediately wash the red cells three
times with large volumes of saline.
Resuspend treated cells to 2% to 5%
in saline.
Label four tubes for each serum to
be tested: untreated, 5 minutes, 10
minutes, 15 minutes.
Add 2 drops of the appropriate serum to each of the four tubes.
Add 1 drop of the appropriate red cell
suspension to each of the labeled
tubes.
Mix and incubate at 37 C for 15 minutes.
Centrifuge and examine for agglutination by gently resuspending the red
cell button.
Proceed with the AHG test described
in Method 3.2.1, steps 6 through 9.

Interpretation
Table 3.5.3-1 shows possible results with
D+, Fy(a+b–) cells and the sera indicated.
In this case, the optimal incubation time
would be 10 minutes. Incubation for only
5 minutes does not completely abolish Fya
activity or maximally enhance anti-D reactivity. Incubation for 15 minutes causes

758

AABB Technical Manual

Table 3.5.3-1. Hypothetical Results with D+, Fy(a+b–) Red Cells
Cells and Enzyme
Untreated
5 minutes
10 minutes
15 minutes

Inert Serum

Anti-D

Anti-Fy

0
0
0
0
0
0
0
w+

0
1+
1+
2+
2+
2+
2+
2+

0
3+
0
1+
0
0
0
w+

37 C incubation
antihuman globulin test
37 C incubation
antihuman globulin test
37 C incubation
antihuman globulin test
37 C incubation
antihuman globulin test

false-positive antiglobulin reactivity with
inert serum.
If incubation for 5 minutes overtreats the
cells, it is preferable to use a more dilute
working solution of enzyme than to reduce
incubation time because it is difficult to accurately monitor very short incubation
times. Additional tests can evaluate a single
dilution at different incubation times, or a
single incubation time can be used for
different enzyme dilutions.

Reagents
1.
2.
3.

Sera known to contain antibody that
will agglutinate enzyme-treated cells.
Sera free of any unexpected antibodies.
Antihuman globulin (AHG) reagent.
Polyspecific or anti-IgG may be used
unless otherwise indicated.
IgG-coated red cells.

Procedure
Method 3.5.4. Evaluating Enzyme-Treated
Red Cells

Principle
After optimal incubation conditions have
been determined for a lot of enzyme solution, treated red cells should be evaluated
before use to demonstrate that they are
adequately, but not excessively, modified.
Satisfactory treatment produces cells that
are agglutinated by an antibody that causes
only indirect antiglobulin test reactivity of
unmodified cells but are not agglutinated
or aggregated by inert serum.

5.
6.

Specimen
Enzyme-treated red cells.

Select an antibody that agglutinates
enzyme-treated red cells positive for
the antigen but gives only AHG reactions with unmodified cells. Many
examples of human source anti-D
behave in this way.
Add 2 drops of the selected antibodycontaining serum to a tube labeled
“positive.”
Add 2 drops of a serum free of unexpected antibodies to a tube labeled
“negative.”
Add 1 drop of 2% to 5% suspension
of enzyme-treated red cells to each
tube.
Mix and incubate 15 minutes at 37 C.
Centrifuge and resuspend the cells by
gentle shaking.

Methods Section 3: Antibody Detection/Identification and Compatibility Testing

7.
8.

Examine macroscopically for the presence of agglutination.
Perform the AHG test described in
Method 3.2.1, steps 6 through 9, on
the tube labeled “negative.”

759

Method 3.5.6. Two-Stage Enzyme
Technique

Specimen
Serum or plasma to be tested.

Reagent
Interpretation
There should be agglutination in the “positive” tube and no agglutination in the
“negative” tube. If agglutination occurs in
the “negative” tube, the cells have been
overtreated; if agglutination does not occur in the “positive” tube, treatment has
been inadequate.

1.
2.

Procedure
1.

Method 3.5.5. One-Stage Enzyme
Technique

Specimen

Serum or plasma to be tested.
4.

Reagent
1.
2.

Reagent red cells.
Antihuman globulin (AHG) reagent.
Polyspecific or anti-IgG may be used
unless otherwise indicated.
IgG-coated red cells.

1.
2.
3.
4.
5.

6.
7.

Procedure
Add 2 drops of serum to an appropriately labeled tube.
Add 2 drops of a 2% to 5% saline suspension of reagent red cells.
Add 2 drops of 0.1% papain solution
and mix well.
Incubate at 37 C for 15 minutes.
Centrifuge; gently resuspend the cells
and observe for agglutination. Grade
and record the results.
Proceed with the AHG test described
in Method 3.2.1, steps 6 through 9.

Reagent red cells.
Antihuman globulin (AHG) reagent.
Polyspecific or anti-IgG may be used
unless otherwise indicated.
IgG-coated red cells.

Prepare a diluted enzyme solution
(papain or ficin) by adding 9 mL of
PBS, pH 7.3, to 1 mL of stock enzyme.
Add one volume of diluted enzyme
to one volume of packed, washed reagent red cells.
Incubate at 37 C for the time determined to be optimal for that enzyme
solution.
Wash treated cells at least three times
with large volumes of saline and resuspend the cells to a 2% to 5% concentration in saline.
Add 2 drops of serum or plasma to
be tested to an appropriately labeled
tube.
Add 1 drop of 2% to 5% suspension
of enzyme-treated cells.
Mix and incubate for 15 minutes at
37 C.
Centrifuge; gently resuspend the cells
and observe for agglutination. Grade
and record the results.
Proceed with the AHG test described
in Method 3.2.1, steps 6 through 9.

Notes
1.

An alternative method for steps 4 and
5 (Method 3.5.5) or steps 7 and 8
(Method 3.5.6) is to incubate the serum and enzyme-treated cells at 37

760

3.
4.

AABB Technical Manual

C for 60 minutes and examine the
settled cells for agglutination without centrifugation. This can be useful for serum with strong cold-reactive agglutinins and can sometimes
prevent the occurrence of false-positive results.
Microscopic examination is not recommended for routine use and is
particularly inappropriate with enzyme enhanced tests; false-positive
reactions will often be detected.
Either papain or ficin may be used in
a two-stage procedure.
Enzyme preparations are available
commercially. The manufacturer’s
directions should be followed for appropriate use and quality control.

Procedure
1.
2.

4.
5.
6.

References
1.

Issitt PD, Anstee DJ. Applied blood group serology, 4th ed. Durham, NC: Montgomery
Scientific, 1998.
Judd WJ. Methods in immunohematology. 2nd
ed. Durham, NC: Montgomery Scientific, 1994.

Method 3.6. Direct
Antiglobulin Test (DAT)

8.
9.

Interpretation
1.

Principle
See Chapter 20 for a discussion of the
principles of direct antiglobulin testing.

Specimen
Red cells from an anticoagulated blood
sample.

Reagents
1.

Antihuman globulin (AHG) reagent:
polyspecific antiglobulin reagent,
anti-IgG, anti-complement antisera.
A control reagent (eg, PBS) is required when all antisera tested give
a positive result.
IgG-coated red cells.

Dispense 1 drop of a 2% to 5% suspension of red cells into each tube.
Wash each tube three or four times
with saline. Completely decant the final wash.
Immediately add antisera and mix. For
the amount of antisera required, refer to the manufacturer’s directions.
Centrifuge according to the manufacturer’s directions.
Examine the cells for agglutination.
Grade and record the reaction.
If using polyspecific AHG or antiC3d, incubate nonreactive tests at
room temperature for 5 minutes, then
centrifuge, and read again.
Confirm the validity of negative tests
by adding IgG-coated red cells to tests
containing anti-IgG.
Centrifuge according to the manufacturer’s directions.
Examine the cells for agglutination and
record the reaction.

The DAT is positive when agglutination is observed either after immediate centrifugation or after the
centrifugation that followed roomtemperature incubation. IgG-coated
red cells usually give immediate reactions, whereas complement coating may be more easily demonstra1,2
ble after incubation. Monospecific
AHG reagents are needed to confirm
which globulins are present.
The DAT is negative when no agglutination is observed at either test phase
and the IgG-coated cells added in step
7 are agglutinated. If the IgG-coated
cells are not agglutinated, the negative DAT result is considered invalid
and the test must be repeated. A
negative DAT does not necessarily

Methods Section 3: Antibody Detection/Identification and Compatibility Testing

mean that the red cells have no attached globulin molecules. Polyspecific and anti-IgG reagents detect
as few as 200 to 500 molecules of IgG
per cell,1 but patients may experience autoimmune hemolytic anemia when IgG coating is below this
2
level.
No interpretation can be made if the
results with all antisera used to perform a DAT and the control are reactive. This indicates spontaneous agglutination, which must be resolved
before further testing is performed.

Notes
1.
2.

Steps 2 through 7 must be performed
without interruption.
Initial testing may be performed with
polyspecific reagent only. If the DAT
is negative with polyspecific reagent,
no further testing is necessary. If the
DAT is positive with polyspecific reagent, perform the DAT test with
monospecific reagents, anti-IgG, and
anticomplement, to determine which
globulins are present.
Verification of negative results with
anti-C3d is recommended. Refer to
the manufacturer’s instructions to
determine appropriate controls.

in a serum sample or to compare the
strength of antigen expression on different red cell samples. The usual applications of titration studies are: 1) estimating
antibody activity in alloimmunized pregnant women to determine whether and
when to perform more complex invasive
investigation of the fetal condition (see
Chapter 23); 2) elucidating autoantibody
specificity (see Chapter 20); 3) characterizing antibodies as high-titer, low-avidity,
traits common in antibodies to antigens
of the Knops and Chido/ Rodgers sysa
tems, Cs , and JMH (see Chapter 15); and
4) observing the effect of sulfhydryl reagents on antibody behavior, to determine immunoglobulin class (IgG or IgM).
See Method 5.3 for titration studies specifically to assist in monitoring clinically
significant antibodies in the pregnant
woman.

Specimen
Serum or plasma antibody to be titrated.

Reagents
1.

References
1.

Mollison PL, Engelfriet CP, Contreras M, eds.
Blood transfusion in clinical medicine. 10th
ed. Oxford, England: Blackwell Scientific Publications, 1997.
Petz LD, Garratty G. Immune hemolytic anemia. Philadelphia: Churchill-Livingstone, 2004.

Method 3.7. Antibody Titration
Principle
Titration is a semiquantitative method used
to determine the concentration of antibody

761

Red cells that express the antigen(s)
corresponding to the antibody specificity (ies), in a 2% to 5% saline suspension. Uniformity of cell suspensions is very important to ensure
comparability of results.
Saline. (Note: Dilutions may be made
with albumin if desired.)

Procedure
The master dilution technique for titration
studies is as follows:
1.
Label 10 test tubes according to the
serum dilution (eg, 1 in 1, 1 in 2, etc).
A 1 in 1 dilution means one volume
of serum undiluted; a 1 in 2 dilution
means one volume of serum in a fi-

762

6.
7.

AABB Technical Manual

nal volume of two, or a 50% solution
of serum in the diluent. See Methods
1.4 and 1.5.
Deliver one volume of saline to all test
tubes except the first (undiluted 1 in
1) tube.
Add an equal volume of serum to
each of the first two tubes (undiluted
and 1 in 2).
Using a clean pipette, mix the contents of the 1 in 2 dilution several
times and transfer one volume into
the next tube (the 1 in 4 dilution).
Continue the same process for all dilutions, using a clean pipette to mix
and transfer each dilution. Remove
one volume of diluted serum from
the final tube and save it for use if
further dilutions are required.
Label 10 tubes for the appropriate
dilutions.
Using separate pipettes for each dilution, transfer 2 drops of each diluted serum into the appropriately
labeled tubes and add 2 drops of a
2% red cell suspension. Alternatively, for convenience, add 1 drop of
a 3%-4% suspension of red cells as
supplied by the reagent manufacturer, although this method is less
precise.
Mix well and test by a serologic technique appropriate to the antibody
(see Chapter 19).
Examine test results macroscopically;
grade and record the reactions. The
prozone phenomenon (see Chapter
12) may cause reactions to be weaker
in the more concentrated serum preparations than in higher dilutions.
To avoid misinterpretation of results,
it may be preferable to examine first
the tube containing the most dilute
serum and proceed through the more
concentrated samples to the undiluted
specimen.

Interpretation
1.

Observe the highest dilution that
produces 1+ macroscopic agglutination. The titer is reported as the reciprocal of the dilution level, eg,
32—not 1 in 32 or 1:32 (see Table
3.7-1). If there is agglutination in the
tube containing the most dilute serum, the endpoint has not been
reached, and additional dilutions
should be prepared and tested.
2.
In comparative studies, a significant
difference in titer is three or more dilutions. Variations in technique and
inherent biologic variability can cause
duplicate tests to give results that
differ by one dilution in either direction. Serum containing antibody at a
true titer of 32 may show, on replicate tests, the endpoint in the 1:32
tube, the 1:64 tube, or the 1:16 tube.
3.
Titer values alone can be misleading
without also evaluating the strength
of agglutination. The observed strength
of agglutination can be assigned a
number and the sum of these numbers for all tubes in a titration study
represents the score, another semiquantitative measurement of antibody
reactivity. The arbitrarily assigned
threshold for significance in comparing scores is a difference of 10 or
more between different test samples.
See Table 3.7-1.
4.
Antibodies with high-titer, low-avidity characteristics generally have a titer greater than 64, with most tubes
showing consistently weak reactivity.
Table 3.7-1 shows the results obtained
with three sera, each of which shows no
more agglutination after 1:256. The differences in score, however, indicate considerable variation in strength of reactivity.

Methods Section 3: Antibody Detection/Identification and Compatibility Testing

763

Table 3.7-1. Examples of Antibody Titers, Endpoints, and Scores
Reciprocal of Serum Dilution
1

16 32 64 128 256 512

Strength:

3+ 3+ 3+ 2+ 2+ 2+ 1+

Score:

10 10 10 8

Strength:

4+ 4+ 4+ 3+ 3+ 2+ 2+ 1+

Score:

12 12 12 10 10 8

Strength:

1+ 1+ 1+ 1+

Score:

Titer*

Score

64(256)

Sample 1
8

64
128(256)

Sample 2
80
8(256)

Sample 3
5

*The titer is often determined from the highest dilution of serum that gives a reaction ≥1+ (score 5). This may differ significantly from the titration endpoint (shown in parentheses), as with the reactions of an antibody with high-titer,
low-avidity characteristics, manifested by Sample 3.

Notes
Titration is a semiquantitative technique.
Technical variables greatly affect the results and care should be taken to achieve
the most uniform possible practices.
1.
Careful pipetting is essential. Pipettes with disposable tips that can
be changed after each dilution are
recommended.
2.
Optimal time and temperature of incubation and time and force of centrifugation must be used consistently.
3.
The age, phenotype, and concentration of the test red cells will influence
the results. When the titers of several
antibody-containing sera are to be
compared, all of them should be
tested against red cells (preferably
freshly collected) from the same donor. If this is not possible, the tests
should use a pool of reagent red cells

from donors of the same phenotype.
When a single serum is to be tested
against different red cell samples, all
samples should be collected and preserved in the same manner and diluted to the same concentration before use.
Completely reproducible results are
virtually impossible to achieve. Comparisons are valid only when specimens are tested concurrently. In tests
with a single serum against different
red cell samples, material from the
master dilution must be used for all
the tests.
Measurements are more accurate with
large volumes than with small volumes; a master dilution technique
(see above) gives more reliable results than individual dilutions for a
single set of tests. The volume needed
for all planned tests should be calcu-

764

AABB Technical Manual

lated and an adequate quantity of
each dilution prepared.
When performing a titration for
anti-D for HDFN, see Method 5.3.

0.01 M dithiothreitol (DTT), prepared
by dissolving 0.154 g of DTT in 100
mL of pH 7.3 PBS. Store at –18 C or
lower.

Procedure

Method 3.8. Use of Sulfhydryl
Reagents to Distinguish IgM
from IgG Antibodies

1.
2.
3.

Principle
Treating IgM antibodies with sulfhydryl
reagents abolishes both agglutinating and
complement-binding activities. Observations of antibody activity before and after
sulfhydryl treatment are useful in determining immunoglobulin class. Sulfhydryl
treatment can also be used to abolish IgM
antibody activity to permit detection of coexisting IgG antibodies. For a discussion of
IgM and IgG structures, see Chapter 11.

4.
5.

Interpretation
1.

Specimen

2 mL of serum or plasma to be treated.

Reagents
1.

Dispense 1 mL of serum or plasma
into each of two test tubes.
To one tube, labeled dilution control,
add 1 mL of pH 7.3 PBS.
To the other tube, labeled test, add 1
mL of 0.01 M DTT.
Mix and incubate at 37 C for 30 to 60
minutes.
Use the DTT-treated and dilution control samples in standard procedures.

Phosphate-buffered saline (PBS) at
pH 7.3.

Reactivity in the dilution control
serum and no reactivity in the DTTtreated serum indicates an IgM antibody.
Reactivity in the dilution control serum and the DTT-treated serum indicates an IgG antibody or an IgG
and IgM mixture. Titration studies
may be necessary to distinguish between them. See Table 3.8-1.

Table 3.8-1. Effect of Dithiothreitol on Blood Group Antibodies
Dilution
Test Sample

1/2

1/4

1/8

1/16

1/32

Interpretation

Serum + DTT
Serum + PBS

3+
3+

2+
2+

1+
1+

0
0

IgG

Serum + DTT
Serum + PBS

0
3+

0
2+

0
1+

0
0

IgM

Serum + DTT
Serum + PBS

2+
3+

1+
2+

0
2+

0
1+

0
0

IgG + IgM*

*May also indicate only partial inactivation of IgM.

Methods Section 3: Antibody Detection/Identification and Compatibility Testing

No reactivity in the dilution control
serum indicates dilution of weak antibody reactivity and an invalid test.

765

Method 3.9. Plasma Inhibition
to Distinguish Anti-Ch and
-Rg from Other Antibodies
with HTLA Characteristics

Control

Principle

A serum or plasma sample known to contain an IgM antibody should be treated and
tested in parallel.

For a discussion of the principles of plasma
inhibition of anti-Ch and -Rg, see Chapter 19.

Specimen
Serum or plasma to be tested.

Notes

Reagents

1.
2.

2-mercaptoethanol can also be used
for this purpose. See Method 2.11 for
preparation.
Sulfhydryl reagents used at low concentration may weaken antigens of
the Kell system. For investigation of
antibodies in the Kell system, it may
be necessary to use other methods.
Gelling of a serum or plasma sample
may be observed during treatment with
DTT. This can occur if the DTT has
been prepared incorrectly and has a
concentration above 0.01 M. Gelling
may also occur if serum and DTT are
incubated too long. An aliquot of the
sample undergoing treatment can be
tested after 30 minutes of incubation;
if the activity thought to be due to IgM
has disappeared, there is no need to
incubate further. Gelled samples cannot be tested for antibody activity because overtreatment with DTT causes
the denaturation of all serum proteins.

3.
4.
5.

Procedure
1.

3.
4.
5.

Reference
Mollison PL, Engelfriet CP, Contreras M, eds. Blood
transfusion in clinical medicine. 10th ed. Oxford,
England: Blackwell Scientific Publications, 1997.

Reactive red cell samples.
A pool of six or more normal plasma
samples.
6% bovine albumin, see Method 1.5.
Anti-IgG.
IgG-coated red cells.

Prepare serial twofold dilutions of test
serum in saline. The dilution range
should be from 1 in 2 to 1 in 512, or
to one tube beyond the known titer
as determined above (Method 3.7). The
volume prepared should be not less
than 0.3 mL for each red cell sample
to be tested.
For each red cell sample to be tested,
place 2 drops of each serum dilution
into each of two sets of appropriately
labeled 10 or 12 × 75-mm test tubes.
To one set, add 2 drops of pooled
plasma to each tube.
To the other set, add 2 drops of 6%
albumin to each tube.
Gently agitate the contents of each
tube and incubate the tubes at room
temperature for at least 30 minutes.
Add 1 drop of a 2% to 5% suspension
of red cells to each tube.

766

10.

AABB Technical Manual

Gently agitate the contents of each
tube and incubate the tubes at 37 C
for 1 hour.
Wash the cells four times in saline,
add anti-IgG, and centrifuge according to the manufacturer’s directions.
Resuspend the cell buttons and examine for agglutination; confirm all
nonreactive tests microscopically.
Grade and record the results.
Confirm the validity of negative tests
by adding IgG-coated red cells.

Interpretation
1.

Inhibition of antibody activity in the
tubes to which plasma has been added
suggests anti-Ch or anti-Rg specificity; this inhibition is often complete.
The presence of partial inhibition
suggests the possibility of additional
alloantibodies. This can be tested by
preparing a large volume of inhibited
serum and testing it against a reagent red cell panel to see if the nonneutralizable activity displays antigenic specificity.
Lack of reactivity in the control (6%
albumin) indicates dilution of weakly
reactive antibody and an invalid test.

Ellisor SS, Shoemaker MM, Reid ME. Adsorption of anti-Chido from serum using autologous red blood cells coated with homologous
C4. Transfusion 1982;22:243-5.

Method 3.10. Dithiothreitol
(DTT) Treatment of Red Cells
Principle
DTT is an efficient reducing agent that
can disrupt the tertiary structure of proteins by irreversibly reducing disulfide
bonds to free sulfhydryl groups. Without
tertiary structure, protein-containing antigens can no longer bind antibodies that
are specific for them. Red cells treated
with DTT will not react with antibodies in
the Kell blood group system, most antibodies in the Knops system, or most examples of anti-LWa, -Yta, -Ytb, -Doa, -Dob,
a
a
-Gy , -Hy, and -Jo . This inhibition technique may be helpful in identifying some
of these antibodies or in determining if a
serum contains additional underlying alloantibodies.

Specimen
Red cells to be tested.

Notes

Reagents

Antibodies to other plasma antigens
may also be partially inhibited by
plasma.1
Adsorption with C4-coated red cells
is an alternative procedure that may
be used for identifying anti-Ch or
anti-Rg and for detecting underlying
alloantibodies.2

2.
3.

References
1.

Reid ME, Lomas-Francis C. The blood group
antigen factsbook. 2nd ed. New York: Academic Press, 2004.

Prepare 0.2 M DTT by dissolving 1 g
of DTT powder in 32 mL of phosphate-buffered saline (PBS), pH 8.0.
Divide it into 1-mL volumes and
freeze aliquots at –18 C or colder.
PBS at pH 7.3, see Method 1.7.
Red cells known to be positive for the
antigen in question and, as a control,
red cells known to be positive for K,
which is consistently disrupted by
DTT.
Anti-K, either in reagent form or
strongly reactive in a serum specimen.

Methods Section 3: Antibody Detection/Identification and Compatibility Testing

767

Procedure

Reference

Branch DR, Muensch HA, Sy Siok Hian S, Petz LD.
Disulfide bonds are a requirement for Kell and
Cartwright (Yt a) blood group antigen integrity. Br J
Haematol 1983;54:573-8.

2.
3.

4.
5.

Wash one volume of the test cells and
the control cells with PBS. After decanting, add four volumes of 0.2 M
DTT, pH 8.0.
Incubate at 37 C for 30 to 45 minutes.
Wash four times with PBS. Slight
hemolysis may occur; if hemolysis is
excessive, repeat the procedure using fresh red cells and a smaller volume of DTT, eg, two or three volumes.
Resuspend the cells to a 2% to 5%
suspension in PBS.
Test DTT-treated cells with serum
containing the antibody in question.
Test K+ red cells with anti-K.

Method 3.11. Urine
a
Neutralization of Anti-Sd
Principle
a

For a discussion of anti-Sd neutralization
by urine, see Chapter 15.

Specimen
Serum or plasma suspected of containing
a
anti-Sd .

Reagents
1.

Interpretation
1.

The control K+ red cells should give
negative reactions when tested with
anti-K; if not, the DTT treatment has
been inadequate. Other antigens in
the Kell system can also serve as the
control.
If reactivity of the test serum is eliminated, the suspected antibody specificity may be confirmed. Enough red
cell samples should be tested to exclude most other clinically significant alloantibodies.

Procedure
1.

Note
Treatment of red cells with 0.2 M DTT, pH
8.0, is optimal for denaturation of all antigens of the Kell, Cartwright, LW, and
Dombrock systems, and most antigens of
the Knops system. Lower concentrations
of DTT may selectively denature particular blood group antigens (ie, 0.002 M DTT
will denature only Jsa and Jsb antigens). This
property may aid in certain antibody investigations.

Urine from a known Sd(a+) individual, or from a pool of at least six individuals of unknown Sda type, prepared as follows: Collect urine and
immediately boil it for 10 minutes.
Dialyze it against phosphate-buffered saline (PBS), pH 7.3, at 4 C for
48 hours. Change PBS several times.
Centrifuge. Dispense supernatant into
aliquots, which can be stored at –20
C until thawed for use.
PBS, pH 7.3. See Method 1.7.

4.
5.

Mix equal volumes of thawed urine
and test serum.
Prepare a dilution control tube containing equal volumes of serum and
PBS.
Prepare a urine control tube by mixing equal volumes of thawed urine
and PBS.
Incubate all tubes at room temperature for 30 minutes.
Mix 1 drop of each test red cell sample with 4 drops from each of the

768

AABB Technical Manual

tubes: neutralized serum, serum with
PBS, and urine with PBS. Test each
one using standard procedures.

Method 3.12. Adsorption
Procedure
Principle
See Chapter 19.

Interpretation
1.

Specimen

Persistent agglutination in the serum
sample incubated with urine means
either that partial or no neutralization was achieved or that underlying
antibodies are present. Microscopic
examination may be helpful; agglutia
nation due to anti-Sd has a refractile,
mixed-field appearance on microscopic examination.
No agglutination in the neutralized
tube with persistent agglutination in
the dilution control tube and absence
of hemolysis and agglutination in
the urine control tube indicate that
the antibody has been neutralized
a
and is quite probably anti-Sd .
The absence of agglutination in the
dilution control tube means that the
dilution in the neutralization step
was too great for the antibody present and the results of the test are invalid. The urine control tube provides
assurance that no substances in the
urine are agglutinating or damaging
the red cells.

Serum or plasma containing antibody to
be adsorbed.

Reagents
Red cells (eg, autologous or allogeneic) that
carry the antigen corresponding to the antibody specificity to be adsorbed.

Procedure
1.
2.

Note
Urine may also contain ABO and Lewis
blood group substances, depending upon
the ABO, Lewis, and secretor status of the
donor.

Reference
Judd WJ. Methods in immunohematology. 2nd ed.
Durham, NC: Montgomery Scientific Publications,
1994.

Wash the selected red cells at least
three times with saline.
After the last wash, centrifuge the red
cells at 800 to 1000 × g for at least 5
minutes and remove as much of the
supernatant saline as possible. Additional saline may be removed by
touching the red cell mass with a
narrow piece of filter paper.
Mix appropriate volumes of the packed red cells and serum and incubate
at the desired temperature for 30 to
60 minutes.
Mix the serum/cell mixture periodically throughout the incubation
phase.
Centrifuge the red cells at 800 to
1000 × g for 5 minutes to pack cells
tightly. Centrifuge at the incubation
temperature, if possible, to avoid
dissociation of antibody from the red
cell membranes.
Transfer the supernatant fluid, which
is the adsorbed serum, to a clean test
tube. If an eluate is to be prepared,
save the red cells.
Test an aliquot of the adsorbed serum, preferably against an addi-

Methods Section 3: Antibody Detection/Identification and Compatibility Testing

tional aliquot of the cells used for
adsorption, to see if all antibody has
been removed.

Interpretation
If reactivity remains, the antibody has not
been completely removed. No reactivity
signifies that antibody has been completely adsorbed.

Notes
1.

Adsorption is more effective if the
area of contact between the red cells
and serum is large; use of a largebore test tube (13 mm or larger) is
recommended.
Multiple adsorptions may be necessary to completely remove an antibody, but each successive adsorption increases the likelihood that the
s e r u m wi l l b e d i l u te d a n d u nadsorbed antibodies weakened.
Repeat adsorptions should use a
fresh aliquot of cells and not the
cells from the prior adsorption.
Enzyme pretreatment of adsorbing
cells can be performed to increase antibody uptake for enzyme-resistant
antigens.

The ARDP maintains a database of rare
donors submitted by immunohematology
reference laboratories that are accredited
by the AABB or the American Red Cross
(ARC). Donors are considered rare due to
the absence of a high-incidence antigen,
absence of multiple common antigens, or
IgA deficiency.
All requests to the ARDP must originate
from an AABB- or ARC-accredited immunohematology reference laboratory to
ensure that the patient in question has
been accurately evaluated and reported. All
shipping and rare unit fees are established
by the shipping institution.

Procedure
1.

Reference
Judd WJ. Methods in immunohematology. 2nd ed.
Durham, NC: Montgomery Scientific Publications,
1994.

Method 3.13. Using the
American Rare Donor
Program
Principle
The American Rare Donor Program (ARDP)
helps to locate blood products for patients requiring rare or unusual blood.

769

A hospital blood bank, transfusion
service, or blood center identifies a
patient who needs rare blood.
The institution contacts the nearest
AABB- or ARC-accredited immunohematology reference laboratory to
supply the needed blood.
If the laboratory cannot supply the
blood, it contacts the ARDP. All requests to the ARDP must come from
an AABB- or ARC-accredited laboratory (or another rare donor program).
Requests received directly from a nonaccredited facility will be referred to
the nearest accredited institution.
The institution contacting the ARDP
(requesting institution) must confirm
the identity of the antibody(ies) by
serologic investigation or by examining the serologic work performed
by another institution.
ARDP staff search their database for
centers that have identified donors
with the needed phenotype and contact the centers for availability of
units. ARDP staff give the name(s) of
the shipping center(s) to the requesting institution.

770

AABB Technical Manual

The requesting and shipping institutions should discuss and agree on
charges and testing requirements
before units are shipped.
If an initial search does not result in
a sufficient number of units, the following mechanisms can be used by
ARDP staff to obtain needed units: 1)

communication to all ARDP participating centers alerting them to search
their inventories and/or recruit donors matching the needed phenotype, 2) contacting other rare donor
files such as those administered by
the World Health Organization, Japanese Red Cross, etc.

Methods Section 4: Investigation of a Positive DAT

Methods Section 4

Section 4

Investigation of a Positive
Direct Antiglobulin Test

Elution Techniques
The objective of all elution techniques is
to interfere with the noncovalent binding
forces that hold antibody-antigen complexes
together on the red cell surface. The cell
membrane can be physically disrupted by
heat, ultrasound, freezing and thawing,
detergents, or organic solvents. The binding forces of antigen-antibody complexes
can be interrupted by alterations in pH or
salt concentration. For a comparison of the
advantages and disadvantages of various
elution methods, see Chapter 20. Selected
elution methods follow, including one example of an organic solvent method. The
cold-acid elution method (Method 4.1) is
the basis of the commercially available acid
elution kits commonly used in the United
States. Because no single elution method
will result in the identification of all
antibodies, use of an alternative elution

method (eg, organic solvent) may be indicated when a nonreactive eluate is not in
agreement with clinical data. The reader
should refer to Chapter 2 for the proper
handling of hazardous chemicals that are
sometimes used in these techniques. Access to a chemical fume hood is desirable
when organic solvents are in use.
Very thorough washing of the red cells
before elution is essential to ensure that antibody in the eluate is only red cell-bound
and does not represent free antibody, eg,
from plasma. A control to show that all free
antibody has been removed by washing can
be obtained by saving the saline from the
last wash and testing it in parallel with the
eluate. Additionally, transferring the red
cells into a clean test tube just before the
elution step eliminates the possibility of
dissociating antibody that may have nonspecifically bound to the glass test tube
during an adsorption or the initial eluate
preparation steps.
771

772

AABB Technical Manual

Method 4.1. Cold-Acid Elution

Principle

Dissociation of antibodies from red cells
enables the identification of auto- or alloantibodies. Elution methods used in conjunction with adsorption techniques are
also useful in detecting weak antigen expression on the adsorbing cells and in
separating mixtures of antibodies against
red cell antigens.

6.
7.

Quickly centrifuge the tube at 900 to
1000 × g for 2 to 3 minutes.
Transfer the supernatant eluate into
a clean test tube and add 0.1 mL of
pH 8.2 phosphate buffer for each 1
mL of eluate (see note 3).
Mix and centrifuge at 900 to 1000 × g
for 2 to 3 minutes.
Transfer the supernatant eluate into
a clean test tube and test it in parallel with the supernatant saline from
the final wash.

Specimen
Red cells positive by the direct antiglobulin
test (DAT) washed six times with large
volumes of saline (save the last wash).

Notes
1.
2.

Reagents
1.

3.
4.

Glycine-HCl (0.1 M, pH 3.0), prepared
by dissolving 3.75 g of glycine and
2.922 g of sodium chloride in 500 mL
of deionized or distilled water. Adjust
the pH to 3.0 with 12 N HCl. Store at
4 C.
Phosphate buffer (0.8 M, pH 8.2),
prepared by dissolving 109.6 g of
Na2HPO4 and 3.8 g of KH2PO4 in approximately 600 mL of deionized or
distilled water and adjusting the final volume to 1 L. Adjust the pH, if
necessary, with either 1 N NaOH or 1
N HCl. Store at 4 C (see note 2).
NaCl, 0.9%, at 4 C.
Supernatant saline from the final
wash of red cells to be tested.

Procedure
1.

2.
3.

Place 1 mL of red cells in a 13 ×
100-mm test tube and chill in an ice
water bath for 5 minutes before adding the glycine-HCl.
Add 1 mL of chilled saline and 2 mL
of chilled glycine-HCl to the red cells.
Mix and incubate the tube in an ice
water bath (0 C) for 1 minute.

Keep glycine-HCl in an ice bath during use, to maintain the correct pH.
Phosphate buffer will crystallize during storage at 4 C. Redissolve it at 37
C before use.
The addition of phosphate buffer restores neutrality to the acidic eluate.
Acidity may cause hemolysis of the
reagent red cells used in testing the
eluate. The addition of 22% bovine
albumin (one part to four parts of
eluate) may reduce such hemolysis.

References
1.

Judd WJ. Methods in immunohematology. 2nd
ed. Durham, NC: Montgomery Scientific Publications, 1994.
Rekvig OP, Hannestad K. Acid elution of blood
group antibodies from intact erythrocytes.
Vox Sang 1977;33:280-5.

Method 4.2.
Glycine-HCl/EDTA Elution
Principle
See Method 4.1.

Specimen
Red cells positive by the direct antiglobulin
test (DAT) washed six times with large
volumes of saline (save the last wash).

Methods Section 4: Investigation of a Positive DAT

Reagents
1.
2.

Disodium EDTA (10% w/v): Na2EDTA,
10 g; distilled water to 100 mL.
Glycine-HCl (0.1 M at pH 1.5): 0.75 g
glycine diluted to 100 mL with 0.9%
NaCl; adjust to pH 1.5 with 12 N HCl.
TRIS-NaCl (1 M): Tris(hydroxymethyl)
aminomethane [TRIS] or TRIZMA
BASE, 12.1 g; 5.25 g NaCl; distilled
water to 100 mL.
Supernatant saline from the final wash
of the red cells to be tested.

2.
3.

6.
7.

In a test tube, mix together 20 volumes (eg, drops) of 0.1 M glycineHCl buffer and 5 volumes of 10%
EDTA. This is the eluting solution.
In a 12 × 75-mm tube, place 10 volumes of packed red cells.
Add 20 volumes of the eluting solution to the red cells, mix well, and incubate at room temperature for 2
minutes. Do not overincubate.
Add 1 volume of TRIS-NaCl, mix,
and immediately centrifuge the tube
at 900 to 1000 × g for 60 seconds.
Transfer the supernatant eluate into
a clean test tube and adjust it carefully dropwise to pH 7.0 to 7.4 with
1 M TRIS-NaCl. The pH can be
checked with pH paper.
Centrifuge at 900 to 1000 × g for 2 to
3 minutes to remove the precipitate.
Transfer the supernatant eluate into
a clean test tube and test it in parallel with the supernatant saline from
the final wash.

Notes
1.

Procedure
1.

Once the red cells have been rendered
DAT negative, they may be tested for
the presence of blood group antigens, except those in the Kell system.
Treatment with glycine-HCl/EDTA

773

denatures Kell system antigens and
Era. Wash the red cells at least three
times in saline before use.
Red cells modified with glycineHCl/EDTA may be treated with a
protease and used in autologous adsorption studies.
Overincubation with the eluting solution (step 3) will irreversibly damage the red cells.
TRIS-NaCl is very alkaline and only a
few drops should be required to attain the desired pH (step 5).
Aliquots of the reagents can be stored
frozen and one tube of each can be
thawed just before use. The 10% EDTA
may precipitate when stored at 2 to 8 C.
Stored eluate (4 C or frozen) may be
more stable if albumin is added (1
volume of 30% bovine albumin for
every 10 volumes of eluate). If albumin is added to the eluate, it should
be added to the last wash.

Reference
Byrne PC. Use of a modified acid/EDTA elution
technique. Immunohematology 1991;7:46-7. [Correction note: Immunohematology 1991;7:106.]

Method 4.3. Heat Elution
Principle
Heat elution uses an increase in temperature to dissociate antibodies from red cells.
This method is best suited for the investigation of ABO hemolytic disease of the fetus and newborn and for the elution of IgM
antibodies from red cells. It should not
routinely be used for the investigation of
abnormalities caused by IgG auto- or alloantibodies.

Specimen
Red cells positive by the direct antiglobulin
test (DAT) washed six times with large

774

AABB Technical Manual

volumes of saline; save the last wash (see
note).

Reagents
1.
2.

6% bovine albumin (see Method 1.5).
Supernatant saline from the final
wash of the red cells to be tested.

of the remaining extracellular fluid, which
then extracts water from the red cells. The
red cells shrink, resulting in lysis. As the
membranes are disrupted, antibody is
dissociated. This method is used primarily for the investigation of ABO hemolytic
disease of the fetus and newborn.

Procedure
1.

Mix equal volumes of washed packed
cells and 6% bovine albumin in a
13 × 100-mm test tube.
Place the tube at 56 C for 10 minutes. Agitate the tube periodically
during this time.
Centrifuge the tube at 900 to 1000 ×
g for 2 to 3 minutes, preferably in a
heated centrifuge.
Immediately transfer the supernatant
eluate into a clean test tube and test
in parallel with the supernatant saline from the final wash.

Specimen
1.
2.

Procedure
1.
2.

Note

For optimal recovery of cold-reactive antibodies, the red cells should be washed in
ice-cold saline to prevent dissociation of
bound antibody before elution.

References

Judd WJ. Methods in immunohematology. 2nd
ed. Durham, NC: Montgomery Scientific Publications, 1994.
Landsteiner K, Miller CP Jr. Serological studies on the blood of primates. II. The blood
groups in anthropoid apes. J Exp Med 1925;
42:853-62.

Method 4.4. Lui
Freeze-Thaw Elution

Mix 0.5 mL of the red cells to be tested
with 3 drops of saline in a test tube.
Cap the tube, then rotate the tube to
coat the tube wall with cells.
Place the tube in a horizontal position in a freezer at –6 C to –70 C for
10 minutes.
Remove the tube from the freezer and
thaw it quickly with warm, running
tap water.
Centrifuge for 2 minutes at 900 to
1000 × g.
Transfer the supernatant to a clean
test tube and test it in parallel with
the supernatant saline from the final
wash.

References
1.

Judd WJ. Methods in immunohematology. 2nd
ed. Durham, NC: Montgomery Scientific Publications, 1994.

Feng CS, Kirkley KC, Eicher CA, et al. The Lui
elution technique: A simple and efficient
method for eluting ABO antibodies. Transfusion 1985;25:433-4.

Principle
As red cells freeze, extracellular ice crystals form that attract water from their surroundings. This increases the osmolarity

Red cells washed six times with large
volumes of saline.
Supernatant saline from the final
wash of the red cells to be tested.

Methods Section 4: Investigation of a Positive DAT

Method 4.5. Methylene
Chloride Elution

775

Reference
Judd WJ. Methods in immunohematology. 2nd ed.
Durham, NC: Montgomery Scientific Publications,
1994.

Principle
Organic solvents can influence antigenantibody dissociation by several mechanisms, including alteration of the tertiary
structure of antibody molecules and disruption of the red cell membrane. This
method is suitable for elution of IgG autoand alloantibodies.

Specimen
DAT-positive red cells washed six times with
large volumes of saline (save last wash).

Immune Hemolytic Anemia
Serum/Plasma Methods
Included in this section are methods used
to remove warm or cold autoantibody reactivity (eg, adsorptions) so that alloantibody detection tests and diagnostic tests
for differentiating the immune hemolytic
anemias can be performed. See Chapter
20 for a discussion of the immune hemolytic anemias.

Reagents
1.
2.

Methylene chloride (dichloromethane).
Supernatant saline from final wash
of the red cells to be tested.

Method 4.6. Cold
Autoadsorption
Principle

Procedure
1.

2.
3.
4.

6.
7.

Mix 1 mL of red cells, 1 mL of saline,
and 2 mL of methylene chloride in a
test tube, eg, 13 × 100 mm.
Stopper the tube and mix by gentle
agitation for 1 minute.
Remove the stopper and centrifuge
the tube at 1000 × g for 10 minutes.
Remove the lower layer of methylene
chloride with a transfer pipette and
discard it.
Place the tube at 56 C for 10 minutes. Stir the eluate constantly with
wooden applicator sticks in the first
several minutes to avoid it boiling
over; thereafter, stir it periodically.
Centrifuge at 1000 × g for 10 minutes.
Transfer the supernatant eluate into
a clean test tube and test it in parallel with the supernatant saline from
the final wash.

Although most cold autoantibodies do not
cause a problem in serologic tests, some
potent cold-reactive autoantibodies may
mask the concomitant presence of clinically significant alloantibodies. In these
cases, adsorbing the serum in the cold
with autologous red cells can remove the
autoantibody, permitting detection of underlying alloantibodies. In the case of most
nonpathologic cold autoantibodies, a
simple quick adsorption of the patient’s
serum with enzyme-treated autologous
red cells will remove most cold antibody.
See Method 3.5.6. A more efficient method
of removing immunoglobulins is the use
of ZZAP reagent, a combination of proteolytic enzyme and a powerful reducing
agent. ZZAP treatment removes IgM and
complement from autologous red cells
and uncovers antigen sites that can be
used to bind free autoantibody in the serum.

776

AABB Technical Manual

Specimen
1.
2.

1 mL of serum or plasma to be adsorbed.
Two 1-mL aliquots of autologous red
cells.

Notes
1.

Reagents
1.
2.
3.

1% cysteine-activated papain or 1%
ficin (see Methods 3.5.1 and 3.5.2).
Phosphate-buffered saline (PBS), pH
7.3 (see Method 1.7).
0.2 M dithiothreitol (DTT) prepared
by dissolving 1 g of DTT in 32.4 mL
of pH 7.3 PBS. Dispense into 3-mL
aliquots and store at –18 C or colder.

To avoid dilution of the serum and
possible loss of weak alloantibody
activity, it is important in step 3 to
remove as much of the residual saline as possible.
If the reactivity of the autoantibody
is not diminished, the target autoantigen may have been destroyed by
either the enzyme or the DTT. The
adsorption should be repeated against
untreated autologous red cells washed
several times in warm saline.

References

Procedure
1.

After the final adsorption, test the
serum with reagent red cells for alloantibody activity.

Prepare ZZAP reagent by mixing 0.5
mL of 1% cysteine-activated papain
with 2.5 mL of 0.2 M DTT and 2 mL
of pH 7.3 PBS. Alternatively, use 1
mL of 1% ficin, 2.5 mL of 0.2 M DTT,
and 1.5 mL of pH 7.3 PBS. The pH
should be between 6.0 and 6.5.
Add 2 mL of ZZAP reagent to 1 mL of
autologous red cells. Mix and incubate at 37 C for 30 minutes.
Wash the cells three times in saline.
Centrifuge the last wash for at least 5
minutes at 900 to 1000 × g and remove as much of the supernatant saline as possible (see note 1).
To the tube of ZZAP-treated red cells,
add 1 mL of the autologous serum.
Mix and incubate at 4 C for 30 minutes.
Centrifuge at 900 to 1000 × g for 4 to
5 minutes and transfer the serum into
a clean tube.
Steps 2 through 5 may be repeated if
the first autoadsorption does not satisfactorily remove the autoantibody
activity.

1.
2.

Branch DR. Blood transfusion in autoimmune
hemolytic anemias. Lab Med 1984;15:402-8.
Branch DR, Petz LD. A new reagent (ZZAP)
having multiple applications in immunohematology. Am J Clin Pathol 1982;78:161-7.

Method 4.7. Determining the
Specificity of Cold-Reactive
Autoagglutinins
Principle
For a discussion of specificity of cold-reacting autoantibodies, see Chapter 20.

Specimen
1.

Serum, separated at 37 C from a blood
sample allowed to clot at 37 C, or
plasma, separated from an anticoagulated sample after periodic inversion at 37 C for approximately 15
minutes.
Autologous red cells.

Reagents
Test red cells of the following phenotypes:

Methods Section 4: Investigation of a Positive DAT

2.
3.

A pool of two examples of adult
group O I adult red cells; they can be
the reagent cells routinely used for
alloantibody detection.
Group O i cord red cells.
The patient’s own (autologous) red
cells, washed at least three times
with 37 C saline.
Red cells of the same ABO group as
the patient, if the patient is not
group O. If the patient is group A or
AB, use both A1 and A2 cells.
Saline or phosphate-buffered saline
(PBS), pH 7.3 (see Method 1.7).

5.
6.

Procedure
1.

Prepare serial twofold dilutions of
the serum or plasma in saline or
PBS. The dilution range should be
from 1 in 2 to 1 in 4096 (12 tubes),
and the volumes prepared should be
more than the total volume needed
to test all of the desired red cells. See
Method 3.7.
Label a set of 12 tubes with the dilution (eg, 2, 4, 8, etc) for each of the
red cells to be tested (eg, adult, cord,
autologous).

Dispense 2 drops of each dilution
into the appropriate tubes.
Add 1 drop of a 3% to 5% saline suspension of each red cell sample to
the appropriate set of tubes.
Mix and incubate at room temperature for 30 to 60 minutes.
Centrifuge for 15 to 20 seconds at
900 to 1000 × g. Examine the tubes
one by one macroscopically for agglutination, starting with the set of tubes
at the highest dilution for each cell
tested (ie, read all the tubes for each
dilution as a set). Grade and record
the results.
Transfer the tubes to 4 C and incubate them at this temperature for 1
to 2 hours.
Centrifuge for 15 to 20 seconds at
900 to 1000 × g. Immediately place
the tubes in a rack in an ice water
bath. Examine the tubes as in step 6.
Grade and record the results.

Interpretation
Table 4.7-1 summarizes the reactions of
the commonly encountered cold-reactive
autoantibodies. In cold agglutinin syn-

Table 4.7-1. Typical Relative Reactivity Patterns of Cold Autoantibodies
Antibody Specificity
Red Cells

777

Anti-I

Anti-i

Anti-I

O I adult

0/↓

O i cord

0/↓

O i adult

Anti-IH

Anti-Pr

0/↓

↓

0/↓

↓

A1 I adult

0/↓

↓

Autologous

0/↓

↓

O I enzyme-treated

↑

+ = reactive; 0 = nonreactive; ↓ = weaker reaction; ↑ = stronger reaction.

778

AABB Technical Manual

drome, anti-I is seen most frequently, but
anti-i may also be encountered. When
cord cells react stronger than adult cells,
the specificity may be anti-i, but adult i
red cells need to be tested to confirm that
these reactions are due to anti-i and not
anti-I T . Some examples of anti-I react
more strongly with red cells that have a
strong expression of H antigen (eg, O and
A2 cells); such antibodies are called antiIH. Rarely, the specificity may be anti-Pr,
which should be suspected if all the cells
tested react equally. Anti-Pr can be confirmed by testing enzyme-treated cells;
anti-Pr does not react with enzymetreated cells, whereas anti-I and anti-i
react better with enzyme-treated cells.
Anti-Pr reacts equally with untreated red
cells of I or i phenotypes.

ferential reactivity may be more
apparent if incubation times are
prolonged and agglutination is evaluated after settling, without centrifugation. Settled readings are more accurate after a 2-hour incubation.
This procedure can be used to determine both the titer and the specificity. If incubations are started at 37 C
(set up prewarmed, and readings are
taken sequentially after incubation
at each temperature—eg, 37 C, 30 C,
room temperature, 4 C), the specificity, titer, and thermal amplitude of
the autoantibody can be determined
with a single set of serum dilutions.
If testing will also be performed at
30 C and 37 C, include a test of the
neat (undiluted) serum.

Notes

Reference

Petz LD, Garratty G. Immune hemolytic anemias.
2nd ed. Philadelphia: Churchill Livingstone, 2004.

It is important to use separate pipettes or pipette tips for each tube
when preparing serum dilutions because the serum carried from one
tube to the next when a single pipette is used throughout may cause
falsely high titration endpoints. The
difference can convert a true titer of
4000 to an apparent titer of 100,000,
when the use of separate pipettes is
compared with the use of a single pipette.
Serum dilutions can be prepared
more accurately with large volumes
(eg, 0.5 mL) than with small volumes.
Potent examples of cold-reactive
autoantibodies generally do not
show apparent specificity until titration studies are performed; this
specificity may not even be apparent
with dilutions at room temperature
or 4 C. In such circumstances, tests
can be incubated at 30 to 37 C. Dif-

Method 4.8. Cold Agglutinin
Titer
Principle
Cold-reactive autoantibodies, if present at
very high titers, may suggest a pathologic
cold agglutinin disease. This may result in
overt hemolysis and systemic symptoms
and may indicate underlying B-cell hematologic neoplasia.

Specimen
Serum, separated at 37 C from a sample
allowed to clot at 37 C, or plasma, separated from an anticoagulated sample after
periodic inversion at 37 C for approximately 15 minutes.

Methods Section 4: Investigation of a Positive DAT

Reagents

Notes

A pool of 2 examples of washed group
O I adult red cells, eg, antibody detection cells.
Phosphate-buffered saline (PBS), pH
7.3 (see Method 1.7).

Procedure
1.

3.
4.

Prepare serial twofold dilutions of the
patient’s serum or plasma in PBS.
The dilution range should be from 1
in 2 to 1 in 4096 (12 tubes). See
Method 3.7.
Mix 2 drops of each dilution with 1
drop of a 3% to 5% cell suspension of
red cells.
Mix and incubate at 4 C for 1 to 2
hours.
Centrifuge the tubes for 15 to 20 seconds at 900 to 1000 × g, then place
the tubes in a rack in an ice water
bath. Examine the tubes one by one
macroscopically for agglutination,
starting with the tube at the highest
dilution. Grade and record the results.

Interpretation
The titer is the reciprocal of the highest
serum dilution at which macroscopic agglutination is observed. Titers above 64
are considered elevated, but hemolytic
anemia resulting from cold-reactive autoagglutinins rarely occurs unless the titer is
>1000. Titers below 1000 may be obtained
when the autoantibody has a different
specificity (eg, anti-i), or if the cold agglutinin is of the less common low-titer,
high-thermal-amplitude type. If the patient has a positive direct antiglobulin test
(DAT) because of complement only and
has clinical signs of hemolytic anemia,
specificity and thermal amplitude studies
should be performed (see Method 4.7).

779

It is important to use separate pipettes for each tube when preparing
serum dilutions because the serum
carried from one tube to the next when
a single pipette is used throughout
may cause falsely high titration endpoints.
Serum dilutions can be prepared
more accurately with large volumes
(eg, 0.5 mL) than with small volumes.

Reference
Petz LD, Garratty G. Immune hemolytic anemias.
2nd ed. Philadelphia: Churchill Livingstone, 2004.

Method 4.9. Autologous
Adsorption of
Warm-Reactive
Autoantibodies
Principle
Warm-reactive autoantibodies in serum
may mask the concomitant presence of
clinically significant alloantibodies. Adsorption of the serum with autologous red
cells can remove autoantibody from the
serum, permitting detection of underlying alloantibodies. However, autologous
red cells in the circulation are coated with
autoantibody. Autologous adsorption of
warm-reactive autoantibodies can be facilitated by dissociating autoantibody from
the red cell membrane, thereby uncovering antigen sites that can bind free autoantibody to remove it from the serum.
Some autoantibody can be dissociated by
a gentle heat elution for 3 to 5 minutes at
56 C. Subsequent treatment of the cells
with enzymes enhances the adsorption
process by removing membrane structures that otherwise hinder the associa-

780

AABB Technical Manual

tion between antigen and antibody. The
most effective procedure involves the use
of ZZAP reagent, a mixture of a proteolytic
enzyme and a sulfhydryl reagent. ZZAP
removes immunoglobulins and complement from the red cells and enhances the
adsorption process. Red cells from patients transfused within the last 3 months
should not be used for autoadsorption
because transfused red cells present in
the circulation are likely to adsorb the
alloantibodies that are being sought (see
Chapter 20).

5.
6.

Specimen
1.
2.

1 mL of serum or plasma (or eluate)
to be adsorbed.
2 mL of autologous red cells.

Reagents
1.
2.
3.

1% cysteine-activated papain or 1%
ficin (see Methods 3.5.1 and 3.5.2).
Phosphate-buffered saline (PBS), pH
7.3 (see Method 1.7).
0.2 M DTT prepared by dissolving 1
g of DTT in 32.4 mL of pH 7.3 PBS.
Dispense into 3-mL aliquots and
store at –18 C or colder.

Procedure
1.

and remove as much supernatant
saline as possible.
Add serum to an equal volume of
ZZAP-treated red cells, mix, and incubate at 37 C for approximately 30
to 45 minutes.
Centrifuge and carefully remove serum.
If the original serum reactivity was
only 1+, proceed to step 7; otherwise,
repeat steps 4 and 5 once more using the once-adsorbed patient’s serum and the second aliquot of ZZAPtreated cells.
Test the serum against a specimen of
group O reagent cells. If reactivity
persists, repeat steps 4 and 5.

Interpretation
One or two adsorptions ordinarily remove
sufficient autoantibody so that alloantibody reactivity, if present, is readily apparent. If the twice-autoadsorbed serum
reacts with defined specificity, as shown
by testing against a small antibody identification panel, then the defined specificity
of the antibody is probably an alloantibody.
If the serum reacts with all cells on the
panel, either additional autoadsorptions
are necessary, the serum contains antibody to a high-incidence antigen, or the
serum contains an autoantibody (eg,
anti-Kpb) that does not react with ZZAPtreated cells and thus will not be adsorbed
by this procedure. To check this latter
possibility, test the reactive autoadsorbed
serum against reagent cells that have
been pretreated with the ZZAP reagent.

Notes
1.

ZZAP treatment destroys all Kell system antigens and all other antigens
that are destroyed by proteases, eg,
M, N, Fya, and Fyb. ZZAP reagent also
denatures the antigens of the LW,
Cartwright, Dombrock, and Knops

Methods Section 4: Investigation of a Positive DAT

2.
3.

systems. If the autoantibody is suspected to have specificity in any of
these latter blood groups, an alternative procedure is to perform autoadsorption with untreated autologous
cells or autologous cells treated only
with 1% ficin or 1% cysteine-activated
papain.
There is no need to wash packed red
cells before treatment with ZZAP.
Cold autoantibodies reactive at room
temperature can also be present in
the serum of about 30% of patients
with warm-reactive autoantibodies.
Removal of these cold antibodies can
be facilitated by placing the serum
and cell mixture at 4 C for about 15
minutes after incubation at 37 C.
As a guide, when the original serum
reactivity is 1+ in the low-ionicstrength saline indirect antiglobulin
test (LISS-IAT), usually only one adsorption would be required. Antibodies with 2+ to 3+ reactivity will generally be removed in two to three
adsorptions. Performing greater
than four adsorptions increases the
risk of diluting alloantibody reactivity.

blood group systems. The specificity of the
antibodies that remain after adsorption
can be confirmed by testing against a
panel of reagent red cells. This procedure
can be used to detect underlying alloantibodies if the patient has been recently
transfused, or if insufficient autologous
red cells are available and the patient’s
phenotype is unknown.
Treating the adsorbing cells with enzyme
or ZZAP typically enhances the adsorption
process. In addition, the treated red cells will
lack the antigens destroyed by dithiothreitol (DTT) and/or enzymes (see Chapter 19).

Specimen
Serum/plasma containing warm-reactive
autoantibodies or eluate from direct antiglobulin test (DAT)-positive cells.

Reagents
1.
2.
3.
4.

Reference
Branch DR, Petz LD. A new reagent (ZZAP) having
multiple applications in immunohematology. Am
J Clin Pathol 1982;78:161-7.

Method 4.10. Differential
Warm Adsorption Using
Enzyme- or ZZAP-Treated
Allogeneic Red Cells

1% cysteine-activated papain or 1%
ficin (see Methods 3.5.1 and 3.5.2).
ZZAP reagent (papain or ficin plus
0.2 M DTT). See Method 4.9.
Phosphate-buffered saline (PBS), pH
7.3 (see Method 1.7).
Group O red cells of the phenotypes
R1R1, R2R2, and rr; one of these cells
should be Jk(a–b+) and one should
be Jk(a+b–). Additionally, if the red
cells are to be only enzyme-treated,
at least one of the cells should also
be K–. They can be reagent cells or
from any blood specimen that will
yield a sufficient volume of red cells.

Procedure
1.

Principle
Adsorption of serum with selected red cells
of known phenotypes will remove autoantibody and leave antibodies to most

781

Wash 1 mL of each red cell specimen
once in a large volume of saline,
centrifuge to pack the cells, and remove the supernatant saline.
To each volume of washed packed
cells, add one volume of 1% enzyme
solution or two volumes of working

782

AABB Technical Manual

ZZAP reagent. Invert several times to
mix them.
Incubate at 37 C: 15 minutes for enzyme or 30 minutes for ZZAP. Mix
periodically throughout incubation.
Wash the red cells three times with
large volumes of saline. Centrifuge
at 900 to 1000 × g for at least 5 minutes and remove the last wash as
completely as possible to prevent dilution of the serum.
For each of the three red cell specimens, mix one volume of treated
cells with an equal volume of the patient’s serum and incubate at 37 C
for 30 minutes, mixing occasionally.
Centrifuge at 900 to 1000 × g for approximately 5 minutes and harvest
the supernatant serum.
Test the three samples of adsorbed
serum against the cells (untreated)
used for adsorption, respectively. If
reactivity is present, repeat steps 5
through 7 with a fresh aliquot of treated red cells until no reactivity remains. The three samples of adsorbed
serum can then be tested against antibody detection/panel cells and the
results compared for demonstration
of persisting and removed alloantibody activity. See section on allogeneic adsorption in Chapter 20.

Notes
1.

If the autoantibody is very strong,
three or more aliquots of adsorbing
cells should be prepared. If the first
adsorption is unsuccessful, the use
of a higher proportion of cells to serum/eluate may enhance effectiveness.
The adsorbing red cells should be
tightly packed to remove residual saline that might dilute the antibodies
remaining in the serum/eluate.

Agitate the serum/cell mixture during incubation to provide maximum
surface contact.
A visible clue to the effectiveness of
adsorption is clumping of the enzyme- or ZZAP-treated cells when they
are mixed with the serum, especially
when strong antibodies are present.
As a guide, when the original serum
reactivity is 1+ in the low-ionicstrength saline indirect antiglobulin
test (LISS-IAT), usually only one adsorption would be required. Antibodies with 2+ to 3+ reactivity will
generally be removed in two to three
adsorptions. Performing greater than
four adsorptions increases the risk of
diluting alloantibody reactivity.
If adsorption with enzyme- or ZZAPtreated cells has no effect on the
autoantibody, adsorption with untreated red cells may be tried.

References
1.

Branch DR, Petz LD. A new reagent (ZZAP)
having multiple applications in immunohematology. Am J Clin Pathol 1982;78:161-7.
Judd WJ. Methods in immunohematology.
2nd ed. Durham, NC: Montgomery Scientific
Publications, 1994.

Method 4.11. One-Cell
Sample Enzyme or ZZAP
Allogeneic Adsorption
Principle
If the Rh and Kidd phenotypes of a recently transfused patient are known or can
be determined, autoantibody activity can
be adsorbed from the serum onto a single
allogeneic red cell sample, leaving serum
that can be evaluated for the presence of
alloantibodies. The red cells used should
have the same Rh and Kidd phenotypes as
the patient; they can be treated with en-

Methods Section 4: Investigation of a Positive DAT

783

zyme or ZZAP to denature antigens (see
Chapter 19). This method is a simplified
version of the previous adsorption procedure, but it should be used only if the patient’s Rh and Kidd phenotypes are known
(see the note).

Specimen

Note

Serum, plasma, or eluate to be tested.

The s antigen may not be denatured by a
particular enzyme or ZZAP solution. The s
antigen status of the adsorbing red cells
may need to be considered.

Reagents
1.
2.
3.

1% cysteine-activated papain or 1%
ficin (see Methods 3.5.1 and 3.5.2).
ZZAP reagent (papain or ficin plus
0.2 M DTT). See Method 4.9.
ABO-compatible red cells of the patient’s Rh and Kidd phenotypes; they
can be reagent cells or cells from any
blood specimen that will yield sufficient cells.

Procedure
1.

Wash the selected allogeneic red cells
once in a large volume of saline and
centrifuge to pack them.
Add one volume of 1% enzyme solution or two volumes of ZZAP reagent
to one volume of these packed cells.
Invert several times to mix them.
Incubate at 37 C: 15 minutes for enzyme or 30 minutes for ZZAP. Mix
periodically throughout incubation.
Wash the cells three times with saline. Centrifuge at 900 to 1000 × g for
at least 5 minutes and remove the
last wash as completely as possible
to prevent dilution of the serum.
To one volume of treated cells, add
an equal volume of the patient’s serum, mix, and incubate at 37 C for 30
minutes, mixing occasionally.
Centrifuge at 900 to 1000 × g for approximately 5 minutes and harvest
the supernatant serum.

Test the adsorbed serum against the
cells (untreated) used for adsorption.
If reactivity persists, repeat steps 5
through 7 with a fresh aliquot of
treated cells until the serum is no
longer reactive.

Method 4.12. Polyethylene
Glycol Adsorption
Principle
Polyethylene glycol (PEG) enhances the
adsorption of antibody by untreated red
cells. Testing the adsorbed aliquot against
a panel of red cells can identify the specificity of antibodies that remain after adsorption. This method can be used for both
autologous and allogeneic adsorption.

Specimen
Serum or plasma to be tested.

Reagents
1.

PEG, 20% (20 g PEG, 3350 MW, in
100 mL of PBS, pH 7.3) or commercial PEG enhancement reagent.
Autologous red cells or ABO-compatible allogeneic red cells of known
phenotype.

Procedure
1.

Wash aliquots of red cells in large
volumes of saline three times and
centrifuge for 5 to 10 minutes at
1000 × g. Remove all residual saline.
To 1 volume (eg, 1 mL) of red cells,
add 1 volume of serum and 1 vol-

784

AABB Technical Manual

ume of PEG. Mix well and incubate
at 37 C for 15 minutes.
Centrifuge the serum/PEG/cell mixture for 5 minutes and harvest the
adsorbed serum/PEG mixture.
To test the adsorbed serum, add 4
drops of serum to 1 drop of test red
cells, incubate for 15 minutes at 37
C, and proceed to the antiglobulin
test with anti-IgG. The larger volume
of serum tested (4 drops) is required
to account for the dilution of the serum by the PEG. See the notes.
To check for completeness of adsorption, test the adsorbed serum
against the red cells used for the adsorption. If positive, repeat the adsorption by adding the adsorbed serum to a fresh aliquot of red cells but
do not add additional PEG. If the test
was negative, test the adsorbed serum with a panel of cells.

Notes
1.

Red cells for adsorption may be chemically modified (eg, with enzymes or
ZZAP) before adsorption if denaturation of antigens is desired.
The adsorbing cells should be thoroughly packed to remove any residual saline that could result in dilution of the antibodies remaining in
the serum.
Test the adsorbed serum on the day
it was adsorbed. Weak antibody reactivity may be lost upon storage of
PEG-adsorbed sera, possibly due to
precipitation of the protein noticeable
after 4 C storage.
Although many laboratories successfully use the PEG adsorption
method, some serologists have reported a weakening or loss of antibody reactivity in some samples
when compared with results ob-

tained using a different technique.
To accommodate this potential
weakening of antibody reactivity,
some serologists test 6 drops of the
PEG-adsorbed serum.
Agglutination of the adsorbing red
cells does not occur when PEG is
used; therefore, there is no visible
clue to the efficiency of the adsorption process. As a guide, when the
original serum reactivity is 1+ in
low-ionic-strength saline indirect
antiglobulin test (LISS-IAT), usually
only one adsorption would be required. Antibodies with 2 to 3+ reactivity will generally be removed in
two adsorptions.

References
1.

Leger RM, Garratty G. Evaluation of methods
for detecting alloantibodies underlying warm
autoantibodies. Transfusion 1999;39:11-6.
Leger RM, Ciesielski D, Garratty G. Effect of
storage on antibody reactivity after adsorption in the presence of polyethylene glycol.
Transfusion 1999;39:1272-3.

Method 4.13. The
Donath-Landsteiner Test
Principle
IgG autoantibodies that cause paroxysmal
cold hemoglobinuria (PCH) act as biphasic hemolysins in vitro. The IgG autoantibodies bind to the red cells at cold
temperatures, and, as the test is warmed
to 37 C, complement is activated and lysis
of the red cells occurs. The patient for whom
this procedure should be considered is
one with a positive direct antiglobulin test
(DAT) resulting from C3; demonstrable
hemoglobinemia, hemoglobinuria, or
both; and no evidence of autoantibody
activity in the serum or the eluate made
from the DAT-positive cells. For a discussion of PCH, see Chapter 20.

Methods Section 4: Investigation of a Positive DAT

Specimen
Serum separated from a freshly collected
blood sample maintained at 37 C.

Reagents
1.

Freshly collected pooled normal sera
known to lack unexpected antibodies, to use as a source of complement.
50% suspension of washed group O
red cells that express the P antigen,
eg, antibody detection cells.

melting ice (ie, tubes B1, B2). The A3, B3,
and C3 tubes serve as a control of the normal sera complement source and should
not manifest hemolysis.

Notes
1.

Procedure
1.

3.
4.

7.
8.

Label three sets of three 10 × 75-mm
test tubes as follows: A1-A2-A3;
B1-B2-B3; C1-C2-C3.
To tubes 1 and 2 of each set, add 10
volumes (eg, drops) of the patient’s
serum.
To tubes 2 and 3 of each set, add 10
volumes of fresh normal serum.
To all tubes, add one volume of the
50% suspension of washed P-positive red cells and mix well.
Place the three “A” tubes in a bath of
melting ice for 30 minutes and then
at 37 C for 1 hour.
Place the three “B” tubes in a bath of
melting ice and keep them in melting ice for 90 minutes.
Place the three “C” tubes at 37 C and
keep them at 37 C for 90 minutes.
Centrifuge all tubes and examine the
supernatant fluid for hemolysis.

Interpretation
The Donath-Landsteiner test is considered
positive when the patient’s serum, with or
without added complement, causes
hemolysis in the tubes that were incubated first in melting ice and then at 37 C
(ie, tubes A1 and A2), and there is no
hemolysis in any of the tubes maintained
throughout at 37 C (ie, tubes C1, C2) or in

785

The biphasic nature of the hemolysin associated with PCH requires
that serum be incubated with cells at
a cold temperature first (eg, melting
ice bath) and then at 37 C.
Active complement is essential for
demonstration of the antibody. Because patients with PCH may have
low levels of serum complement,
fresh normal serum should be included in the reaction medium as a
source of complement.
To avoid loss of antibody by cold
autoadsorption before testing, the
patient’s blood should be allowed to
clot at 37 C, and the serum separated from the clot at this temperature.
If a limited amount of blood is available (eg, from young children), set
up tubes A-1, A-2, A-3 and C-1, C-2;
if there is only enough serum for two
tests (ie, 20 drops), set up tubes A-2,
A-3, and C-2.
To demonstrate the P specificity of
the Donath-Landsteiner antibody,
ABO-compatible p red cells should
be tested in a second set of tubes
A-1, A-2, and A-3. No lysis should
develop in these tubes, confirming
the P specificity of the antibody.

References
1.

Judd WJ. Methods in immunohematology.
2nd ed. Durham, NC: Montgomery Scientific
Publications, 1994.

Dacie JV, Lewis SM. Practical hematology. 7th
ed. New York: Churchill Livingstone, 1991:
500-1.

786

AABB Technical Manual

Method 4.14. Detection of
Antibodies to Penicillin or
Cephalosporins by Testing
Drug-Treated Red Cells
Principle

See Chapter 20 for a discussion of the
mechanisms by which drugs cause a positive direct antiglobulin test (DAT). The
preparations of drugs used should, to the
extent possible, be the same as those
given to the patient. For drugs other than
penicillin and the cephalosporins, refer to
published reports for the method used to
treat the red cells.

Specimen

Serum or plasma and eluate (and last
wash) to be studied.
4.

Reagents
1.

2.
3.
4.
5.
6.

0.1 M sodium barbital-buffer (BB) at
pH 9.6 to 9.8, prepared by dissolving
2.06 g of sodium barbital in 80 mL of
distilled or deionized H20. Adjust the
pH to between 9.6 and 9.8 with 0.1 N
HCl. Bring total volume to 100 mL.
Store at 2 to 8 C.
Phosphate-buffered saline (PBS), pH
7.3 (see Method 1.7).
Drug, eg, penicillin, cephalosporin.
Washed, packed, group O red cells.
Normal sera/plasma.
IgG-coated red cells.

by adding 1 mL of untreated red
cells (without the drug) to 15 mL of
the same buffer. Incubate both tubes
for 1 hour at room temperature with
occasional mixing. Wash three times
in saline and store in PBS at 2 to 8 C
for up to 1 week. See note 1.
For cephalosporin-treated cells, dissolve 400 mg of the drug in 10 mL of
PBS, pH 7.3. Add 1 mL of red cells. In
a separate tube, prepare control cells
by adding 1 mL of untreated red cells
(without the drug) to 10 mL of PBS.
Incubate both tubes for 1 hour at 37
C with occasional mixing. Wash three
times in saline and store in PBS for
up to 1 week at 2 to 8 C. See notes 1
and 2.
Mix 2 or 3 drops of each specimen
(serum, eluate, and last wash) and
controls with 1 drop of 5% saline
suspension of drug-treated red cells.
In parallel, test each specimen and
control with the untreated red cells.
See notes 3 and 4.
Incubate the tests at 37 C for 60 minutes. Centrifuge and examine for
hemolysis and agglutination. Record
the results.
Wash the cells four times in saline
and test by an indirect antiglobulin
technique using polyspecific antihuman globulin or anti-IgG reagent.
Centrifuge and examine for agglutination. Record the results.
Confirm the validity of negative tests
by adding IgG-coated red cells.

Procedure
1.

For penicillin-treated cells, dissolve
600 mg of penicillin in 15 mL of BB.
This high pH is optimal, but if the
buffer is unavailable, PBS, pH 7.3,
can be used. Add 1 mL of red cells. In
a separate tube, prepare control cells

Interpretation
Reactivity (hemolysis, agglutination, and/
or positive indirect antiglobulin test) with
drug-treated cells, but not with untreated
cells, indicates that drug antibodies are
present (see notes 3 and 4). No hemolysis

Methods Section 4: Investigation of a Positive DAT

will be seen in tests with plasma or the
eluate. Antibodies to either penicillin or
cephalothin may cross-react with cells
treated with the other drug (ie, penicillin
antibodies may attach to cephalothintreated cells and vice versa). Antibodies to
other cephalosporins may react with
cephalothin-treated cells. It is best to treat
cells with the drug that is suspect.
Negative results without a positive control can only be interpreted to mean that
drug antibodies were not detected. The
drug may or may not be bound to the test
red cells.

Notes
1.

The volume of drug-treated red cells
can be scaled down as long as the ratio of the 40 mg/mL drug solution to
red cells is constant; eg, 120 mg penicillin in 3 mL BB plus 0.2 mL red
cells or 100 mg cephalosporin in 2.5
mL PBS plus 0.25 mL red cells.1
Cephalosporins do not require a
high pH for optimal coating of red
cells. In fact, a lower pH, ie, pH 6 to
7, decreases nonspecific protein adsorption seen when a high pH buffer
is used. The least amount of nonspecific protein adsorption by drugtreated red cells will occur if a pH 6.0
buffer is used, but this leads to a
slight decrease in coating by the
drug.
Test normal pooled serum and PBS
as negative controls and, when available, a specimen known to contain
an antibody to the drug being investigated as a positive control.
To control for nonspecific protein
adsorption and nonspecific agglutination of normal sera observed with
some cephalosporins (eg, cephalothin), test the normal serum and the

8.
9.

10.

787

test serum at a 1 in 20 dilution in PBS.
Normal sera diluted 1 in 20 generally
do not react nonspecifically. Thus,
reactivity of the diluted serum with
the drug-treated cells but not with
the untreated cells indicates that
drug antibody is present.
When testing cefotetan-treated red
cells, test the serum at a 1 in 100 dilution in PBS to prevent a false-positive test result. In addition to the
nonspecific uptake of protein onto
cefotetan-treated red cells, some
normal sera appear to have a “naturally occurring” anticefotetan,2 a few
of which react weakly at a 1 in 20 dilution. Cases of cefotetan-induced
immune hemolytic anemia are associated with very high antibody titers
(eg, mean antiglobulin test titer =
3
16,000).
About 30% of patients with anticefotetan also have a drug-independ3
ent antibody ; in these cases, the
serum and/or eluate, when tested
undilute, may react with both the
cefotetan-treated and untreated red
cells.
The last wash control can sometimes react with cefotetan-treated
red cells when antibodies to cefotetan are present, regardless of the
wash solution used (commercial
acid eluate kit wash solution, 4 C
LISS, or 4 C PBS) or the number of
washes performed.3
Prepare drug solutions just before
use.
Drug-treated red cells may be kept
in PBS at 4 C for up to 1 week; however, there may be some weakening
of drug coating upon storage. Drugtreated and untreated red cells may
also be stored frozen.
When antibodies are not detected
with drug-treated red cells, test by

788

AABB Technical Manual

the immune complex method (Method
4.15). Antibodies to some third-generation cephalosporins (eg, ceftriaxone)
do not react with drug-treated red cells.

Petz LD, Garratty G. Immune hemolytic
anemias. 2nd ed. Philadelphia: Churchill
Livingstone, 2004.
Ar ndt P, Garratty G. Is severe immune
hemolytic anemia, following a single dose of
cefotetan, associated with the presence of
“naturally occurring” anti-cefotetan? (abstract) Transfusion 2001;41(Suppl):24S.
Arndt PA, Leger RM, Garratty G. Serology of
antibodies to second- and third-generation
cephalosporins associated with immune
hemolytic anemia and/or positive direct
antiglobulin tests. Transfusion 1999;39:123946.

Method 4.15. Demonstration
of Immune-Complex
Formation Involving Drugs
Principle
For a discussion of the mechanism of
drug-induced immune-complex formation, see Chapter 20.

Specimen
The patient’s serum.

Reagents
1.

2.
3.

Polyspecific antihuman globulin reagent.
IgG-coated red cells.

Procedure

References
1.

The drug under investigation, in the
same form (powder, tablet, capsules)
that the patient is receiving.
Phosphate-buffered saline (PBS) at
pH 7.3 (see Method 1.7).
Fresh, normal serum known to lack
unexpected antibodies, as a source
of complement.
Pooled group O reagent red cells, 5%
suspension, one aliquot treated with
a proteolytic enzyme (see Method
3.5.6) and one untreated.

5.
6.

8.
9.

Prepare a 1 mg/mL solution of the
drug in PBS. Centrifuge to remove
any particulate matter and adjust
the pH of the supernatant fluid to
approximately 7 with either 1 N
NaOH or 1 N HCl, as required, if the
pH is below 5 or above 8.
Label two sets of tubes for the following test mixtures:
a.
Patient’s serum + drug.
b.
Patient’s serum + PBS.
c.
Patient’s serum + complement
(normal serum) + drug.
d.
Patient’s serum + complement
(normal serum) + PBS.
e.
Normal serum + drug.
f.
Normal serum + PBS.
Add 2 volumes (eg, 2 drops) of each
component in the appropriate tubes
(eg, 2 drops of serum + 2 drops of
drug).
Add 1 drop of a 5% saline suspension of untreated group O reagent
red cells to one set of tubes. Add 1
drop of a 5% saline suspension of
enzyme-treated group O reagent red
cells to the second set of tubes.
Mix and incubate at 37 C for 1 to 2
hours, with periodic gentle mixing.
Centrifuge, examine for hemolysis
and agglutination, and record the results.
Wash the cells four times in saline
and test with a polyspecific antiglobulin reagent.
Centrifuge, examine for agglutination, and record the results.
Confirm the validity of negative tests
by adding IgG-coated red cells.

Methods Section 4: Investigation of a Positive DAT

Interpretation
Hemolysis, direct agglutination, or positive indirect antiglobulin tests can occur
together or separately. Reactivity in any of
the tests containing the patient’s serum to
which the drug was added, and absence
of reactivity in the corresponding control
tests containing PBS instead of the drug,
indicate that antibody to the drug is present. See note 4.

789

ment may increase the sensitivity of
the test.
If tests for drug antibodies by the
immune-complex method and drug
adsorption method are noninformative, consider testing with ex-vivo
antigen (see Method 4.16).

Reference
Petz LD, Garratty G. Immune hemolytic anemias.
2nd ed. Philadelphia: Churchill Livingstone, 2004.

Notes
1.

The drug may be more easily dissolved by incubation at 37 C and vigorous shaking of the solution. If the
drug is in tablet form, crush it with a
mortar and pestle and remove any
visible outer tablet coating material
before adding PBS.
Not all drugs will dissolve completely in PBS. Consult the manufacturer or a reference such as the Merck
Index for the solubility of the drug in
question. A previous report of druginduced immune hemolytic anemia
resulting from the drug in question
may provide information on the
drug solution preparation.
When available, a serum/plasma
known to contain antibody with the
drug specificity being evaluated
should be included as a positive
control.
Tests without the drug added may be
positive if autoantibodies or circulating drug-antibody immune complexes are present in the patient’s
sample. Autoantibody reactivity would
be persistent over time, whereas circulating immune complexes are
transient.
Testing with enzyme-treated red
cells and the addition of fresh normal serum as a source of comple-

Method 4.16. Ex-Vivo
Demonstration of
Drug/Anti-Drug Complexes
Principle
Immune drug/anti-drug complexes can
activate complement and cause hemolysis
in vivo. These immune complexes may be
demonstrable by serologic testing in the
presence of the drug, but with some drugs
(notably nomifensine), antibodies are directed against metabolites of the drug,
rather than the native drug. Serum and/or
urine from volunteers who have ingested
therapeutic levels of the drug can be used
as a source of these metabolites. See note
1.
This procedure is used to investigate
drug-associated immune hemolysis, particularly when use of the preceding methods
has been noninformative.

Specimen
Patient’s serum.

Reagents
1.
2.

Polyspecific antihuman globulin
(AHG) reagent.
Drug metabolites from volunteer
drug recipients. See note 2.

790

AABB Technical Manual

4.
5.

6.
7.

Volunteer serum (VS) obtained
immediately before (VS0), at 1
hour (VS1), and 6 hours (VS6)
after drug administration. Divide serum into 1-mL aliquots
and store them at 2 to 8 C for a
few hours or at –20 C or colder
until use.
b.
Volunteer urine (VU) obtained
immediately before (VU0), at 1
hour (VU1), 3.5 hours (VU3.5), 7
hours ( VU 7 ), and 16 hours
(VU 16 ) after drug administration. Divide into 1-mL aliquots
and store them at 2 to 8 C for a
few hours or at –20 C or colder
until use.
Fresh normal serum, known to lack
unexpected antibodies, as a source of
complement.
Phosphate-buffered saline (PBS), pH
7.3 (see Method 1.7).
Pooled group O reagent red cells
washed three times with saline and
resuspended to a 5% concentration
with PBS.
Pooled, enzyme-treated, group O red
cells, 5% suspension in PBS.
IgG-coated red cells.

7.
8.

Add 1 drop of a 5% saline suspension of the untreated group O reagent red cells to one set of tubes.
Add 1 drop of a 5% saline suspension of enzyme-treated reagent red
cells to the second set of tubes.
Mix the contents of each tube and
incubate at 37 C for 1 to 2 hours, with
periodic mixing.
Centrifuge, examine for agglutination and/or hemolysis, and record
the results.
Wash the cells four times with saline
and test with a polyspecific antiglobulin reagent.
Centrifuge, examine for agglutination, and record the results.
Confirm the validity of negative tests
by adding IgG-coated red cells.

Interpretation
Hemolysis, direct agglutination, or reactivity with AHG in any of the tubes containing test serum and VS or VU, and absence of reactivity in all the control tubes,
indicate antibody against a metabolite of
the drug in question.

Procedure
1.

For each volunteer serum and/or
volunteer urine sample collected, label two sets of the following test
mixtures:
a.
Patient’s serum + VS (or VU).
b.
Patient’s serum + PBS.
c.
Patient’s serum + complement
+ VS (or VU).
d.
Patient’s serum + complement
+ PBS.
e.
Complement + VS (or VU).
f.
Complement + PBS.
Add 0.1 mL of each component to the
appropriate tubes.

Notes
1.

Approval of the institutional ethics
committee should be obtained for
the use of volunteers for obtaining
drug metabolites.
The urine sample collection times
given are those optimal for antibodies to nomifensine metabolites; different collection times may be required for other drugs.
Complement may be omitted from
step 1 if the VS samples have been
kept on ice and are used for testing
within 8 hours of collection.

Methods Section 4: Investigation of a Positive DAT

Testing with enzyme-treated red
cells and the addition of fresh normal serum as a source of complement
may increase the sensitivity of the
test.

791

References
1.

Judd WJ. Methods in immunohematology.
2nd ed. Durham, NC: Montgomery Scientific
Publications, 1994.
Salama A, Mueller-Eckhardt C. The role of metabolite-specific antibodies in nomifensinedependent immune hemolytic anemia. N
Engl J Med 1985;313:469-74.

Methods Section 5: Hemolytic Disease of the Fetus and Newborn

Methods Section 5

Method 5.1. Indicator Cell
Rosette Test for
Fetomaternal Hemorrhage
Principle
This test detects D+ red cells in the blood
of a D– woman whose fetus or recently
delivered infant is D+. When reagent
anti-D is added to maternal blood containing D+ fetal cells, fetal red cells become coated with anti-D. When D+ reagent cells are subsequently added, easily
visible rosettes are formed, with several
red cells clustered around each antibody-coated D+ red cell.
Although the number of rosettes is
roughly proportional to the number of D+
red cells present in the original mixture,
this test provides only qualitative information about fetal-maternal admixture. Specimens giving a positive result should be subjected to further testing to quantify the

number of fetal cells. The acid-elution procedure given below and flow cytometry are
acceptable choices. If a commercial test is
available, the directions in the package insert should be followed.

Specimen
A 2% to 5% saline suspension of washed
red cells from a maternal blood sample.

Reagents
Prepared reagents are commercially available. The steps below can be used for inhouse preparation.
1.
Negative control: a 2% to 5% saline
suspension of washed red cells known
to be D–.
2.
Positive control: a 2% to 5% saline
suspension of a mixture containing
approximately 0.6% D+ red cells and
99.4% D– red cells. The positive control can be prepared by adding 1 drop
793

Section 5

Hemolytic Disease of the
Fetus and Newborn

794

AABB Technical Manual

of a 2% to 5% suspension of D+ control cells to 15 drops of a 2% to 5%
suspension of washed D– control cells.
Mix well, then add 1 drop of this cell
suspension to 9 drops of the 2% to
5% suspension of D– red cells. Mix well.
Indicator red cells: a 2% to 5% saline
suspension of group O, R2R2 red cells.
Either enzyme-treated or untreated
cells in an enhancing medium can be
used.
Chemically modified or high-protein
reagent anti-D serum. Some monoclonal/polyclonal blended reagents
are unsuitable for use in this method.
The antisera selected for use should
be evaluated for suitability before incorporation into the test procedure.

Procedure
1.

6.
7.

To each of three test tubes, add 1 drop
(or volume specified in the manufacturer’s instructions) of reagent anti-D.
Add 1 drop of maternal cells, negative control cells, and positive control
cells to the appropriately labeled tubes.
Incubate at 37 C for 15 to 30 minutes, or as specified by the manufacturer’s instructions.
Wash cell suspensions at least four
times with large volumes of saline, to
remove all unbound reagent anti-D.
Decant saline completely after last
wash.
To the dry cell button, add 1 drop of
indicator cells and mix thoroughly to
resuspend them.
Centrifuge the tubes for 15 seconds
at 900 to 1000 × g.
Resuspend cell button and examine
the red cell suspension microscopically at 100 to 150 × magnification.
Examine at least 10 fields and count
the number of red cell rosettes in each
field.

Interpretation
The absence of rosettes is a negative result. With enzyme-treated indicator cells,
up to one rosette per three fields may occur in a negative specimen. With untreated indicator cells and an enhancing
medium, there may be up to six rosettes
per five fields in a negative test. The presence of more rosettes than these allowable maxima constitutes a positive result,
and the specimen should be examined
using a test that quantifies the amount of
fetal blood present.
The presence of rosettes or agglutination
in the negative control tube indicates inadequate washing after incubation, allowing
residual anti-D to agglutinate the D+ indicator cells. A strongly positive result is seen
with red cells from a woman whose Rh
phenotype is weak D rather than D–; massive fetomaternal hemorrhage may produce an appearance difficult to distinguish
from that caused by a weak D phenotype,
and a quantitative test for fetal cells should
be performed. If the infant’s cells are shown
to be weak D, a negative result on the
mother’s specimen should be interpreted
with caution. In this situation, a quantitative test that does not rely on D antigen
expression should be performed.

Reference
Sebring ES, Polesky HF. Detection of fetal maternal hemorrhage in Rh immune globulin candidates. Transfusion 1982;22:468-71.

Method 5.2. Acid-Elution Stain
(Modified Kleihauer-Betke)
Principle
Fetal hemoglobin resists elution from red
cells under acid conditions, whereas adult
hemoglobin is eluted. When a thin blood
smear is exposed to an acid buffer, hemo-

Methods Section 5: Hemolytic Disease of the Fetus and Newborn

globin from adult red cells is leached into
the buffer so that only the stroma remains;
fetal cells retain their hemoglobin and
can be identified by a positive staining
pattern. The approximate volume of fetomaternal hemorrhage can be calculated
from the percentage of fetal red cells in
the maternal blood film.

2.
3.
4.

Specimen

5.
6.

Maternal anticoagulated whole blood
sample.

Reagents

Prepared reagents are commercially available in kits. The steps below can be used
for in-house preparations.
1.
Stock solution A (0.1 M of citric acid).
C6H8O7•H2O, 21.0 g, diluted to 1 liter
with distilled water. Keep it in the refrigerator.
2.
Stock solution B (0.2 M of sodium
phosphate). Na2HPO4•7H2O, 53.6 g,
diluted to 1 liter with distilled water.
Keep it in the refrigerator.
3.
McIlvaine’s buffer, pH 3.2. Add 75
mL of stock solution A to 21 mL of
stock solution B. Prepare fresh mixture for each test. This buffer mixture should be brought to room temperature or used at 37 C.
4.
Erythrosin B, 0.5% in water.
5.
Harris hematoxylin (filtered).
6.
80% ethyl alcohol.
7.
Positive control specimen. Ten parts
of anticoagulated adult blood, mixed
with one part of anticoagulated ABOcompatible cord blood.
8.
Negative control specimen. Anticoagulated adult blood.

Procedure
1.

Prepare very thin blood smears, diluting blood with an equal volume of
saline. Air dry.

10.

11.

795

Fix the smears in 80% ethyl alcohol
for 5 minutes.
Wash the smears with distilled water.
Immerse the smears in McIlvaine’s
buffer, pH 3.2, for 11 minutes at room
temperature or 5 minutes at 37 C. This
reaction is temperature-sensitive.
Wash the smears in distilled water.
Immerse the smears in erythrosin B
for 5 minutes.
Wash the smears completely in distilled water.
Immerse the smears in Harris hematoxylin for 5 minutes.
Wash the smears in running tap water for 1 minute.
Examine dry using 40× magnification, count a total of 2000 red cells,
and record the number of fetal cells
observed.
Calculate the percent of fetal red cells
in the total counted.

Interpretation
1.

Fetal cells are bright pink and refractile; normal adult red cells appear as very pale ghosts.
The conversion factor used to indicate the volume (as mL of whole
blood) of fetomaternal hemorrhage
is the percent of fetal red cells observed times 50.

Note
The accuracy and precision of this procedure are poor, and decisions regarding Rh
Immune Globulin (RhIG) dosage in massive fetomaternal hemorrhage should be
made accordingly. If there is a question
regarding the need for additional RhIG, it
is preferable to administer another dose
to prevent the risks of undertreatment. (See
Table 23-1 for dosage.)

796

AABB Technical Manual

Reference

Materials

Sebring ES. Fetomaternal hemorrhage—incidence
and methods of detection and quantitation. In:
Garratty G, ed. Hemolytic disease of the newborn.
Arlington, VA: AABB, 1984:87-118.

Method 5.3. Antibody
Titration Studies to Assist in
Early Detection of Hemolytic
Disease of the Fetus and
Newborn
Principle

2.
3.

Antibody titration is a semiquantitative
method of determining antibody concentration. Serial twofold dilutions of serum
are prepared and tested for antibody activity. The reciprocal of the highest dilution of plasma or serum that gives a 1+ reaction is referred to as the titer (ie, 1 in
128 dilution; titer = 128).
In pregnancy, antibody titration is performed to identify women with significant
levels of antibodies that may lead to
hemolytic disease of the fetus and newborn
(HDFN) and, for low-titer antibodies, to establish a baseline for comparison with
titers found later in pregnancy. Titration of
non-Rh antibodies should be undertaken
only after discussion with the obstetrician
about how the data will be used in the clinical management of the pregnancy. The significance of titers has been sufficiently established only for anti-D (using a saline
technique).

Specimen

Quality Control
1.
2.

Test the preceding sample in parallel
with the most recent sample.
Prepare the dilutions using a separate pipette for each tube. Failure to
do so will result in falsely high titers
because of carryover.
Confirm all negative reactions with
IgG-coated red cells (see step 9 below).

Procedure
1.

2.
3.

Serum for titration (containing potentially
significant unexpected antibodies to red
cell antigens, 1 mL). If possible, test the
current sample in parallel with the most
recent previously submitted (preceding)
sample from the current pregnancy.

Antihuman IgG: need not be heavychain-specific.
Isotonic saline.
Volumetric pipettes, or equivalent:
0.1- to 0.5-mL delivery, with disposable tips.
Red cells: group O reagent red cells,
2% suspension. (See note 1 regarding the selection of red cells for testing.) Avoid using Bg+ red cells because they may result in falsely high
values, especially with sera from
multiparous women.
IgG-coated red cells.

Using 0.5-mL volumes, prepare serial
twofold dilutions of serum in saline.
The initial tube should contain undiluted serum and the doubling dilution range should be from 1 in 2 to
1 in 2048 (total of 12 tubes). (See
Method 3.7.)
Place 0.1 mL of each dilution into
appropriately labeled test tubes.
Add 0.1 mL of the 2% suspension of
red cells to each dilution. Alternatively, for convenience, add 1 drop of
a solution of a 3% to 4% suspension
of red cells as supplied by the reagent manufacturer, although this
method is less precise.

Methods Section 5: Hemolytic Disease of the Fetus and Newborn

4.
5.

7.
8.
9.

Gently agitate the contents of each
tube; incubate at 37 C for 1 hour.
Wash the red cells four times with
saline; completely decant the final
wash supernatant.
To the dry red cell buttons thus obtained, add anti-IgG according to the
manufacturer’s directions.
Centrifuge as for hemagglutination
tests.
Examine the red cells macroscopically;
grade and record the reactions.
Add IgG-coated red cells to all negative tests; recentrifuge and examine
the tests for macroscopic agglutination; repeat the testing if the tests with
IgG-coated red cells are nonreactive.

Interpretation
The titer is reported as the reciprocal of
the highest dilution of serum at which 1+
agglutination is observed. A titer ≥16 (this
value may vary according to the laboratory) is considered significant and may
warrant further monitoring for HDFN.

Notes
1.

The selection of the most suitable
phenotype of red cells to use when
performing titration studies for HDFN
is controversial. Some workers select
red cells that have the strongest expression of antigen, such as R2R2 for
anti-D. Others select red cells with the
phenotype that would be expected
in fetal circulation—ie, red cells that
express a single dose of the antigen,
such as R 1 r for testing for anti-D.
Whichever viewpoint is followed, it
is important that the laboratory be
consistent and use red cells of the
same phenotype for future titrations
to test the same patient’s serum.
Titration studies should be performed
upon initial detection of the anti-

797

body; save an appropriately labeled
aliquot of the serum (frozen at –20 C
or colder) for comparative studies
with the next submitted sample.
When the titer (eg, >16) and the antibody specificity have been associated with HDFN, it is recommended
that repeat titration studies be performed every 2 to 4 weeks, beginning
at 18 weeks’ gestation; save an aliquot of the serum (frozen at –20 C or
colder) for comparative studies with
the next submitted sample.
When invasive procedures (eg, amniocentesis) have demonstrated fetal
compromise and are being used to
monitor the pregnancy, use the optimal method for follow-up of fetal
well-being. However, if initial studies
do not show fetal compromise or the
Liley curve result is borderline, additional titrations may be helpful as a
means of following the pregnancy in
a less invasive manner.
Each institution should develop a
policy to ensure a degree of uniformity in reporting and interpreting
antibody titers.
For antibodies to low-incidence antigens, consider using putative paternal red cells, having established
that they express the antigen in question.
Do not use enhancement techniques
[albumin, polyethylene glycol, lowionic-strength saline (LISS)] or enzyme-treated red cells because falsely
elevated titers may be obtained. Gel
testing is not recommended.
LISS should not be used as a diluent
in titration studies; nonspecific uptake of globulins may occur in serumLISS dilutions.
Failure to obtain the correct results
may be caused by 1) incorrect technique, notably, failure to use sepa-

798

AABB Technical Manual

rate pipette tips for each dilution or
2) failure to adequately mix thawed
frozen serum.

References
1.

Issitt PD, Anstee DJ. Applied blood group serology. 4th ed. Durham, NC: Montgomery
Scientific Publications, 1998:1067-9.
Judd WJ, Luban NLC, Ness PM, et al. Prenatal
and perinatal immunohematology: Recom-

mendations for serologic management of the
fetus, newborn infant, and obstetric patient.
Transfusion 1990;30:175-83.
Judd WJ. Methods in immunohematology.
2nd ed. Durham, NC: Montgomery Scientific
Publications, 1994.
Judd WJ. Practice guidelines for prenatal and
perinatal immunhematology, revisited. Transfusion 2001;41:1445-52.

Methods Section 6: Blood Collection, Storage, and Component Preparation

Methods Section 6

Blood Collection, Storage,
and Component Preparation

Principle
This method estimates the hemoglobin
content of blood from its specific gravity.
A drop of blood in contact with copper
sulfate solution of specific gravity 1.053
becomes encased in a sac of copper proteinate, which prevents dispersion of the
fluid or any change in specific gravity for
about 15 seconds. If the specific gravity of
the blood is higher than that of the solution, the drop will sink within 15 seconds;
if not, the drop will hesitate and remain
suspended or rise to the top of the solution. A specific gravity of 1.053 corresponds to a hemoglobin concentration of
12.5 g/dL.
This is not a quantitative test; it shows
only whether the prospective donor’s hemoglobin is below or above the acceptable
level of 12.5 g/dL. False-positive reactions

are rare; donors whose drop of blood sinks
nearly always have an acceptable hemoglobin level. False-negative reactions occur
fairly commonly and can cause inappropriate deferral.1,2 Measuring hemoglobin by
another method or determining hematocrit
sometimes reveals that the prospective donor is acceptable.

Reagents and Materials
1.

2.
3.
4.

Copper sulfate solution at specific
gravity 1.053, available commercially. Store it in tightly capped containers to prevent evaporation. The
solution should be kept at room temperature or brought to room temperature before it is used.
Sterile gauze, antiseptic wipes, and
sterile lancets.
Containers for the disposal of sharps
and other biohazardous materials.
Capillary tubes and dropper bulbs or
a device to collect capillary blood
without contact.
799

Section 6

Method 6.1. Copper Sulfate
Method for Screening Donors
for Anemia

800

AABB Technical Manual

at an acceptable level for blood donation. If time and equipment permit,
it is desirable to perform a quantitative measurement of hemoglobin or
hematocrit.

Procedure
1.

6.
7.

Into a labeled, clean, dry tube or
bottle, dispense a sufficient amount
(at least 30 mL) of copper sulfate solution to allow the drop to fall approximately 3 inches. Change the solution daily or after 25 tests. Be sure
that the solution is adequately mixed
before beginning each day’s determinations.
Clean the site of the skin puncture
thoroughly with antiseptic solution
and wipe it dry with sterile gauze.
Puncture the finger firmly, near the
end but slightly to the side, with a
sterile, disposable lancet or springloaded, disposable needle system. A
good free flow of blood is important.
Do not squeeze the puncture site repeatedly because this may dilute the
drop of blood with tissue fluid and
lower the specific gravity.
Collect the blood in a capillary tube
without allowing air to enter the
tube.
Let one drop of blood fall gently
from the tube at a height about 1 cm
above the surface of the copper sulfate solution.
Observe for 15 seconds.
Dispose of lancets and capillary tubes
in appropriate biohazard containers.
Dispose of gauze appropriately;
gauze contaminated with droplets of
blood that subsequently dry such
that the item is stained but not
soaked or caked may be considered
nonhazardous.

Interpretation
1.

If the drop of blood sinks, the donor’s hemoglobin is at an acceptable
level for blood donation.
If the drop of blood does not sink,
the donor’s hemoglobin may not be

Notes
1.

A certificate of analysis from the manufacturer should be obtained with
each new lot of copper sulfate solution.
Used solution should be disposed of
as biohazardous or chemical material because of the blood in the container. Refer to local and state laws
regarding disposal.
Use care to prevent blood from contaminating work surfaces, the donor’s
clothing, or other persons or equipment.
Cover the container between uses to
prevent evaporation.

References
1.

Lloyd H, Collins A, Walker W, et al. Volunteer
blood donors who fail the copper sulfate
screening test: What does failure mean, and
what should be done? Transfusion 1988;28:
467-9.

Morris MW, Davey FR. Basic examination of
blood. In: Henry JB, ed. Clinical diagnosis
and management by laboratory methods.
20th ed. Philadelphia: WB Saunders, 2001:
479-519.

Method 6.2. Arm Preparation
for Blood Collection
Detailed instructions are specific to each
manufacturer and should be followed as
indicated. The following procedure is
written in general terms as an example.

Methods Section 6: Blood Collection, Storage, and Component Preparation

Principle
Iodophor compounds, or other sterilizing
compounds, are used to sterilize the venipuncture site before blood collection.

Materials
1.

Scrub solution: Disposable povidoneiodine scrub 0.75% or disposable
povidone-iodine swabstick 10%; available in prepackaged single-use form.
Preparation solution: 10% povidoneiodine; available in prepackaged single-use form.
Sterile gauze.

Procedure
1.

Apply tourniquet or blood pressure
cuff; identify venipuncture site, then
release tourniquet or cuff.
Scrub area at least 4 cm (1.5 inches)
in all directions from the intended
site of venipuncture (ie, 8 cm or 3
inches in diameter) for a minimum
of 30 seconds with 0.7% aqueous solution of iodophor compound. Excess
foam may be removed, but the arm
need not be dry before the next step.
Starting at the intended site of venipuncture and moving outward in a
concentric spiral, apply “prep” solution; let stand for 30 seconds or as
indicated by manufacturer.
Cover the area with dry, sterile gauze
until the time of venipuncture. After
the skin has been prepared, it must
not be touched again. Do not repalpate the vein at the intended venipuncture site.

For donors sensitive to iodine (tincture or povidone preparations), another method (eg, ChloraPrep 2%

chlorhexidine and 70% isopropyl alcohol) should be designated by the
blood bank physician. Green soap
should not be used.
For donors sensitive to both iodine
and chlorhexidine, a method using
only isopropyl alcohol could be considered. The preferred procedure is
the use of a 30-second up-and-down
scrub, followed by enough time for
the skin to dry. A second scrub is
then applied. This method may require a variance from the Food and
Drug Administration.
Arm preparation methods approved
by the Food and Drug Administration are available at http://www.fda.
gov/cber/infosheets/armprep.htm.

Reference
Goldman M, Roy G, Frechette N, et al. Evaluation
of donor skin disinfection methods. Transfusion
1997;37:309-12.

Method 6.3. Phlebotomy and
Collection of Samples for
Processing and Compatibility
Tests
Principle
Blood for transfusion and accompanying
samples is obtained from prominent veins
on the donor’s arm, usually in the area of
the antecubital fossa.

Materials
1.

2.
3.

Notes

801

Sterile collection bag containing anticoagulant, with integrally attached
tubing and needle.
Metal clips and hand sealers.
Balance system to monitor volume
of blood drawn.
Sterile gauze and clean instruments
(scissors, hemostats, forceps).

802

5.
6.
7.

AABB Technical Manual

Test tubes for sample collection.
Device for stripping blood in the
tubing.
Dielectric sealer (optional).

Procedure
1.
2.

3.
4.

Ask donor to confirm his or her identification.
Ensure that all labeling on blood container, processing tubes, retention
segment, and donor records is correct.
Prepare donor’s arm as described in
Method 6.2.
Inspect bag for any defects and discoloration. The anticoagulant and
additive solutions should be inspected for particulate contaminants.
Position bag below the level of the
donor’s arm.
a.
If a balance system is used, be
sure the counterbalance is level
and adjusted for the amount of
blood to be drawn. Unless metal
clips and a hand sealer are used,
make a very loose overhand knot
in the tubing. Hang the bag and
route the tubing through the
p i n c h c l a m p. A h e m o s t a t
should be applied to the tubing
before the needle is uncapped
to prevent air from entering the
line.
b.
If a balance system is not used,
be sure to monitor the volume
of blood drawn.
Reapply tourniquet or inflate blood
pressure cuff. Ask the donor to open
and close hand until previously selected vein is again prominent.
Uncover sterile needle and perform
the venipuncture immediately. A
clean, skillful venipuncture is essential for collection of a full, clot-free
unit. Once the bevel has penetrated

10.

11.

12.

13.

the skin, palpation of the skin above
the needle stem may be performed
with a gloved finger, provided the
needle is not touched. When the
needle position is acceptable, tape
the tubing to the donor’s arm to hold
the needle in place and cover the
site with sterile gauze.
Release the hemostat. Open the temporary closure between the interior
of the bag and the tubing.
Ask the donor to open and close hand
slowly every 10 to 12 seconds during
collection.
Keep the donor under observation
throughout the donation process.
The donor should never be left unattended during or immediately after
donation.
Mix blood and anticoagulant gently
and periodically (approximately every 45 seconds) during collection.
Mixing may be done by hand or by
continuous mechanical mixing.
Be sure blood flow remains fairly
brisk, so that coagulation activity is
not triggered. If there is continuous,
adequate blood flow and constant
agitation, rigid time limits are not
necessary. However, units requiring
more than 15 minutes to draw may
not be suitable for preparation of
Platelets, Fresh Frozen Plasma, or
Cryoprecipitated AHF. The time required for collection can be monitored by indicating the time of phlebotomy or the maximal allowable
time (start time plus 15 minutes) on
the donor record.
Monitor volume of blood being drawn.
If a balance is used, the device will
interrupt blood flow after the proper
amount has been collected. One mL
of blood weighs at least 1.053 g, indicated by the minimum allowable
specific gravity for donors. A conve-

Methods Section 6: Blood Collection, Storage, and Component Preparation

14.

nient figure to use is 1.06 g/mL; a unit
containing 405 to 550 mL should
weigh 429 to 583 g plus the weight of
the container and anticoagulant. For
a 500-mL bag, this is 565 to 671 g.
Clamp the tubing near the venipuncture using a hemostat, metal
clip, or other temporary clamp. Release the blood pressure cuff/tourniquet to 20 mm Hg or less and fill the
tube(s) for blood processing sample(s) by a method that prevents
contamination of the contents of the
bag. This can be done in several ways.
a.
If the blood collection bag contains an inline needle, make an
additional seal with a hemostat, metal clip, hand sealer, or
a tight knot made from previously prepared loose knot just
distal to the inline needle. Open
the connector by separating
the needles. Insert the proximal
needle into a processing test
tube, remove the hemostat, allow the tube to fill, and reclamp the tubing. The donor
needle is now ready for removal.
b.
If the blood collection bag contains an inline processing tube,
be certain that the processing
tube, or pouch, is full when the
collection is complete and the
original clamp is placed near
the donor needle. The entire
assembly may now be removed
from the donor.
c.
If a straight-tubing assembly
set is used, the following procedure should be followed. Place
a hemostat on the tubing, allowing about four segments between the hemostat and the
needle. Pull tight the loose
overhand knot made in step 3.
Release the hemostat and strip

15.

16.

17.

18.

803

a segment of the tubing free of
blood between the knot and
the needle (about 1 inch in
length). Reapply the hemostat
and cut the tubing in the stripped area between the knot and
the hemostat. Fill the required
tube(s) by releasing the hemostat and then reclamp the tubing with the hemostat. Because
this system is open, Biosafety
Level 2 precautions should be
followed.
Deflate the cuff and remove the
tourniquet. Remove the needle from
the donor’s arm, if not already removed. Apply pressure over the gauze
and ask the donor to raise his or her
arm (elbow straight) and hold the
gauze firmly over the phlebotomy
site with the other hand.
Discard the needle assembly into a
biohazard container designed to prevent accidental injury to, and contamination of, personnel.
Strip donor tubing as completely as
possible into the bag, starting at the
seal. Work quickly, to prevent the
blood from clotting in the tubing. Invert the bag several times to mix the
contents thoroughly; then allow the
tubing to refill with anticoagulated
blood from the bag. Repeat this procedure a second time.
Seal the tubing attached to the collection bag into segments, leaving a
segment number clearly and completely readable. Attach a unit identification number to one segment to
be stored as a retention segment.
Knots, metal clips, or a dielectric
sealer may be used to make segments suitable for compatibility
testing. It must be possible to separate segments from the unit without
breaking sterility of the bag. If a di-

804

19.
20.

21.

AABB Technical Manual

electric sealer is used, the knot or
clip should be removed from the distal end of the tubing after creating a
hermetic seal.
Reinspect the container for defects.
Recheck numbers on the container,
processing tubes, donation record,
and retention segment.
Place blood at appropriate temperature. Unless platelets are to be removed, whole blood should be placed
at 1 to 6 C immediately after collection. If platelets are to be prepared,
blood should not be chilled but
should be stored in a manner intended to reach a temperature of 20
to 24 C until platelets are separated.
Platelets must be separated within 8
hours after collection of the unit of
Whole Blood.

Method 6.4. Preparation of
Red Blood Cells
Principle
Red Blood Cells (RBCs) are obtained by
removal of supernatant plasma from centrifuged Whole Blood. The volume of
plasma removed determines the hematocrit of the component. When RBCs are
preserved in CPDA-1, maximal viability
during storage requires an appropriate ratio of cells to preservative. A hematocrit of
80% or lower ensures the presence of adequate glucose for red cell metabolism for
up to 35 days of storage.

Materials
1.

Notes
1.

If the needle is withdrawn and venipuncture is attempted again, preparation of the site must be repeated as
in Method 6.2.
In addition to routine blood donor
phlebotomy, this procedure may be
adapted for use in therapeutic phlebotomy.

2.
3.
4.
5.
6.
7.

Freshly collected Whole Blood, obtained by phlebotomy as described
in Method 6.3. Collect blood in a collection unit with integrally attached
transfer container(s).
Plasma extractor.
Metal clips and hand sealer.
Clean instruments (scissors, hemostats).
Dielectric sealer (optional).
Refrigerated centrifuge.
Scale.

Procedure

References
1.

Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.

Smith LG. Blood collection. In: Green TS,
Steckler D, eds. Donor room policies and procedures. Arlington, VA: AABB, 1985:25-45.

Huh YO, Lightiger B, Giacco GG, et al. Effect
of donation time on platelet concentrates
and fresh frozen plasma. Vox Sang 1989;56:
21-4.

Sataro P. Blood collection. In: Kasprisin CA,
Laird-Fryer B, eds. Blood donor collection
practices. Bethesda, MD: AABB, 1993:89-103.

Centrifuge whole blood using a “heavy”
spin (see Method 7.4), with a temperature setting of 4 C.
Place the primary bag containing
centrifuged blood on a plasma expressor, and release the spring, allowing the plate of the expressor to
contact the bag.
Temporarily clamp the tubing between the primary and satellite bags
with a hemostat or, if a mechanical

Methods Section 6: Blood Collection, Storage, and Component Preparation

sealer will not be used, make a loose
overhand knot in the tubing.
If two or more satellite bags are attached, apply the hemostat to allow
plasma to flow into only one of the
satellite bags. Penetrate the closure
of the primary bag. A scale, such as a
dietary scale, may be used to measure the expressed plasma. Remove
the appropriate amount of plasma
to obtain the desired hematocrit.
Reapply the hemostat when the desired amount of supernatant plasma
has entered the satellite bag. Seal the
tubing between the primary bag and
the satellite bag in two places.
Check that the satellite bag has the
same donor number as that on the
primary bag and cut the tubing between the two seals.

Table 6.4-1. Removing Plasma from
Units of Whole Blood (To Prepare RBCs
in Anticoagulant-Preservative with a
Known Hematocrit)
Hematocrit of Volume of
Final
Segment from Plasma
Hematocrit of
Whole Blood
to Be
Red Blood Cell
Unit
Removed
Unit

Notes
1.

If blood was collected in a single bag,
modify the above directions as follows: after placing the bag on the
expressor, apply a hemostat to the
tubing of a sterile transfer bag, aseptically insert the cannula of the transfer bag into the outlet port of the bag
of blood, release the hemostat, and
continue as outlined above. The expiration date will change, however.
Collection of blood in an additive solution allows removal of a greater
volume of plasma in step 4. After the
plasma has been removed, the additive solution is allowed to flow from
the attached satellite bag into the red
cells. This will result in a hematocrit
of 55% to 65%. Be sure that an appropriate label and dating period are
used.
The removal of 230 to 256 g (225 to
250 mL) of plasma and preservation

805

40%

150 mL

56%

39%

150 mL

55%

38%

160 mL

55%

37%

165 mL

54%

36%

170 mL

54%

35%

180 mL

54%

34%

195 mL

55%

33%

200 mL

55%

of the red cells in the anticoagulantpreservative solution will generally
result in a red cell component with a
hematocrit between 70% and 80%.
See Table 6.4-1 to prepare Red Blood
Cells with a specific (desired) hematocrit.

Method 6.5. Preparation of
Prestorage Red Blood Cells
Leukocytes Reduced
Principle
The general principle and materials of
Method 6.4 apply, except that the red cells
are filtered using a special leukocyte reduction filter. All red cell leukocyte reduction filters licensed in the United States
remove platelets to some degree. Anticoagulated whole blood may be filtered,
from which only platelet-poor plasma

806

AABB Technical Manual

(leukocyte reduced) and red cells may be
made. Alternatively, the red cells may be
filtered in additive solution, potentially
allowing the preparation of platelets,
plasma, and red cells. Nonleukocyte-reduced red cells may also undergo leukocyte reduction after preparation by attaching a leukocyte reduction filter connected to a storage container using a sterile connection device.

Notes
1.

If the collection system does not include an in-line filter, a sterile connection device can be used to attach
a leukocyte reduction filter to the
collection system. The filter should
be used according to the manufacturer’s directions.
Whole-blood-derived platelets can
be manufactured only before leukocyte reduction (see Method 6.14).

Procedure
1.

Before centrifugation, the anticoagulated Whole Blood may be filtered by hanging the container upside down and allowing the blood to
flow through an in-line filter by gravity into a secondary container. The
steps in Method 6.4 are then followed (see note 2, for addition of additive solution).
The anticoagulated Whole Blood
may be centrifuged with the in-line
filter attached. After centrifugation,
the plasma is expressed. The additive solution is added, and the red
cells in the additive solution are filtered by gravity, as in step 1 above.
A red cell component prepared using Method 6.4 either in residual
anticoagulated plasma or in additive
solution (AS-1, AS-3, AS-5) may have
a secondary container with an inline filter attached using a sterile
connection device. Filtration can
proceed according to the manufacturer’s directions using gravity, as in
step 1. The timing of this filtration is
often within 24 hours of collection
but can be up to 5 days.
Red cells that are leukocyte reduced
are labeled “Red Blood Cells Leukocytes Reduced.” There is no specific
label for prestorage leukocyte reduction.

Method 6.6. Rejuvenation of
Red Blood Cells
Principle
Rejuvenation is a process to restore depleted metabolites and improve the function and posttransfusion survival of stored
red cells. The rejuvenating solution is not
intended for intravenous administration;
after warm incubation with the solution,
the red cells are washed and either glycerolized for frozen storage or kept at 1 to
6 C for transfusion within 24 hours.
The rejuvenating solution approved by
the Food and Drug Administration contains
pyruvate, inosine, phosphate, and adenine.
Its use is permitted only with RBCs prepared from Whole Blood collected into
CPD, CP2D, or CPDA-1, and it may be
added at any time between 3 days after collection of the blood and 3 days after the expiration of the unit. However, the use of the
rejuvenation solution with RBC units before 14 days of storage is not routinely accepted because the treated cells may develop
supranormal levels of 2,3-diphosphoglycerate, which impairs oxygen uptake.

Reagents and Materials
1.

RBCs stored at 1 to 6 C and prepared
from Whole Blood collected in CPD
or CPDA-1. After collection, RBCs

Methods Section 6: Blood Collection, Storage, and Component Preparation

3.
4.
5.

suspended in CPD from day 3 to day
24 (or in CPDA-1 from day 3 to day
38) may be used. The solution is not
approved for use with cells stored in
additive solutions.
Red Blood Cell rejuvenation solution, in 50-mL sterile vial (Rejuvesol,
Cytosol Laboratories, Braintree, MA);
also called rejuvenating solution.
Waterproof plastic bag.
Metal clips and hand sealer.
Sterile airway.

Procedure
1.

Connect the container of rejuvenating solution to the RBCs, using a
transfer set and aseptic technique.
Allow 50 mL of rejuvenating solution
to flow by gravity into the container
of red cells. Gently agitate the cell/
solution mixture during this addition.
Note: A sterile airway is required if
the solution is in a bottle.
Seal the tubing near the blood bag
and incubate the mixture for 1 hour
at 37 C. Either a dry incubator or circulating waterbath can be used. If
placed in a waterbath, the container
should be completely immersed; use
of a waterproof overwrap is essential
to prevent contamination.
For use within 24 hours, wash the rejuvenated cells with saline (2 L unbuffered 0.9% NaCl) by the use of an
approved protocol. Storage of the
washed cells from the start of the
wash procedure should be at 1 to 6 C
for no longer than 24 hours.
If the rejuvenated cells are to be cryopreserved, the standard glycerolization protocol adequately removes
the rejuvenation solution from the
processed cells. Expiration date remains 10 years from the date of collection.

807

Be sure that units are appropriately
labeled and that all applicable records are complete.

Reference
Valeri CR, Zaroules CG. Rejuvenation and freezing
of outdated stored human red cells. N Engl J Med
1972;287:1307-13.

Method 6.7. Red Cell
Cryopreservation Using
High-Concentration
Glycerol—Meryman Method
Principle
Cryoprotective agents make possible the
long-term (10 or more years) preservation
of red cells in the frozen state. High-concentration glycerol is particularly suitable
for this purpose. A practical method for
RBCs collected in a 450-mL bag is described below.

Materials
(See Chapter 8 for additional information
on frozen cellular components.)
1.
Donor blood, collected into CPD,
CD2D, CPDA-1, or AS.
a.
Complete all blood processing
on units intended for freezing.
b.
RBCs preserved in CPD or CPDA-1
may be stored at 1 to 6 C for up
to 6 days before freezing.
c.
RBCs preserved in AS-1 and AS-3
may be stored at 1 to 6 C for up
to 42 days before freezing.
d.
RBCs that have undergone rejuvenation (see Method 6.6) may
be processed for freezing up to
3 days after their original expiration.
e.
RBCs in any preservative solution that have been entered for

808

2.
3.
4.
5.
6.
7.
8.
9.

10.
11.

AABB Technical Manual

processing must be frozen within 24 hours of puncturing the
seal.
Storage containers, either polyvinyl
chloride or polyolefin bags.
6.2 M of glycerol lactate solution
(400 mL).
Cardboard or metal canisters for
freezing.
Hypertonic (12%) sodium chloride
solution.
1.6% NaCl, 1 liter for batch wash.
Isotonic (0.9%) NaCl with 0.2% dextrose solution.
37 C waterbath or 37 C dry warmer.
Equipment for batch or continuousflow washing, to deglycerolize cells
frozen in high-concentration glycerol.
Freezer tape.
Freezer (–65 C or colder).

Glycerolization
1.

Procedure

Preparing RBCs for Glycerolization
1.

Prepare RBCs from Whole Blood
units by removal of supernatant anticoagulant-preservative or additive
solution. Weigh the RBC unit to be
frozen and obtain the net weight of
the RBCs. The combined weight of
the cells and the collection bag
should be between 260 g and 400 g.
Underweight units can be adjusted
to approximately 300 g either by the
addition of 0.9% NaCl or by the removal of less plasma than usual. Record the weight and, if applicable,
document the amount of NaCl added.
Record the Whole Blood number,
ABO group and Rh type, anticoagulant, date of collection, date frozen,
expiration time, and the identification of the person performing the

procedure. If applicable, document
the lot number of the transfer bag.
Warm the red cells and the glycerol
to at least 25 C by placing them in a
dry warming chamber for 10 to 15
minutes or by allowing them to remain at room temperature for 1 to 2
hours. The temperature must not
exceed 42 C.
Apply a “Red Blood Cells Frozen” label to the freezing bag in which the
unit will be frozen. The label must
also include: name of the facility
freezing the unit; Whole Blood number; ABO group and Rh type; date
collected; date frozen; the cryoprotective agent used; and the expiration date.

4.
5.

Document the lot numbers of the
glycerol, the freezing bags, and, if
used, the 0.9% NaCl.
Place the container of red cells on a
shaker and add approximately 100
mL of glycerol as the red cells are
gently agitated.
Turn off the shaker and allow the
cells to equilibrate, without agitation, for 5 to 30 minutes.
Allow the partially glycerolized cells
to flow by gravity into the freezing bag.
Add the remaining 300 mL of glycerol slowly in a stepwise fashion,
with gentle mixing. Add smaller volumes of glycerol for smaller volumes
of red cells. The final glycerol concentration is 40% w/v. Remove any
air from the bag.
Allow some glycerolized cells to flow
back into the tubing so that segments can be prepared.
Maintain the glycerolized cells at
temperatures between 25 and 32 C
until freezing. The recommended in-

Methods Section 6: Blood Collection, Storage, and Component Preparation

terval between removing the RBC
unit from refrigeration and placing
the glycerolized cells in the freezer
should not exceed 4 hours.

Freezing and Storage
1.

3.
4.
5.

Place the glycerolized unit in a cardboard or metal canister and place it
in a freezer at –65 C or colder.
Label the top edge of the canister
with freezer tape marked with the
Whole Blood number, ABO group
and Rh type, the date frozen, and the
expiration date.
Do not bump or handle the frozen
cells roughly.
The freezing rate should be less than
10 mL/minute.
Store the frozen RBCs at –65 C or
colder for up to 10 years. For blood
of rare phenotypes, a facility’s medical director may wish to extend the
storage period. The unusual nature
of such units and the reason for retaining them past the routine 10year storage period must be documented.

Thawing and Deglycerolizing
1.

Put an overwrap on the protective
canister containing the frozen cells
and place it in either a 37 C waterbath or 37 C dry warmer.
Agitate it gently to speed thawing.
The thawing process takes at least 10
minutes. Thawed cells should be at
37 C.
After the cells have thawed, use a
commercial instrument for batch or
continuous-flow washing to deglycerolize cells. Follow the manufacturer’s instructions.
Record the lot numbers and manufacturer of all the solutions and software used. Apply a “Red Blood Cells

809

Deglycerolized” label to the transfer
pack and be sure that the label includes identification of the collecting facility, the facility preparing the
deglycerolized cells, the ABO group
and Rh type of the cells, the Whole
Blood number, and the expiration
date and time.
Dilute the unit with a quantity of
hypertonic (12%) NaCl solution appropriate for the size of the unit. Allow it to equilibrate for approximately 5 minutes.
Wash the cells with 1.6% NaCl until
deglycerolization is complete. Approximately 2 liters of wash solution
are required. To check for residual
glycerol, see Method 6.8.
Suspend the deglycerolized cells in
isotonic (0.9%) saline with 0.2% dextrose.
Fill the integrally attached tubing
with an aliquot of cells sealed in
such a manner that it will be available for subsequent compatibility
testing.
Deglycerolized RBCs must be stored
at 1 to 6 C for no longer than 24 hours.
(A closed system has been licensed
that allows storage of deglycerolized
RBCs at 1 to 6 C for 2 weeks.)

Notes
1.

An aliquot of the donor’s serum or
plasma should be frozen and stored
at –65 C or colder for possible future
use if new diagnostic tests are implemented.
When new diagnostic tests have
been implemented and stored units
do not have aliquots available for
testing, the units may have to be issued with a label stating that the test
has not been performed. The reason
for distributing an untested compo-

810

AABB Technical Manual

nent should be documented. If a
specimen from the donor is obtained
and tested after the unit was stored,
the date of testing should be noted
on the unit when it is issued.

10.

11.

Reference
Meryman HT, Hornblower M. A method for freezing and washing RBCs using a high glycerol concentration. Transfusion 1972;12:145-56.

12.
13.
14.
15.

Method 6.8. Red Cell
Cryopreservation Using
High-Concentration
Glycerol—Valeri Method

Procedure

Preparing RBCs for Glycerolization
1.

Principle
RBCs collected in an 800-mL primary collection bag in CPDA-1 and stored at 1 to 6
C for 3 to 38 days can be biochemically rejuvenated and frozen with 40% w/v glycerol in the 800-mL primary container. See
Method 6.6 for additional information.

Materials
1.
2.
3.

4.
5.
6.
7.

8.
9.

Quadruple plastic bag collection
system with 800-mL primary bag.
Hand sealer clips.
Empty 600-mL polyethylene cryogenic vials (eg, Corning 25702 or
Fisher 033746).
Sterile connection device with wafers.
Freezer tape.
600-mL transfer bag.
50 mL of Red Blood Cell Processing
Solution (Rejuvesol, Cytosol Laboratories, Braintree, MA).
Heat-sealable 8" × 12" plastic bags.
Rejuvenation harness (Fenwal 4C1921
or Cutter 98052).

Sterile filtered airway needle (BD
5200), for Fenwal rejuvenation harness only.
500 mL of glycerolyte 57 solution
(Fenwal 4A7833) or 500 mL of 6.2 M
glycerolization solution (Cytosol
PN5500).
Labels—Red Blood Cells Frozen Rejuvenated.
Corrugated cardboard storage box
(7" × 5.5" × 2" outside dimensions).
Heat sealing device.
Plastic bag for overwrapping.

Collect 450 mL of Whole Blood in
the primary bag. Invert the bag, fold
it about 2 inches from the base, secure the fold with tape, and place
the bag upright in a centrifuge. Centrifuge and remove all visible supernatant plasma. The hematocrit of
the RBC unit must be 75% ± 5%.
Store RBCs at 1 to 6 C in the 800-mL
primary bag, along with the adapter
port on the tubing that connects the
primary bag and transfer pack.
Centrifuge the stored cells to remove
all visible plasma before undertaking
rejuvenation. The gross and net
weights of the RBCs should not exceed 352 g and 280 g, respectively.
Transfer the plasma to the integrally
connected transfer pack, fold the integral tubing, and replace the hand
sealer clip (not crimped).
Attach an empty 600-mL transfer
pack to the integral tubing of the primary collection bag, using a sterile
connection device.
Transfer 1 mL of plasma to each of
three cryogenic vials to be used for
future testing.

Methods Section 6: Blood Collection, Storage, and Component Preparation

nected empty transfer pack, and the
coupler of the Y-type harness and
incubate them in a 37 C waterbath
for 1 hour.

Biochemical Modification of the Cells
1.

Using the Fenwal Rejuvenation Harness: Aseptically insert the needle of
the Y-type Fenwal Harness into the
rubber stopper of a 50-mL Red
Blood Cell Processing Solution bottle and the coupler of the set into the
adapter port of the primary collection bag. Insert the filtered airway
needle into the rubber stopper of the
Red Blood Cell Processing Solution
bottle.
Using the Cutter Rejuvenation Harness: Aseptically insert the vented
white spike with the drip chamber
into the rubber stopper of the Red
Blood Cell Processing Solution bottle and the nonvented spike into the
special adapter port on the primary
collection bag.
With gentle manual agitation, allow
50 mL of Red Blood Cell Processing
Solution to flow directly into the red
cells.
Heat-seal the tubing of the harness
set that connects the Red Blood Cell
Processing Solution to the adapter
port. The second tubing of the harness Y-set is used to add glycerol
(see below).
Completely overwrap the 800-mL
primary bag, the integrally con-

811

Glycerolization
1.

Remove the numbered crossmatch
segments, leaving the initial segment and number attached to the
collection bag. Weigh the unit.
Determine the amount of glycerol to
be added, based on the gross or net
weight of the unit, from the values
shown in Table 6.8-1.
Aseptically insert the coupler of the
rejuvenation harness into the outlet
port of the rubber stopper on the
glycerol solution bottle. For the
Fenwal harness only, insert a filtered
airway needle into the vent portion
of the glycerol bottle stopper.
Place the bag on a shaker. Add the
amount of glycerol shown in Table
6.8-1 for the first volume while the
bag is shaking at low speed (180 oscillations/minute).
Equilibrate the mixture for 5 minutes without shaking and add the
second volume. Equilibrate it for 2
minutes. Add the third volume of
glycerol, using vigorous manual
shaking.

Table 6.8-1. Amount of Glycerol Needed for Different Weights of Red Cell Units
Gross Weight
of Unit
(grams)*

Net Weight
of Unit
(grams)

222-272

150-200

250

350

273-312

201-240

350

450

313-402

241-330

400

500

Initial
Second
Third
Total
Addition of
Addition of
Addition of
Glycerol
Glycerol (mL) Glycerol (mL) Glycerol (mL) Added (mL)

*Weight of the empty 800-mL primary bag with the integrally attached transfer pack and the adapter port is 72 grams
(average).

812

10.
11.

12.

13.

14.

AABB Technical Manual

Heat-seal the tubing between the
empty bottle of glycerol and the tubing proximal to the adapter port. Ensure that the transfer pack remains
integrally attached to the primary
collection bag.
Centrifuge the mixture of red cells
and glycerol and transfer all visible
supernatant glycerol to the transfer
pack, resuspend, and mix. Note: This
step differs from Method 6.7.
Seal the tubing 4" from the primary
collection bag, detach the transfer
pack containing the supernatant fluid,
and discard it.
Affix an overlay blood component
label, the facility label, and an ABO/
Rh label. Record the expiration date
on the label.
Weigh the unit just before freezing
and record the weight.
Fold over the top portion of the primary bag (approximately 2"). Place
the primary bag into a plastic bag
overwrap and heat-seal the outer
bag across the top so that there is as
little air as possible between the bags.
Place one vial of plasma and the
plastic bag containing the glycerolized red cells in the cardboard box.
Store the other two vials, suitably
identified, at –65 C or colder for future testing, if needed.
Affix a “Red Blood Cells Frozen Rejuvenated” label, an ABO/Rh label, a
facility label, and the original unit
number on the outside of the box.
Record separately or affix on the
cardboard box the collection, freezing, and expiration dates.
Freeze the unit in a –80 C freezer. No
more than 4 hours should be allowed to elapse between the time
the unit was removed from the 4 C
refrigerator and the time the cells
are placed in the –80 C freezer.

Thawing and Deglycerolization
See Method 6.7. Note, however, that the
supernatant glycerol is removed before
freezing. Therefore, only two salt solutions (the hypertonic 12% saline and the
0.9% saline-0.2% dextrose solution) are
used in the deglycerolization process.

References
1.
2.

Rejuvesol Package insert. Braintree, MA:
Cytosol Laboratories, 2002.
Valeri CR, Ragno G, Pivacek LE, et al. A
multicenter study of in vitro and in vivo values in human RBCs frozen with 40% (wt/vol)
glycerol and stored after deglycerolization for
15 days at 4 C in AS-3: Assessment of RBC
processing in the ACP 215. Transfusion 2001;
41:933-9.

Method 6.9. Checking the
Adequacy of
Deglycerolization of Red
Blood Cells
Principle
Glycerolization of red cells for frozen storage creates a hyperosmolar intracellular
fluid, which must be restored to physiologically compatible levels before the cells
are transfused. Inadequately deglycerolized
red cells will be hemolyzed by contact
with normal saline, or with serum or
plasma if subjected to crossmatching.
During deglycerolization, the last solution
in contact with the cells is normal saline.
The easiest way to determine adequacy of
glycerol removal is to determine the level
of free hemoglobin (mg/dL) in the final
wash. An adequate estimate of hemolysis
can be achieved by comparing the color
of the final wash fluid with the blocks in a
commercially available color comparator.
Alternatively, normal saline can be added
to an aliquot of deglycerolized cells and

Methods Section 6: Blood Collection, Storage, and Component Preparation

the color of the supernatant fluid evaluated against the color comparator.

Materials and Equipment
1.
2.

time frame required for the anticoagulant
or collection process.

Materials

Semiautomated instrument for deglycerolizing cryopreserved RBCs.
Transparent tubing, as part of disposable material used to deglycerolize individual unit.
Color comparator, available commercially.

Procedure
1.

813

Interrupt the last wash cycle at a
point when wash fluid is visible in
the tubing leading to the disposal bag.
Hold the comparator block next to
an accessible segment of tubing,
against a well-lighted white background.
Note coloration of the wash fluid,
which should be no stronger than
the block, indicating 3% hemolysis
(3% of the red cells are hemolyzed).
If the level of hemolysis is excessive,
continue the wash process until the
color is within acceptable limits.
Record observation for the individual unit and for the quality assurance
program.
If unacceptable hemolysis occurs repeatedly, document corrective action.

Method 6.10. Preparation of
Fresh Frozen Plasma from
Whole Blood
Principle

2.
3.
4.
5.
6.
7.
8.

Procedure
1.

Plasma is separated from cellular blood
elements and frozen to preserve the activity of labile coagulation factors. Plasma
must be placed in the freezer within the

Freshly collected Whole Blood, obtained by phlebotomy as described
in Method 6.3, in a collection unit
with integrally attached transfer container(s).
Metal clips and hand sealer.
Clean instruments (scissors, hemostats).
Dielectric sealer (optional).
Plasma extractor.
Freezing apparatus.
Refrigerated centrifuge.
Scale.

Centrifuge blood soon after collection,
using a “heavy” spin (see Method 7.4).
Use a refrigerated centrifuge at 1 to 6
C unless also preparing platelets (see
Method 6.13).
Place the primary bag containing
centrifuged blood on a plasma extractor and place the attached satellite bag on a scale adjusted to zero.
Express the plasma into the satellite
bag and weigh the plasma.
Seal the transfer tubing with a dielectric sealer or metal clips but do
not obliterate the segment numbers
of the tubing. Place another seal
nearer the transfer bag.
Label the transfer bag with the unit
number before it is separated from
the original container. Record the
volume of plasma on the label.
Cut the tubing between the two seals.
The tubing may be coiled and taped
against the plasma container, leaving
the segments available for any testing desired.

814

AABB Technical Manual

Place the plasma at –18 C or colder
within the time frame required for the
anticoagulant or collection process.

Procedure
1.

Method 6.11. Preparation of
Cryoprecipitated AHF from
Whole Blood

Principle
Coagulation Factor VIII (antihemophilic
factor, AHF) can be concentrated from
freshly collected plasma by cryoprecipitation. Cryoprecipitation is accomplished
by slow thawing, at 1 to 6 C, plasma that
has been prepared for freezing within the
time frame required for the anticoagulant
or collection process.

Materials
1.

2.
3.
4.
5.
6.
7.

8.
9.

Freshly collected Whole Blood, obtained by phlebotomy as described
in Method 6.3, in a collection unit
with at least two integrally attached
transfer containers.
Metal clips and hand sealer.
Clean instruments (scissors, hemostats).
Dielectric sealer (optional).
Plasma extractor.
Refrigerated centrifuge.
Freezing apparatus: suitable freezing
devices include blast freezers or mechanical freezers capable of maintaining temperatures of –18 C or
colder; dry ice; or an ethanol dry ice
bath. In a bath of 95% ethanol and
chipped dry ice, freezing will be
complete in about 15 minutes.
1 to 6 C circulating waterbath or refrigerator.
Scale.

Collect blood in a collection unit
with two integrally attached transfer
containers.
Centrifuge blood shortly after collection at 1 to 6 C, using a “heavy” spin
(see Method 7.4). Collect at least 200
mL (205 g) of cell-free plasma for
processing into cryoprecipitate.
Promptly place plasma in a freezing
device so that freezing is started
within the time frame required for
the anticoagulant or collection process. Plasma containers immersed in
liquid must be protected with a plastic overwrap.
Allow the frozen plasma to thaw at 1
to 6 C by placing the bag in a 1 to 6 C
circulating waterbath or in a refrigerator. If thawed in a waterbath, use
a plastic overwrap (or other means)
to keep container ports dry.
When the plasma has a slushy consistency, separate liquid plasma from
the cryoprecipitate by one of the
procedures below:
a.
Centrifuge the plasma at 1 to 6
C using a “heavy” spin. Hang the
bag in an inverted position and
allow the separated plasma to
flow rapidly into the transfer
bag, leaving the cryoprecipitate
adhering to the sides of the primary bag. Separate the cryoprecipitate from the plasma
promptly, to prevent the cryoprecipitate from dissolving and
flowing out of the bag. Ten to
15 mL of supernatant plasma
may be left in the bag for resuspension of the cryoprecipitate
after thawing. Refreeze the cryoprecipitate immediately.
b.
Place the thawing plasma in a
plasma expressor when appro-

Methods Section 6: Blood Collection, Storage, and Component Preparation

ximately one-tenth of the contents is still frozen. With the
bag in an upright position, allow the supernatant plasma to
flow slowly into the transfer
bag, using the ice crystals at
the top as a filter. The cryoprecipitate paste will adhere to
the sides of the bag or to the ice.
Seal the bag when about 90% of
the cryoprecipitate-reduced
plasma has been removed and
refreeze the cryoprecipitate immediately.
The cryoprecipitate should be refrozen within 1 hour of thawing.
Store at –18 C or colder, preferably
–30 C or colder, for up to 12 months
from the date of blood collection.

2.
3.
4.

3.
Cryoprecipitated AHF may be prepared
from Fresh Frozen Plasma at any time
within 12 months of collection. The expiration date of Cryoprecipitated AHF is 12
months from the date of phlebotomy, not
from the date it was prepared.
4.

Principle
Cryoprecipitated AHF should be rapidly
thawed at 30 to 37 C but should not remain at this temperature once thawing is
complete. The following method permits
rapid thawing and pooling of this product.

are available commercially, as are
specially designed dry heat devices).
Medication injection ports.
Sterile 0.9% sodium chloride for injection.
Syringes and needles.

Procedure

Note

Method 6.12. Thawing and
Pooling Cryoprecipitated
AHF

815

Cover the container with a plastic
overwrap to prevent contamination
of the ports with unsterile water, or
use a device to keep the containers
upright with the ports above water.
Resuspend the thawed precipitate
carefully and completely, either by
kneading it into the residual 10 to 15
mL of plasma or by adding approximately 10 mL of 0.9% sodium chloride and gently resuspending.
Pool by inserting a medication injection site into a port of each bag. Aspirate contents of one bag into a syringe and inject into the next bag. Use
the ever-increasing volume to flush
each subsequent bag of as much dissolved cryoprecipitate as possible,
until all contents are in the final bag.
Thawed Cryoprecipitated AHF must
be stored at room temperature. If
pooled, it must be administered
within 4 hours. Thawed single units,
if not entered, must be administered
within 6 hours of thawing if intended for replacement of Factor VIII.
Pools of thawed individual units may
not be refrozen.

Method 6.13. Preparation of
Platelets from Whole Blood
Principle

Materials
1.

Circulating waterbath at 37 C (waterbaths designed for thawing plasma

Platelet-rich plasma is separated from
Whole Blood by “light-spin” centrifugation
and the platelets are concentrated by

816

AABB Technical Manual

“heavy-spin” centrifugation, with subsequent removal of supernatant plasma (see
Method 7.4).

Materials
1.

2.
3.
4.
5.
6.
7.
8.

Freshly collected Whole Blood, obtained by phlebotomy as described
in Method 6.3, in a collection unit
with two integrally attached transfer
containers. The final container must
be a plastic approved for platelet
storage. Keep blood at room temperature (20 to 24 C) before separating
platelet-rich plasma from the red cells.
This separation must take place
within 8 hours of phlebotomy.
Metal clips and hand sealer.
Scissors, hemostats.
Plasma extractor.
Dielectric sealer (optional).
Centrifuge, calibrated as in Method
7.4.
Scale.
Rotator.

Procedure
1.

Do not chill the blood at any time
before or during platelet separation.
If the temperature of the centrifuge
is 1 to 6 C, set the temperature control of the refrigerated centrifuge at
20 C and allow the temperature to
rise to approximately 20 C. Centrifuge the blood using a “light” spin
(see Method 7.4).
Express the platelet-rich plasma into
the transfer bag intended for platelet
storage. Seal the tubing twice between the primary bag and Y connector of the two satellite bags and
cut between the two seals. Place the
red cells at 1 to 6 C.
Centrifuge the platelet-rich plasma
at 20 C using a “heavy” spin (see
Method 7.4).

Express the platelet-poor plasma
into the second transfer bag and seal
the tubing. Some plasma should remain on the platelet button for storage, but no exact volume can be designated. AABB Standards for Blood
Banks and Transfusion Services requires that sufficient plasma remain
with the platelet concentrate to maintain the pH at 6.2 or higher for the
entire storage period. This usually
requires a minimum of 35 mL of
plasma when storage is at 20 to 24 C,
but 50 to 70 mL is preferable.
The platelet concentrate container
should be left stationary, with the label side down, at room temperature
for approximately 1 hour.
Resuspend the platelets in either of
the following ways:
a.
Manipulate the platelet container gently by hand to achieve
uniform resuspension.
b.
Place the container on a rotator
at room temperature. The slow,
gentle agitation should achieve
uniform resuspension within 2
hours.
Maintain the platelet suspensions at
20 to 24 C with continuous gentle
agitation.
Platelets should be inspected before
issue to ensure that no platelet aggregates are visible.

Notes
The platelet-reduced plasma may be frozen
promptly and stored as Fresh Frozen
Plasma (FFP), if the separation and freezing are completed within the time frame
required for the anticoagulant or collection process. The volume of FFP prepared
after platelet preparation will be substantially less than that prepared directly from
Whole Blood.

Methods Section 6: Blood Collection, Storage, and Component Preparation

Reference
Silva MA, ed. Standards for blood banks and transfusion services. 23rd ed. Bethesda, MD: AABB,
2005:30.

Method 6.14. Preparation of
Prestorage Platelets
Leukocytes Reduced from
Whole Blood
Principle
Prestorage leukocyte-reduced platelets
may be prepared from whole blood using
in-line filtration of the platelet-rich plasma
(PRP). The resulting intermediate product
is a filtered PRP, from which a leukocytereduced platelet concentrate and leukocyte-reduced plasma may be manufactured. Materials and procedures are the
same as for Method 6.13, except that the
PRP is expressed through an in-line filter.

References
1.

Sweeney JD, Holme S, Heaton WAL, Nelson E.
Leukodepleted platelet concentrates prepared by in-line filtration of platelet rich
plasma. Transfusion 1995;35:131-6.
Sweeney JD, Kouttab N, Penn LC, et al. A
comparison of prestorage leukoreduced
whole blood derived platelets with bedside
filtered whole blood derived platelets in
autologous stem cell transplant. Transfusion
2000;40:794-800.

Method 6.15. Removing
Plasma from Platelet
Concentrates (Volume
Reduction)

817

centrifuged and much of the plasma removed shortly before transfusion, but
appropriate resuspension is essential.
The platelets must remain at room temperature, without agitation, for 20 to 60
minutes before resuspension into the remaining plasma. Transfusion must take
place within 4 hours of the time the
platelet bag was entered. Volume reduction can be performed on individual or
pooled units.
No consensus exists regarding the optimal centrifugation rate. One study1 found
35% to 55% platelet loss in several units
centrifuged at 500 × g for 6 minutes, compared with 5% to 20% loss in units centrifuged at 5000 × g for 6 minutes or 2000 × g
for 10 minutes. The authors recommend
2000 × g for 10 minutes, to avoid any risk
that a higher centrifugal force might inflict
on the plastic container. A study by Moroff
2
et al found mean platelet loss to be less
than 15% in 42 units centrifuged at 580 × g
for 20 minutes. High g forces are of theoretical concern because they may damage the
platelets when they are forced against the
wall of the container and also increase the
possibility of container breakage.

Materials
1.
2.
3.
4.
5.
6.

Platelet concentrate(s), prepared as
described in Method 6.13.
Metal clips and hand sealer.
Scissors, hemostats.
Dielectric sealer (optional).
Centrifuge, calibrated as in Method
7.4.
Plasma extractor.

Principle

Procedure

Although optimal storage of platelets requires an adequate volume of plasma, a
few patients may not tolerate large-volume infusion. Stored platelets may be

Pool platelets, if desired, into a transfer pack, using standard technique.
Single platelet concentrates may
need volume reduction for pediatric

818

AABB Technical Manual

recipients. Apheresis components may
be processed directly.
Centrifuge at 20 to 24 C, using one of
the following protocols:
a.
580 × g for 20 minutes.
b.
2000 × g for 10 minutes.
c.
5000 × g for 6 minutes.
Without disturbing the contents,
transfer the bag to a plasma extractor. Remove all but 10 to 15 mL plasma
from single units, or somewhat more
volume, proportionately, from a pool
or from a component prepared by
apheresis.
Mark expiration time on bag as 4
hours after the time the unit was entered.
Leave bag at 20 to 24 C without agitation for 20 minutes if centrifuged
at 580 × g, or for 1 hour if centrifuged
at 2000 or 5000 × g.
Resuspend platelets as described in
Method 6.13.

References
1.

Notes
1.

If a sterile connection device is used
for removing plasma from a hema-

pheresis component or individual
platelet concentrate, the unit can be
considered sterile and it is not necessary to impose the 4-hour expiration interval required for entered
Platelets. However, no data exist to
support storage of reduced volume
platelet concentrates; therefore, it is
preferable to transfuse them as soon
as possible.
Reduced-volume platelet concentrates
may not be distributed as a licensed
product.
Platelets that have been pooled must
be used within 4 hours of entering
the units, whether or not they have
been volume-reduced. Pooled platelets may not be distributed as a licensed product.

Simon TL, Sierra ER. Concentration of platelet units into small volumes. Transfusion
1984;24:173-5.
Moroff G, Friedman A, Robkin-Kline L, et al.
Reduction of the volume of stored platelet
concentrates for use in neonatal patients.
Transfusion 1984;24:144-6.

Methods Section 7: Quality Control

Methods Section 7

Quality Control

Procedure
1.

Either of the two methods presented below is acceptable for quality control of copper sulfate solution.
2.

Method 7.1.1. Functional Validation of
Copper Sulfate Solution

Principle

Copper sulfate solution can be checked for
suitability in donor screening by observing
the behavior (sinking or floating) of drops
of blood of known hemoglobin concentration.

Materials
1.
2.
3.

Copper sulfate—specific gravity 1.053.
Capillary tubes.
Worksheet for recording results.

Obtain several (three to six, if possible) blood samples with known hemoglobin levels. Samples should include hemoglobin levels slightly
above and below 12.5 g/dL.
Gently place a drop of each blood
sample into a vial of copper sulfate
solution of stated specific gravity of
1.053.
Drops of all blood samples with hemoglobin at or above 12.5 g/dL must
sink and those with hemoglobin levels below 12.5 g/dL must float.
Record the date of testing; the manufacturer, lot number, and expiration
date of the copper sulfate; sample
identity; the results; and the identity
of the person performing the test.
Document the corrective action taken
if the results are outside acceptable
limits.
819

Section 7

Method 7.1. Quality Control
for Copper Sulfate Solution

820

AABB Technical Manual

Method 7.1.2. Use of Measurement
Instruments for Specific Gravity, Density,
or Refractive Index of Copper Sulfate
Solution

Principle
The specific gravity, density, or refractive
index of the copper sulfate solution can
be measured directly and the result compared with the value stated by the manufacturer. An error in the specific gravity
reading of ±0.0001 corresponds to ±0.06
g/dL hemoglobin in whole blood.1 Therefore, a copper sulfate solution with a specific gravity of 1.053 ± 0.0003 would result
in a corresponding hemoglobin range of
12.5 ± 0.18 g/dL.

Materials
1.
2.

3.
4.

5.
6.

Copper sulfate—specific gravity 1.053.
Measurement instruments
•
For specific gravity—high-precision hydrometer, with gradations
of 0.0005 or smaller.
•
For refractive index—refractometer.
Pipette.
For specific gravity method—graduated cylinder or other container suitable for use with a hydrometer.
Alcohol for cleaning hydrometer.
Worksheet for recording results.

stead of an ellipse. Read at the point
where the line intersects the scale on
the instrument.
Refractometer method:
1.
Follow manufacturer’s instructions for
operation and maintenance of the instrument.
2.
Add a drop of copper sulfate to the
refractometer.
3.
Observe refractive index.
Note: Some refractometers designed
for urine analysis provide both a refractive index scale and a specific
gravity scale. Do not read specific
gravity directly from this type of
refractometer.
4.
A copper sulfate solution with a specific gravity of 1.053 will have a refractive index of about 1.3425 at room
temperature.2
Both methods:
1.
Record the results; the date of testing; the manufacturer, lot number,
and expiration date of the copper
sulfate solution; the identity of the
person performing the test; and the
identification of the instrument used
for measurement.
2.
Document the corrective action taken
if the results are outside the acceptable limits.

Procedure

Note

Hydrometer method:
1.
Wipe the hydrometer clean with alcohol and dry it.
2.
Gently lower the hydrometer into the
solution until it floats on its own.
Drops of solution on the stem will
cause inaccurate readings.
3.
To read, observe a point just below
the plane of the liquid surface and
then raise the line of vision until the
surface is seen as a straight line in-

A liquid densitometer may be used to
measure density directly. At 4 C, the density of the solution will be exactly the
same as the specific gravity (specific
gravity = density of solution/density of
water; density of water at 4 C = 1 g/mL).
When density is measured at room temperature, a conversion factor of 0.9970
(the density of water at 25 C) is used to
calculate specific gravity.2 Specific gravity
of copper sulfate solution at 25 C = the

Methods Section 7: Quality Control

observed density in g/mL divided by
0.9970 g/mL.

References
1.

Phillips RA, Van Slyke DD, Hamilton PB, et al.
Measurement of specific gravities of whole
blood and plasma by standard copper sulfate
solutions. J Biol Chem 1950;183:305-30.
Blood hemoglobin screening (specific gravity
method). Technical Reference Document 12.
Arlington, TX: Ricca Chemical Company,
2002.

3.
4.
5.
6.
7.

Procedure

Method 7.2. Standardization
and Calibration of
Thermometers
Principle

Materials
1.

National Institute of Standards and
Technology (NIST)-certified thermometer or thermometer with NISTtraceable calibration certificate.
Thermometer to be calibrated.

Suitable container, eg, 250-500 mL
beaker.
Water.
Crushed ice.
37 C waterbath.
Worksheet for recording results.

Method 7.2.1. Liquid-in-Glass Laboratory
Thermometers

Thermometers used during laboratory
testing and in the collection (donor suitability), processing, and storage of blood
components and reagents should be calibrated and standardized to ensure accurate indication of temperatures. Calibration should be performed at temperatures
close to the temperature at which the
thermometers will be used. Over time,
liquid-in-glass thermometers may give a
different reading at a given temperature
because of permanent changes in the volume of the bulb related to relaxation of
the glass.1 Each thermometer should be
calibrated before initial use and periodically thereafter, as well as any time there
is reason to suspect change or damage.
Calibration must be verified for all electronic thermometers, even those described as “self-calibrating.”

821

Before choosing a thermometer for a
particular application, consider all
the governing factors; be sure that
the thermometer will be used at its
proper immersion; and follow the
manufacturer’s instructions for its
proper use. When using a certified
thermometer, read and follow the
applicable notes. Be sure to include
any correction factors noted on the
certificate for the NIST-traceable
thermometer and apply them in
calculations.
Categorize the thermometers by key
factors, such as immersion, increments, and temperature of intended
use. Test them in groups, comparing
similar thermometers. Do not attempt
to compare dissimilar thermometers
in a single procedure.
Number each thermometer being
tested (eg, place a numbered piece
of tape around the top of each thermometer or use the manufacturer’s
serial number).
Perform calibration with water at a
temperature close to that which the
thermometer will monitor.
a.
To calibrate at 37 C, place the
thermometers to be tested and
the NIST thermometer at a uniform depth in a standard 37 C
waterbath, making sure that the
tips of all devices are at the same
level in the liquid.

822

AABB Technical Manual

To calibrate at 1 to 6 C, fill a
suitable container with water.
Add crushed ice until the approximate desired temperature
is reached. Place the thermometers to be tested and the NIST
thermometer at a uniform depth
in the water/ice mixture, making sure that the tips of all devices are at the same level and
are in the liquid, not the upper
ice.
Stir constantly in a random motion
until the temperature equilibrates,
approximately 3 to 5 minutes.
Observe temperatures. Record each
thermometer’s identification and results. Acceptance criteria depend on
the level of precision required, but,
for most blood banking applications,
agreement within 1 C between the two
thermometers may be considered
acceptable. If the reading varies by
more than one degree from the standard, the thermometer may be returned to the distributor (if newly
purchased), labeled with the correction factor (degrees different from
the NIST thermometer) that must be
applied to each reading, or discarded.
Complete the calibration record
with the date of testing and identity
of the person who performed the
test.

References
1.

Wise JA. A procedure for the effective recalibration of liquid-in-glass thermometers.
NIST Special Publication 819. Gaithersburg,
MD: National Institute of Standards and
Technology, 1991.

Temperature calibration of water baths, instruments, and temperature sensors. 2nd ed;
approved standard I2-A2 Vol. 10 No. 3. Wayne,
PA: National Committee for Clinical Laboratory Standards, 1990.

Method 7.2.2. Electronic Oral
Thermometers

Procedure
1.

Notes
1.

If a thermometer is to be used for
temperatures over a range greater
than a few degrees (eg, 10 degrees), a
three-point calibration should be
performed. Use water of appropriate
temperature. Test at temperatures
just below, just above, and at the
midway point of intended use. All

results should be within 1 C of the
NIST thermometer to be considered
acceptable.
Thermometers should be observed
routinely for any split in the column
because this will cause inaccurate
readings. The methods for reuniting
the separation can be found in NCCLS
Standard I2-A2.2 When this occurs,
document corrective action and
recalibrate the thermometer.

Use any of the following methods to
verify calibration:
a.
Follow manufacturer’s instructions for verifying calibration.
b.
Use a commercially available
calibration device by following
the instructions provided by
the device’s manufacturer.
c.
Calibrate the thermometer by
inserting the probe in a 37 C
waterbath with a NIST-certified
thermometer.
A result is acceptable if the reading
on the thermometer agrees with the
NIST thermometer within 0.1 C. If
expected results are not achieved,
unsatisfactory thermometers should
be returned to the distributor. Document corrective actions.

Methods Section 7: Quality Control

Record the date of testing, thermometer identification numbers, temperature readings, and the identity of the
person performing the test.

823

manufacturer or other equipment storage
expert. The facility procedures manual
must include a detailed description of the
method(s) in local use. (See Appendix 10 for
quality control testing intervals.)

Method 7.3. Testing Blood
Storage Equipment Alarms

Method 7.3.1. Testing Refrigerator Alarms

Blood storage refrigerators and freezers
must be equipped with a system for continuous temperature monitoring and an
audible alarm. If a storage unit goes into
alarm, it is essential that personnel know
the appropriate actions to take. Directions for such events should be available
in a conspicuous location, and personnel
should be trained to initiate these actions
if the temperature cannot be corrected
rapidly. The alarm on each storage unit
should be checked periodically for proper
functioning. Monthly checks are appropriate until consistent behavior of a particular storage unit has been demonstrated. Thereafter, alarms should be
tested regularly and frequently enough to
achieve and maintain personnel competency as well as to detect malfunctions.
For equipment in good condition, quarterly checks are usually sufficient. Because alarms may be disconnected or silenced during repairs, it is also prudent to
verify alarm functioning after repairs.
The high and low temperatures of activation must be checked and the results recorded. AABB Standards for Blood Banks
and Transfusion Services1(p5) requires that
the alarm be set to activate at a temperature that will allow appropriate intervention before blood or components reach an
undesirable temperature. Because of the diversity of equipment available, it is not possible to give specific instructions for all applicable alarm systems. If the equipment
user’s manual does not provide suitable directions for testing the alarm, consult the

Refrigerator temperatures may increase
beyond acceptable limits for several reasons, including:
1.
Improperly closed door.
2.
Insufficient refrigerant.
3.
Compressor failure.
4.
Dirty or blocked heat exchanger.
5.
Loss of electrical power.

Principle

Materials
1.
2.
3.
4.
5.
6.

Calibrated thermometer.
Pan large enough to hold the thermocouple container.
Water.
Crushed ice.
Table salt.
Worksheet for recording results.

Procedure
1.

Verify that the alarm circuits are operating, the alarm is switched on, and
the starting temperature is 1 to 6 C.
Immerse an easy-to-read calibrated
thermometer in the container with
the alarm thermocouple.
For low activation:
2.
Place the container with the thermocouple and thermometer in a pan
containing an ice and water slush at
a temperature of –4 C or colder. To
achieve this temperature, add several
spoonfuls of table salt to the slush.
3.
Close the refrigerator door to avoid
changing the temperature of the
storage compartment. Keep the container in the pan of cold slush, and

824

AABB Technical Manual

gently agitate it periodically until the
alarm sounds.
Record this temperature as the lowactivation temperature.
For high activation:
Place the container with thermocouple and thermometer in a pan containing cool water (eg, 12 to 15 C).
Close the refrigerator door. Allow the
fluid in the container to warm slowly,
with occasional agitation.
Record the temperature at which the
alarm sounds as the high-activation
temperature.
Record the date of testing, the refrigerator identification, the thermometer identification, and the identity of
the person performing the test.
If temperatures of activation are too
low or too high, take appropriate
corrective actions such as those suggested by the manufacturer, record
the nature of the corrections, and repeat the alarm check to document
that the corrections were effective.

Notes
1.

The thermocouple for the alarm should
be easily accessible and equipped
with a cord long enough so that it
can be manipulated easily.
The thermocouple for the continuous temperature monitor need not
be in the same container as that of
the alarm. If it is in the same container, a notation should be made in
the records that explains any outof-range temperature registered as a
result of the alarm check.
When the temperatures of alarm activation are checked, the temperature
change should occur slowly enough
so that the measurements and recording are accurate. Too rapid a

change in temperature may give the
false impression that the alarm does
not sound until an inappropriate
temperature is registered.
The low temperature of activation
should be greater than 1 C (eg, 1.5 C);
the high temperature of activation
should be less than 6 C (eg, 5.5 C).
Alarms should sound simultaneously at the site of the refrigerator and
at the location of remote alarms,
when employed. If remote alarms
are used, the alarm check should include a verification that the alarm
sounded at the remote location.
The amount of fluid in which the
thermocouple is immersed must be
no larger than the volume of the
smallest component stored in that
refrigerator. The thermocouple may
be immersed in a smaller volume,
but this means that the alarm will
go off with smaller temperature
changes than those registered in a
larger volume of fluid. Excessive sensitivity may create a nuisance.
With the one-time assistance of a
qualified electrician, the required refrigerator alarm checks of units with
virtually inaccessible temperature
probes can be performed with an
electrical modification cited by Wenz
and Owens.2

References
1.

Silva MA, ed. Standards for blood banks and
transfusion services. 23rd ed. Bethesda, MD:
AABB, 2005.
Wenz B, Owens RT. A simplified method for
monitoring and calibrating refrigerator alarm
systems. Transfusion 1980;20:75-8.

Method 7.3.2. Testing Freezer Alarms

Principle
Freezer temperatures may rise to unacceptable levels for a variety of reasons.

Methods Section 7: Quality Control

Common causes of rising temperatures
include:
1.
Improperly closed freezer door or lid.
2.
Low level of refrigerant.
3.
Compressor failure.
4.
Dirty or blocked heat exchanger.
5.
Loss of electrical power.

5.
6.

825

Return the freezer and the alarm system to their normal conditions.
If the alarm sounds at too high a
temperature, take appropriate corrective actions such as those suggested by the manufacturer, record
the nature of the correction, and repeat the alarm check to document
that the corrections were effective.

Materials
1.
2.

3.
4.

Protection for the freezer contents,
eg, a blanket.
Calibrated thermometer or thermocouple independent from that built
into the system.
Warm water or an oven mitt.
Worksheet for recording results.

Procedure
1.

Notes
1.

Protect frozen components from exposure to elevated temperatures during the test.
Use a thermometer or thermocouple, independent from that built into
the system, that will accurately indicate the temperature of alarm activation. Compare these readings with
the temperatures registered on the
recorder.
Warm the alarm probe and thermometer slowly (eg, in warm water, by
an oven-mitt-covered hand, exposure to air). The specific temperature
of activation cannot be determined
accurately during rapid warming,
and the apparent temperature of activation will be too high.
Record the temperature at which the
alarm sounds, the date of testing,
the identity of the person performing the test, the identity of the
freezer and calibrating instrument,
and any observations that might
suggest impaired activity.

Alarms should sound simultaneously at the site of the freezer and at
the location of the remote alarms,
when employed. If remote alarms
are used, the alarm check should include a verification that the alarm
sounded at the remote location.
Test battery function, electrical circuits, and power-off alarms more
frequently than the activation temperature. Record function, freezer
identification, date, and identity of
person performing the testing.
For units with the sensor installed in
the wall or in air, apply local warmth
to the site or allow the temperature
of the entire compartment to rise to
the point at which the alarm sounds.
Remove the frozen contents or protect them with insulation while the
temperature rises.
For units with the thermocouple located in antifreeze solution, pull the
container and the cables outside the
freezer chest for testing, leaving the
door shut and the contents protected.
For units with a tracking alarm that
sounds whenever the temperature
reaches a constant interval above
the setting on the temperature controller, set the controller to a warmer
setting and note the temperature interval at which the alarm sounds.

826

AABB Technical Manual

Liquid nitrogen freezers must have
alarm systems that activate at an unsafe level of contained liquid nitrogen.

Method 7.4. Functional
Calibration of Centrifuges for
Platelet Separation

Procedure

Preparation of Platelet-Rich Plasma
1.

Principle
Successful preparation of platelet concentrates requires adequate but not excessive
centrifugation; the equipment used must
perform in a consistent and dependable
manner. Each centrifuge used to prepare
platelets should be calibrated upon receipt and after adjustment or repair.
Functional calibration of the centrifuge
for both the preparation of platelet-rich
plasma (PRP) from whole blood and subsequent preparation of platelet concentrate from PRP can be performed during
the same procedure.

Materials
1.

3.
4.
5.
6.
7.

Freshly collected whole blood, obtained by phlebotomy into a bag with
two integrally attached transfer containers.
A specimen of blood from the donor,
anticoagulated with EDTA and collected in addition to the specimens
drawn for routine processing.
Metal clips and hand sealer or dielectric sealer.
Clean instruments (scissors, hemostats, tubing stripper).
Plasma extractor.
Centrifuge suitable for preparation
of platelet concentrates.
Worksheet for recording results.

Perform a platelet count on the anticoagulated specimen. If the platelet
count is below 133,000/µL, do not
use this donor’s blood for calibration.
Calculate and record the number of
platelets in the Whole Blood unit: platelets/µL × 1000 × mL of whole blood
= number of platelets in whole blood.
Prepare PRP at a selected speed and
time. (See “light spin” in Table 7.4-1
or guidance provided by the centrifuge manufacturer.)
Place a temporary clamp on the tubing so that one satellite bag is closed
off. Express the PRP into the other
satellite bag. Seal the tubing close to
the primary bag, leaving a long section of tubing, the “tail.” Disconnect
the two satellite bags from the primary bag. Do not remove the temporary clamp between the satellite
bags until the platelets are prepared
(see next section).
Strip the tubing and “tail” several
times so that they contain a representative sample of PRP.
Seal off a segment of the “tail” and
disconnect it, so that the bag of PRP
remains sterile.
Perform a platelet count on the sample of PRP in the sealed segment.
Calculate and record the number of
platelets in the bag of PRP: platelets/
µL × 1000 × mL of PRP = number of
platelets in PRP.
Calculate and record percent yield:
(number of platelets in PRP × 100)
divided by (number of platelets in
whole blood) = % yield.
Repeat the above process three or four
times with different donors, using
different speeds and times of centrifugation, and compare the yields

Methods Section 7: Quality Control

10.

11.

achieved under each set of test conditions.
Select the shortest time/lowest speed
combination that results in the highest percent of platelet yield without
unacceptable levels of red cell content in the PRP.
Record the centrifuge identification,
the calibration settings selected, the
date, and the identity of the person
performing the calibration.

3.
4.

Preparation of Platelets
1.

Centrifuge the PRP (as prepared
above) at a selected time and speed
to prepare platelets. (See “heavy spin”
in Table 7.4-1 or guidance provided
by the centrifuge manufacturer.)
Remove the temporary clamp between the two satellite bags and express the platelet-reduced plasma
into the second attached satellite
bag, leaving approximately 55 to 60
mL volume in the platelet bag. Seal
the tubing, leaving a long section of
tubing attached to the platelet bag.

6.
7.

8.
9.

827

Allow the platelets to rest for approximately 1 hour.
Place the platelets on an agitator for
at least 1 hour to ensure that they are
evenly resuspended. Platelet counts
performed immediately after centrifugation will not be accurate.
Strip the tubing several times, mixing tubing contents well with the
contents of the platelet bag. Seal off
a segment of the tubing and disconnect it, so that the platelet bag remains sterile.
Perform a platelet count on the contents of the segment.
Calculate and record the number of
platelets in the concentrate: platelets/
µL × 1000 × mL of platelets = number
of platelets in platelet concentrate.
Calculate and record percent yield.
Repeat the above process with PRP
from different donors, using different speeds and times of centrifugation, and compare the yields achieved
under each set of test conditions.

Table 7.4-1. Centrifugation for Component Preparation
Heavy Spin
Red cells
Platelets from whole blood

Cell- free plasma
Cryoprecipitate

5000 × g, 5 minutes*
5000 × g, 7 minutes*

Light Spin
2000 × g, 3 minutes*

Platelet-rich plasma

To calculate relative centrifugal force in g:
rcf (in g) = 28.38 × R† × (rpm/1000)2
*Times include acceleration but not deceleration times. Times given are approximations only. Each individual centrifuge
must be evaluated for the preparation of the various components.
†
R = radius of centrifuge rotor in inches.

828

10.

11.

AABB Technical Manual

Select the shortest time/lowest speed
combination that results in the highest percent of platelet yield in the
platelet concentrate.
Record the centrifuge identification,
the calibration settings selected, the
date, and the identity of the person
performing the calibration.

Notes
1.

McShine R, Das P, Smit Sibinga C, Brozovic B.
Effect of EDTA on platelet parameters in
blood and blood components collected with
CPDA-1. Vox Sang 1991;61:84-9.

Method 7.5. Functional
Calibration of a Serologic
Centrifuge
Principle

It is not necessary to perform functional recalibration of a centrifuge
unless the instrument has undergone adjustments or repairs, or
component quality control indicates
that platelet counts have fallen below acceptable levels. However,
timer, speed, and temperature calibrations of the centrifuge should occur on a regularly scheduled basis
(see Appendix 10).
Each centrifuge used for preparing
platelets must be calibrated individually. Use the conditions determined
to be optimal for each instrument.
When counting platelet samples on
an instrument intended for whole
blood, it may be necessary to use a
correction factor to obtain accurate
results.
When determining the appropriate
time and speed of centrifugation,
consideration should also be given to
other products that will be prepared
from the whole blood. Final size and
hematocrit of red cell and plasma
volume made available for further
processing are important factors to
consider.

References
1.

Kahn R, Cossette I, Friedman L. Optimum
centrifugation conditions for the preparation
of platelet and plasma products. Transfusion
1976;16:162-5.

Each centrifuge should be calibrated
upon receipt, after adjustments or repairs,
and periodically. Calibration evaluates the
behavior of red cells in solutions of different viscosities, not the reactivity of different antibodies.

For Immediate Agglutination

Materials
1.

2.
3.

Test tubes, 10 × 75 mm or 12 × 75
mm (whichever size is routinely
used in the laboratory)
Worksheet for recording results.
For saline-active antibodies:
■
Serum from a group A person
(anti-B) diluted with 6% albumin to give 1+ macroscopic agglutination (3 mL of 22% bovine
albumin + 8 mL of normal saline = 6% bovine albumin). See
Method 1.5.
■
Positive control: Group B red cells
in a 2% to 5% saline suspension.
■
Negative control: Group A red cells
in a 2% to 5% saline suspension.
For high-protein antibodies:
■
Anti-D diluted with 22% or 30%
albumin to give 1+ macroscopic agglutination.
■
Positive control: D+ red cells in
a 2% to 5% saline suspension.
■
Negative control: D– red cells in
a 2% to 5% saline suspension.

Methods Section 7: Quality Control

Procedure
1.

For each set of tests (saline and highprotein antibodies), label five test
tubes for positive reactions and five
for negative reactions.
In quantities that correspond to routine use, add diluted anti-B to each
of 10 tubes for the saline test and
add diluted anti-D to each of 10 tubes
for the high-protein test. Add serum
and reagents in quantities that correspond to routine use.
Add the appropriate control cell suspension to one set of tubes (one positive and one negative tube for the
saline test, and one positive and one
negative tube for the high-protein
antibody test). Centrifuge immediately for the desired time interval
(eg, 10 seconds).
Observe each tube for agglutination
and record observations. (See example in Table 7.5-1.)
Repeat steps 2 and 3 for each time
interval (eg, 15, 20, 30, and 45 seconds). Do not allow cells and sera to
incubate before centrifugation.

829

Select the optimal time of centrifugation, which is the shortest time required to fulfill the following criteria:
a.
Agglutination in the positive
tubes is as strong as determined
in preparing reagents.
b.
There is no agglutination or
ambiguity in the negative tubes.
c.
The cell button is clearly delineated and the periphery is
sharply defined, not fuzzy.
d.
The supernatant fluid is clear.
e.
The cell button is easily resuspended.
In the example shown in Table 7.5-1, these criteria are met
by the 30-second and the 45second spins; the optimal time
for these tests in this centrifuge
is 30 seconds.
Record centrifuge identification, the
times selected, the date, and the
identity of the person performing
the calibration.

For Washing and Antiglobulin Testing
Tests in which antihuman globulin (AHG)
serum is added to red cells may require

Table 7.5-1. Example of Serologic Centrifuge Test Results*
Time in Seconds
Criteria

Supernatant fluid is clear

Yes

Cell button is clearly delineated

Yes

Cells are easily resuspended

Yes

Agglutination is observed

Negative tube is negative

Yes

Resuspends
roughly

*The optimal time for centrifugation in this example is 30 seconds.

830

AABB Technical Manual

centrifugation conditions different from
those for immediate agglutination. Centrifugation conditions appropriate for
both washing and AHG reactions can be
determined in one procedure. Note that
this procedure does not monitor the completeness of washing; use of IgG-coated cells
to control negative AHG reactions provides
this check. The following procedure addresses only the mechanics of centrifugation.

4.
5.

AHG reagent, unmodified.
Saline, large volumes.
Test tubes, 10 × 75 mm or 12 × 75
mm (whichever size is routinely used
in the laboratory).
Worksheet for recording results.
Positive control: a 2% to 5% saline
suspension of D+ red cells incubated
for 15 minutes at 37 C with anti-D
diluted to give 1+ macroscopic agglutination after addition of AHG.
Negative control: a 2% to 5% suspension of D+ red cells incubated for 15
minutes at 37 C with 6% albumin.
[Note: D– red cells incubated with
diluted anti-D may also be used as a
negative control.]

Procedure
1.

4.
5.

Materials
1.
2.
3.

Prepare five test tubes containing 1
drop of positive cells and five tubes
containing 1 drop of negative control cells.
Fill tubes with saline and centrifuge
them in pairs, one positive and one
negative, for different times (eg, 30,
45, 60, 90, and 120 seconds). The red
cells should form a clearly delineated button, with minimal cells
trailing up the side of the tube. After
the saline has been decanted, the
cell button should be easily resus-

8.
9.

pended in the residual fluid. The
optimal time for washing is the
shortest time that accomplishes
these goals.
Repeat washing process on all pairs
three more times, using time determined to be optimal.
Decant supernatant saline thoroughly.
Add AHG to one positive control test
tube and one negative control test tube.
Centrifuge immediately for the desired interval (eg, 10 seconds).
Observe each tube for agglutination
and record observations.
Repeat steps 5 and 6 for each interval (eg, 15, 20, 30, and 45 seconds).
Do not allow cells and AHG to incubate before centrifugation.
Select optimal time as in immediate
agglutination procedure.
Record centrifuge identification, the
times selected, the date, and the
identity of the person performing
the calibration.

Notes
Periodic recalibration is performed to verify that the timing in use continues to be
the optimal timing. This may be accomplished by using a shortened version of
the procedures outlined above. For example, use the current timing for a particular
centrifuge and each medium and those
times just above and just below the current timing.

Method 7.6. Performance
Testing of Automatic Cell
Washers
Principle
Antihuman globulin (AHG) is inactivated
readily by unbound immunoglobulin. The
red cells to which AHG will be added

Methods Section 7: Quality Control

must be washed free of all proteins and
suspended in a protein-free medium. A
properly functioning cell washer must add
large volumes of saline to each tube, resuspend the cells, centrifuge them adequately to avoid excessive red cell loss, and
decant the saline to leave a dry cell button.

5.
6.

7.
8.

Materials
1.
2.
3.
4.

5.
6.
7.

Test tubes routinely used in the laboratory, 10 × 75 mm or 12 × 75 mm.
Additive routinely used to potentiate
antigen-antibody reactions.
Human serum, from patient or donor.
IgG-coated red cells, known to give a
1 to 2+ reaction in antiglobulin testing.
Normal saline.
AHG reagent, anti-IgG or polyspecific.
Worksheet for recording results.

10.

831

Continue the washing cycle.
After addition of saline in the third
cycle, stop the cell washer and inspect tubes as above. Record observations.
Complete the wash cycle.
At the end of the wash cycle, inspect
all tubes to see that saline has been
completely decanted and that each
tube contains a dry cell button. Record observations.
Add AHG according to the manufacturer’s directions, centrifuge, and examine all tubes for agglutination. If
the cell washer is functioning properly, the size of the cell button
should be the same in all tubes. All
tubes should show the same degree
of agglutination. Record observations.
Record identity of centrifuge, the date
of testing, and the identity of the
person performing the testing.

Procedure
1.

To each of 12 tubes, add potentiator
and human serum in quantities that
correspond to routine use and 1 drop
of IgG-coated red cells.
Place the tubes in a centrifuge carrier, seat the carrier in the cell washer,
and start the wash cycle.
After addition of saline in the second
cycle, stop the cell washer. Inspect
the contents of all tubes. There
should be an approximately equal
volume of saline in all tubes; some
variation is acceptable. Tubes should
be approximately 80% full, to avoid
splashing and cross-contamination.
(Refer to manufacturer’s instructions
for specific requirements.) Record
observations.
Observe all tubes to see that the red
cells have been completely resuspended. Record observations.

Notes
1.

Further investigation is needed if:
a.
The amount of saline varies
significantly from tube to tube
or cycle to cycle.
b.
The cell button is not resuspended completely after being
filled with saline.
c.
Any tube has weak or absent
agglutination in the antiglobulin phase.
d.
Any tube has a significant decrease in the size of the cell
button.
Cell washers that automatically add
AHG should also be checked for uniform addition of AHG. In step 9
above, AHG would be added automatically, and failure of addition
would be apparent by absence of agglutination. The volume of AHG

832

AABB Technical Manual

should be inspected and found to be
equal in all tubes. The volume of
AHG delivered automatically by cell
washers should be checked monthly
to ensure that it is as specified in the
manufacturer’s directions and that
delivery is uniform in all tubes.
Some manufacturers market AHG
colored with green dye for use in automated cell washers so that it will
be immediately obvious if no reagent has been added.

Method 7.7. Monitoring Cell
Counts of Apheresis
Components

4.
5.

Principle
When cellular components are prepared
by apheresis, it is essential to determine
cell yields without compromising the sterility of the component.

Materials
1.
2.
3.
4.
5.
6.
7.

Component collected by apheresis.
Metal clips and hand sealer or dielectric sealer.
Tubing stripper.
Clean instruments (scissors, hemostats).
Test tubes.
Cell-counting equipment.
Worksheet for recording results.

Seal a 5- to 8-cm (2- to 3-inch) segment distal to the collection bag.
There should be approximately 2 mL
of fluid in the segment. Double-seal
the end of the tubing next to the
component bag and detach the segment.
Empty the contents of the segment
into a suitably labeled tube.
Determine and record cell counts in
cells/mL.
a.
For results reported as cells/µL,
change values to cells/mL by
multiplying by 1000 (or 103).
b.
For results reported as cells/L,
change values to cells/mL by
dividing by 1000 (or 103).
Multiply cells/mL by the volume of
the component, in mL, to obtain total cell count in the component.
Record component’s identity, the date,
and the identity of the person performing the testing.

Note
Refer to manufacturer’s directions for any
additional requirements.

Method 7.8. Manual Method
for Counting Residual White
Cells in Leukocyte-Reduced
Blood and Components
Principle

Procedure
1.
2.

Ensure that the contents of the apheresis component bag are well mixed.
Strip the attached tubing at least four
times, mixing the contents of the
tubing with the contents of the bag,
to ensure that the contents of the
tubing accurately represent the entire contents of the bag.

The residual white cell content of leukocyte-reduced whole blood and components
can be determined using a large-volume
hemocytometer. For red-cell-containing
components, the red cells in the aliquot to
be counted are first lysed. Crystal violet is
used to stain the leukocyte nuclei. The
Nageotte counting chamber has a volume
56 times that of the standard hemocyto-

Methods Section 7: Quality Control

meter. Accuracy of counting is improved
by examining a larger volume of minimally diluted specimen, compared with
standard counting techniques.

Materials
1.

4.
5.
6.
7.

Hemocytometer chamber with 50 µL
counting volume (eg, Nageotte Brite
Line Chamber).
Crystal violet stain: 0.01% w/v crystal violet in 1% v/v acetic acid (eg,
Turks solution).
Red cell lysing agent (eg, Zapoglobin,
Coulter Electronics, Hialeah, FL), for
red-cell-containing components
only.
Pipettor (40 µL and 100 µL) with disposable tips.
Talc-free gloves, clean plastic test
tubes, plastic petri dish, filter paper.
Light microscope with 10× ocular
lens and 20× objective.
Worksheet for recording results.

Procedure
1.

Dilute and stain leukocyte-reduced
blood and component samples as
follows:
a.
For Red-Cell-Containing Components
1)
Pipette 40 µL of lysing
agent into a clean test tube.
2)
Place a representative
sample of the component
to be tested in a clean test
tube. The hematocrit of the
sample to be tested should
not exceed 60%.
3)
Pipette 100 µL of the sample into the tube containing 40 µL of lysing agent.
Rinse the pipette several
times to mix the two fluids, until the pipette tip is

833

no longer coated with intact red cells.
4)
Pipette 360 µL of crystal
violet stain into the mixture and mix fluids by
pipetting up and down
several times. The final
volume is now 500 µL.
b.
For Platelets
1)
Place a representative sample of the platelet in a clean
test tube.
2)
Pipette 100 µL of the platelet sample into a clean
test tube.
3)
Pipette 400 µL of crystal
violet stain into the 100 µL
of platelets and mix fluids
by pipetting up and down
several times. The final
volume is now 500 µL.
Fit the hemocytometer with a coverslip and, using a pipette, load the
mixture until the counting area is
completely covered but not overflowing.
Cover the hemocytometer with a
moist lid to prevent evaporation (a
plastic petri dish into which a piece
of damp filter paper has been placed
works well) and let it rest undisturbed
for 10 to 15 minutes, to allow the
white cells to settle in the counting
area of the chamber.
Remove the moist lid, place the
hemocytometer on the microscope
and, using a 20× objective, count the
white cells present in the entire
50-µL volume of the counting chamber.
Calculate and record results.
a.
White cell concentration:

leukocytes/µL

= (cells counted/50 µL) × 5
= cells counted/10

834

AABB Technical Manual

where 50 µL is the volume
counted and 5 is the dilution
factor resulting from the addition of lysing agent and stain.
Total white cell content of the
leukocyte-reduced component:

leukocytes/µL ×
leukocytes/
1000
µL /mL ×
=
component
volume in mL
of the component
6.

Record the component’s identity, the
date, and the identity of the person
performing the testing.

Notes
1.

White cells deteriorate during refrigerated storage; counts on stored blood
or red cell components may give inaccurate results.
Use of talc-free gloves is recommended because talc particles that
contaminate the counting chamber
can be misread as white cells.
Experience identifying crystal-violetstained white cells can be obtained
by examining samples from components that have not been leukocyte
reduced.

The accuracy of the counting method
can be validated from a reference
sample with a high white cell content that has been quantified by another means. This reference sample
can be used for serial dilutions in
blood or a component that has been
rendered extremely leukocyte reduced by two passages through a
leukocyte reduction filter. Counts
obtained on the serially diluted samples can be compared with the expected concentration derived by calculation.
This counting technique is not known
to be accurate at concentrations lower
than 1 white cell/µL.

References
1.

Lutz P, Dzik WH. Large-volume hemocytometer chamber for accurate counting of
white cells (WBCs) in WBC-reduced platelets;
validation and application for quality control
of WBC-reduced platelets prepared by
apheresis and filtration. Transfusion 1993;33:
409-12.
Dzik WH, Szuflad P. Method for counting
white cells in white cell-reduced red cell concentrates (letter). Transfusion 1993;33:272.

Appendices

Appendices
Appendix 1. Normal Values in Adults
Determination

SI Units

Conventional Units

Alanine aminotransferase
Bilirubin, total
Haptoglobin
Hematocrit
Males
Females
Hemoglobin
Males
Females
Hemoglobin A2
Hemoglobin F
Hemoglobin (plasma)
Immunoglobulins
IgG
IgA
IgM
IgD
IgE
Methemoglobin
Platelet count
Red cells
Males
Females
Reticulocyte count
Viscosity, relative
White cells

4-36 U/L at 37 C
2-21 µmol/L
0.6-2.7 g/L

4-36 U/L at 37 C
0.1-1.2 mg/dL
60-270 mg/dL

0.40-0.54
0.38-0.47

40-54%
38-47%

135-180 g/L
120-160 g/L
0.015-0.035 total Hb
0-0.01 total Hb
5-50 mg/L

13.5-18.0 g/dL
12.0-16.0 g/dL
1.5-3.5% total Hb
<1% total Hb
0.5-5.0 mg/dL

8.0-18.0 g/L
1.1-5.6 g/L
0.5-2.2 g/L
5.0-30 mg/L
0.1-0.4 mg/L
<0.01 total Hb
150-450 × 109/L

800-1801 mg/dL
113-563 mg/dL
54-222 mg/dL
0.5-3.0 mg/dL
0.01-0.04 mg/dL
<1% total Hb
150-450 × 103/µL

4.6-6.2 × 1012/L
4.2-5.4 × 1012/L
25-75 × 109/L
1.4-1.8 × water
4.5-11.0 × 109/L

4.6-6.2 × 106/µL
4.2-5.4 × 106/µL
25-75 × 103/µL
1.4-1.8 × water
4.5-11.0 × 103/µL
835

836

AABB Technical Manual

Appendix 2. Selected Normal Values in Children
SI Units

Conventional Units

Preterm

<30 mmol/L

<1.8 mg/dL

Term

<30 mmol/L

<1.8 mg/dL

Preterm

<137 mmol/L

<8 mg/dL

Term

<103 mmol/L

<6 mg/dL

Preterm

<205 mmol/L

<12 mg/dL

Term

<137 mmol/L

<8 mg/dL

Preterm

<274 mmol/L

<16 mg/dL

Term

<205 mmol/L

<12 mg/dL

Preterm

<205 mmol/L

<12 mg/dL

Term

<120 mmol/L

<7 mg/dL

Preterm

<34 mmol/L

<2 mg/dL

Term

<17 mmol/L

<1 mg/dL

Bilirubin (total)
Cord
0-1 day
1-2 days
3-7 days
7-30 days
Thereafter

26-30 weeks’ gestation
Term
1-3 days

Hemoglobin

WBC

11.0-15.8 g/dL

1.7-7.1 × 10'/L

13.5-19.5 g/dL
14.5-22.5 g/dL

Platelets

192,000/µL (mean)

252,000/µL (mean)

9-30 × 10 /L
9.4-34 × 10 /L
'

2 weeks

13.4-19.8 g/dL

5-20 × 10 /L

1 month

10.7-17.1 g/dL

4-19.5 × 10'/L

2 months

9.4-13.0 g/dL

6 months

11.1-14.1 g/dL

6-17.5 × 10'/L

6 months-2 years

10.5-13.5 g/dL

6-17 × 10'/L

2-6 years
6-12 years

11.5-13.5 g/dL

180,000-327,000/µL

150,000-350,000/µL

5-15.5 × 10 /L

11.5-15.5 g/dL

4.5-13.5 × 10 /L

150,000-350,000/µL

13.0-16.9 g/dL

4.5-13.5 × 10'/L

150,000-350,000/µL

12-18 years
Male
Female

12.0-16.0 g/dL

4.5-13.5 × 10 /L

150,000-350,000/µL

Appendices

837

Appendix 2. Selected Normal Values in Children (cont’d)
IgG
Newborn

IgM

IgA

831-1231

mg/dL

6-16

mg/dL

1-3 months

312-549

mg/dL

19-41

mg/dL

8-34

mg/dL

4-6 months

241-613

mg/dL

26-60

mg/dL

10-46

mg/dL

7-12 months

442-880

mg/dL

31-77

mg/dL

19-55

mg/dL

13-24 months

553-971

mg/dL

35-81

mg/dL

26-74

mg/dL

25-36 months

709-1075

mg/dL

42-80

mg/dL

34-108

mg/dL

3-5 years

701-1157

mg/dL

38-74

mg/dL

66-120

mg/dL

6-8 years

667-1179

mg/dL

40-80

mg/dL

79-169

mg/dL

9-11 years

889-1359

mg/dL

46-112

mg/dL

71-191

mg/dL

12-16 years

822-1070

mg/dL

39-79

mg/dL

85-211

mg/dL

Activated Partial Thromboplastin Time
Preterm

70 seconds

Full-term

45-65 seconds

Prothrombin Time
Preterm

12-21 seconds

Full-term

13-20 seconds

Reprinted with permission from The Harriet Lane Handbook. 15th ed. St. Louis, MO: Mosby, 2000.

Appendix 3. Typical Normal Values in Tests of Hemostasis and Coagulation
(Adults)
Test

Normal Value

Activated partial thromboplastin time
Bleeding time
Coagulation factors
Fibrin degradation products
Fibrinogen
Plasma D-dimers
Protein C
Protein S (total)
Prothrombin time
Thrombin time

25-35 seconds
2-8 minutes
500-1500 U/L
<10 mg/L
2.0-4.0 g/L
<200 mg/L
70-1400 U/L
70-1400 U/L
10-13 seconds
17-25 seconds

Reprinted with permission from Henry JB. Clinical diagnosis and management by laboratory methods. 20th ed. Philadelphia: WB Saunders, 2001.

838

AABB Technical Manual

Appendix 4. Coagulation Factor Values in Platelet Concentrates
Factor/
Protein

Normal
Range

Day 0

Day 1

Day 2

Day 3

Day 4

Day 5

II %
V%
VII %
VIII %
IX %
X%
XI %
XII %
C%
S%
Antithrombin %
Plasminogen %
Fibrinogen
mg/dL
Ristocetin
cofactor %

78-122
47-153
51-168
48-152
62-138
58-142
52-148
46-126
57-128
83-167
88-126
60-140
198-434

104
78-98
108
68-126
72-105
66-101
91-111
117
106
95
103
140
217-308

91-96
69-78
93-117
85-99
100-106
93-94
106-108
107-112
102
75
99
133
278-313

96
50
88
76
95
92
103
116
101
61
101
126
310

85-94
36-47
80-103
68-76
91-98
85-88
96-98
106-123
98
40
102
122
265-323

90
28
75
75
93
84
101
123
99
32
103
124
302

90
24-35
72
39-70
63-97
60-83
86-110
131
100
31
97
117
221-299

50-150

106

124

125

133

116

127

Note: Coagulation factor % = 100 x coagulation factor units/mL.
Reproduced with permission from Brecher ME, ed. Collected questions and answers. 6th ed. Bethesda, MD:
AABB, 2000.

Appendices

839

Appendix 5. Approximate Normal Values for Red Cell, Plasma, and Blood
Volumes
Infant

Adult

Premature

Term Birth at
72 hours

Male

Female

Red Cell Volume mL/kg

Plasma Volume mL/kg

108

Blood Volume mL/kg

The adult values should be modified to correct
for:
1. Below age 18: increase values by 10%.
2. Weight loss:
a. Marked loss within 6 months—calculations made at original weight.
b. Gradual loss over a longer time—calculations made at present weight and
raised 10% to 15%.
3. Obese and short: values are reduced by
10%.
4. Elderly: values are reduced by 10%.
5. Pregnancy3:

Estimation of Body Surface Area :
BSA(m 2 ) =

Ht(cm) × Wt(kg)
or
3600

Ht(in) × Wt(lb)
3131

Blood Volume (BV) :
BV = 2740 mL/m2—males
BV = 2370 mL/m2—females
6
Hematocrit :
Venous hematocrit = Hv (blood obtained by
vein or finger puncture)
Whole-body hematocrit = HB
HB = (Hv) × (0.91)

References
1.

2.
3.
4.
5.

Miller D. Normal values and examination of the
blood: Perinatal period, infancy, childhood and adolescence. In: Miller DR, Baehner RL, McMillan CW,
Miller LP, eds. Blood diseases of infancy and childhood. St. Louis: C.V. Mosby, 1984:21,22.
Albert SN. Blood volume. Springfield, IL: Charles C.
Thomas, 1963:26.
Peck TM, Arias F. Hematologic changes associated
with pregnancy. Clin Obstet Gynecol 1979;22:788.
Mosteller RD. Simplfied calculation of body-surface
area. N Engl J Med 1987;317:1098.
Shoemaker WC. Fluids and electrolytes in the
acutely ill adult. In: Shoemaker WC, Ayres S,
Grenvik A, et al, eds. Textbook of critical care. 2nd
ed. Philadelphia: WB Saunders Co., 1989:1130.
Mollison PL, Englefriet CP, Contreras M. Blood
transfusion in clinical medicine. 9th ed. Oxford:
Blackwell Scientific Publications, 1993.

840

AABB Technical Manual

Appendix 6. Blood Group Antigens Assigned to Systems
In 1980, the International Society of Blood Transfusion (ISBT) formed a Working Party on Terminology
for Red Cell Surface Antigens. The task of this group was to devise a uniform nomenclature that would
be both eye- and machine-readable. The numeric system proposed by this group was not intended to replace traditional terminology but, instead, to enable communication using computer systems where
numbers are necessary. ISBT terminology uses uppercase letters and Arabic numbers for system and
antigen codes. Each system, collection, or series of antigens is given a number (eg, ABO system = 001),
and each antigen within the system is given a number (eg, A = 001, B = 002). Sinistral zeros may be
omitted. Thus, in ISBT terminology, the A antigen would be written using computer code as 001001 or
1.1, or using the system symbol, as ABO1.
Periodically, the Working Party meets to update assignment of antigens to systems, collections, and series. The table below lists the blood group systems and the antigens assigned to those systems. Other
red cell antigens are assigned to collections and to series of high- and low-incidence antigens. Although
all terms in the table are acceptable, the Technical Manual and TRANSFUSION choose to use traditional
terminology in most cases. Further information on blood group terminology, which antigens are assigned
to the collections, and the series of high- and low-incidence antigens can be found in the references.

System (ISBT
Symbol/Number)

Antigen (ISBT Number)

ABO (ABO/001)

A(ABO1)
B (ABO2)
A,B (ABO3)
A1 (ABO4)

MNSs or MNS or
MN (MNS/002)

M (MNS1)
N (MNS2)
S (MNS3)
s (MNS4)
U (MNS5)
He (MNS6)
Mia (MNS7)
Mc (MNS8)
Vw (MNS9)
Mur (MNS10)
Mg (MNS11)
Vr (MNS12)

P (P/003)

P1 (P1)

Me (MNS13)
Mta (MNS14)
Sta (MNS15)
Ria (MNS16)
Cla (MNS17)
Nya (MNS18)
Hut (MNS19)
Hil (MNS20)
Mv (MNS21)
Far (MNS22)
sD (MNS23)
Mit (MNS24)

Dantu (MNS25)
Hop (MNS26)
Nob (MNS27)
Ena (MNS28)
ENKT (MNS29)
‘N’ (MNS30)
Or (MNS31)
Dane (MNS32)
TSEN (MNS33)
MINY (MNS34)
MUT (MNS35)
SAT (MNS36)

ERIK (MNS37)
Osa (MNS38)
ENEP (MNS39)
ENEH (MNS40)
HAG (MNS41)
ENAV (MNS42)
MARS (MNS43)

Appendices

841

Appendix 6. Blood Group Antigens Assigned to Systems (cont’d)
System (ISBT
Symbol/Number)

Antigen (ISBT Number)

Rh (RH/004)

D (RH1)
C (RH2)
E (RH3)
C (RH4)
e (RH5)
f (RH6)
Ce (RH7)
CW (RH8)
CX (RH9)
V (RH10)
EW (RH11)
G (RH12)

Hro (RH17)
Hr (RH18)
hrS (RH19)
VS (RH20)
CG (RH21)
CE (RH22)
DW (RH23)
c-like (RH26)
cE (RH27)
hrH (RH28)
Rh29 (RH29)
Goa (RH30)

hrB (RH31)
Rh32 (RH32)
Rh33 (RH33)
HrB (RH34)
Rh35 (RH35)
Bea (RH36)
Evans (RH37)
Rh39 (RH39)
Tar (RH40)
Rh41 (RH41)
Rh42 (RH42)
Crawford (RH43)

Nou (RH44)
Riv (RH45)
Sec (RH46)
Dav (RH47)
JAL (RH48)
STEM (RH49)
FPTT (RH50)
MAR (RH51)
BARC (RH52)
JAHK (RH53)
DAK (RH54)
LOCR (RH55)
CENR (RH56)

Lutheran (LU/005)

Lua (LU1)
Lub (LU2)
Lu3 (LU3)
Lu4 (LU4)
Lu5 (LU5)

Lu6 (LU6)
Lu7 (LU7)
Lu8 (LU8)
Lu9 (LU9)
Lu11 (LU11)

Lu12 (LU12)
Lu13 (LU13)
Lu14 (LU14)
Lu16 (LU16)
Lu17 (LU17)

Aua (LU18)
Aub (LU19)
Lu20 (LU20)
Lu21 (LU21)

Kell (KEL/006)

K (KEL1)
k (KEL2)
Kpa (KEL3)
Kpb (KEL4)
Ku (KEL5)
Jsa (KEL6)
Jsb (KEL7)

Ula (KEL10)
K11 (KEL11)
K12 (KEL12)
K13 (KEL13)
K14 (KEL14)
K16 (KEL16)
Wka (KEL17)

K18 (KEL18)
K19 (KEL19)
Km (KEL20)
Kpc (KEL21)
K22 (KEL22)
K23 (KEL23)
K24 (KEL24)

VLAN (KEL25)
TOU (KEL26)
RAZ (KEL27)
VONG (KEL28)

Lewis (LE/007)

Lea (LE1)
Leb (LE2)
Leab (LE3)

LebH (LE4)
ALeb (LE5)
BLeb (LE6)

Duffy (FY/008)

Fya (FY1)
Fyb (FY2)
Fy3 (FY3)

Fy4 (FY4)
Fy5 (FY5)
Fy6 (FY6)

Kidd (JK/009)

Jka (JK1)
Jkb (JK2)
Jk3 (JK3)

(cont’d)

842

AABB Technical Manual

Appendix 6. Blood Group Antigens Assigned to Systems (cont’d)
System (ISBT
Symbol/Number)

Antigen (ISBT Number)

Diego (DI/010)

Dia (DI1)
Dib (DI2)
Wra (DI3)
Wrb (DI4)
Wda (DI5)
Rba (DI6)

Yt or Cartwright
(YT/011)

Yta (YT1)
Ytb (YT2)

Xg (XG/012)

Xga (XG1)

CD99 (XG2)

Scianna (SC/013)

Sc1 (SC1)
Sc2 (SC2)
Sc3 (SC3)

Rd (SC4)
STAR (SC5)

WARR (DI7)
ELO (DI8)
Wu (DI9)
Bpa (DI10)
Moa (DI11)
Hga (DI12)

Vga (DI13)
Swa (DI14)
BOW (DI15)
NFLD (DI16)
Jna (DI17)
KREP (DI18)
Tra (DI19)*

Dombrock (DO/014) Doa (DO1)
Dob (DO2)
Gya (DO3)
Hy (DO4)
Joa (DO5)
Colton (CO/015)

Coa (CO1)
Cob (CO2)
Co3 (CO3)

LW or LandsteinerWiener (LW/016)

LWa (LW5)
LWab (LW6)
LWb (LW7)

Chido/Rodgers
(CH/RG /017)

Ch1 (CH/RG1)
Ch2 (CH/RG2)
Ch3 (CH/RG3)
Ch4 (CH/RG4)
Ch5 (CH/RG5)
Ch6 (CH/RG6)
WH (CH/RG7)

H (H/018)

H (H1)

Kx (XK/019)

Kx (XK1)

Rg1 (CH/RG11)
Rg2 (CH/RG12)

Fra (DI20)
SW1 (DI21)

Appendices

843

Appendix 6. Blood Group Antigens Assigned to Systems (cont’d)
System (ISBT
Symbol/Number)

Antigen (ISBT Number)

Gerbich (GE/020)

Ge2 (GE2)
Ge3 (GE3)
Ge4 (GE4)

Wb (GE5)
Lsa (GE6)
Ana (GE7)

Dha (GE8)
GEIS (GE9)

Cromer (CR/021)

Cra (CROM1)
Tca (CROM2)
Tcb (CROM3)
Tcc (CROM4)

Dra (CROM5)
Esa (CROM6)
IFC (CROM7)
WESa (CROM8)

WESb (CROM9)
UMC (CROM10)
GUTI (CROM11)
SERF (CROM12)

ZENA (CROM13)

Knops (KN/022)

Kna (KN1)
Knb (KN2)

McCa (KN3)
Sla (KN4)

Yka (KN5)
McCb (KN6)

Sl2 (KN7)
Sl3 (KN8)*

Indian (IN/023)

Ina (IN1)
Inb (IN2)

Ok (OK/024)

Oka (OK1)

Raph (RAPH/025)

MER2 (RAPH1)

JMH or John Milton JMH (JMH1)
Hagen
(JMH/026)
I (I/027)

I (I1)

Globoside
(GLOB/028)

P (GLOB1)

GIL (GIL/029)

GIL (GIL1)

*Provisional.
Daniels GL, Anstee DJ, Cartron JP, et al. International Society of Blood Transfusion working party on terminology for red cell surface antigens. Vox Sang 2001;80:193-6.
Daniels GL, Fletcher A, Garratty G, et al. Blood group terminology 2004. From the ISBT committee on terminology for red cell surface antigens. Vox Sang 2004;87:304-16.
Garratty G, Dzik W, Issitt PD, et al. Terminology for blood group antigens and genes—historical origins and
guidelines in the new millennium. Transfusion 2000;40:477-89.
Issitt PD, Anstee DJ. Applied blood group serology. 4th ed. Durham, NC: Montgomery Scientific, 1998.

844

AABB Technical Manual

Appendix 7. Examples of Gene, Antigen, and Phenotype Terms
System

Genes

Antigens

Phenotypes

ABO
Rh
MN
P
Lewis
Kell
Kell
Scianna
Kidd

A A1 A2 B
DCEce
MNSs
P1
Le le
K k Kpa Jsa
K1 K2 K3
Sc1 Sc2 Sc
Jka Jkb Jk3

A A1 A2 B
DCEce
MNSs
P1
Lea Leb
K k Kpa Jsa
K1 K2 K3
Sc1 Sc2
Jka Jkb Jk3

A A1 A2 B
D+C+E–c+e+
M+N+S–s+
P1+ P1–
Le(a+)Le(a–b+)
K–k+Kp(a+)Js(a–)
K:–1,2,–3
Sc:–1,–2,–3
Jk(a+)Jk(a+b+)Jk:3

Modified from:
Denomme G, Lomas-Francis C, Storry J, Reid ME. Approaches to blood group molecular genotyping and its applications. In: Stowell C, Dzik W, eds. Emerging diagnostic and therapeutic technologies in transfusion medicine. Bethesda, MD: AABB Press, 2003:95-129.
Garratty G, Dzik W, Issitt PD, et al. Terminology for blood group antigens and genes—historical origins and
guidelines in the new millennium. Transfusion 2000;40:477-89.
Zelinski T. Chromosomal localization of human blood group genes. In: Silberstein LE, ed. Molecular and functional aspects of blood group antigens. Bethesda, MD: AABB, 1995:41-73.

Appendix 8. Examples of Correct and Incorrect Terminology*
Term Description

Correct Terminology

Incorrect Terminology

Phenotype

Fy(a+)

Fya+, Fy(a+), Fya(+), Fya+, Fya(+), Duffya+,
Duffya-positive

Phenotype

Fy(a+b–)

Fya+b–, Fy(a+b–), Fya(+)b(–), Fya(+)b(–)

Antibody

Anti-Fya

Anti Fya, Anti-Duffy

Antigen

Kell (name of system)

Antibody

Anti-k

Anti-Cellano

Phenotype

K:1, K:–1

K1+, K:1+, K(1), K:(1), K1–, K:1–, K1-negative

Phenotypes

A Rh+, B Rh–

A+ (means positive for A antigen)
B– (means negative for B antigen)

Phenotype

M+N–

M(+), MM (implies unproved genotype)

Phenotype

Rh:–1,–2,–3,4,5

Rh:–1,–2,–3,+4,+5
Rh:1–,2–,3–,4+,5+

Note: The examples shown may not represent the only correct terminologies. In the Rh system, for example,
use of CDE terminology is also acceptable and is more commonly used. The example demonstrates the correct
usage if numeric terminology is used.
*Issitt L. Blood group nomenclature. In: Blood groups: Refresher and updates. Bethesda, MD: AABB, 1995.

Race or
Ethnicity

Number

O Rh+

O Rh–

A Rh+

A Rh–

White non-Hispanic

2,215,623

37.2

8.0

33.0

6.8

259,233

52.6

3.9

28.7

236,050

46.6

3.6

126,780

39.0

19,664
3,086,215

Hispanic

‡

Black non-Hispanic
Asian

North American Indian
All donors

B Rh+

B Rh–

AB Rh+

AB Rh–

9.1

1.8

3.4

0.7

2.4

9.2

0.7

2.3

0.2

24.0

1.9

18.4

1.3

4.0

0.3

0.7

27.3

0.5

25.0

0.4

7.0

0.1

50.0

4.7

31.3

3.8

7.0

0.9

2.2

0.3

39.8

6.9

31.5

5.6

10.6

1.6

3.5

0.6

*Used with permission from Garratty G, Glynn SA, McEntire R, et al for the Retrovirus Epidemiology Donor Study. ABO and Rh(D) phenotype frequencies of different racial/ethnic
groups in the United States. Transfusion 2004;44:703-6.
†
Percentages may not add up to 100.0% because of rounding.
‡
Hispanic includes Mexican (68.8%), Puerto Rican (5.0%), Cuban (1.6%), and other Hispanic donors (24.6%).
§
Asian includes Chinese (29.8%), Filipino (24.1%), Indian (13.8%), Japanese (12.7%), Korean (12.5%), and Vietnamese (7.1%) donors.

Appendices
845

846

AABB Technical Manual

Appendix 10. Suggested Quality Control Performance Intervals
Equipment and Reagents
I.

II.

Frequency

Refrigerators/Freezers/Platelet Incubators
A. Refrigerators
1. Recorder
2. Manual temperature
3. Alarm system board (if applicable)
4. Temperature charts (review daily)
5. Alarm activation
B. Freezers
1. Recorder
2. Manual temperature
3. Alarm system board (if applicable)
4. Temperature charts (review daily)
5. Alarm activation
C. Platelet incubators
1. Recorder
2. Manual temperature
3. Temperature charts (review daily)
4. Alarm activation
D. Ambient platelet storage area
Laboratory Equipment
A. Centrifuges/cell washers
1. Speed
2. Timer
3. Function
4. Tube fill level (serologic)
5. Saline fill volume (serologic)
6. Volume of antihuman globulin dispensed
(if applicable)
7. Temperature check (refrigerated centrifuge)
8. Temperature verification (refrigerated centrifuge)
B. Heating blocks/Waterbaths/View boxes
1. Temperature
2. Quadrant/area checks
C. Component thawing devices
D. pH meters
E. Blood irradiators
1. Calibration
2. Turntable (visual each time of use)
3. Timer
4. Source decay
5. Leak test
6. Dose delivery check (with indicator)
7. Dose delivery verification
a. Cesium-137
b. Cobalt-60
c. Other source

Daily
Daily
Daily
Weekly
Quarterly
Daily
Daily
Daily
Weekly
Quarterly
Daily
Daily
Weekly
Quarterly
Every 4 hours

Quarterly
Quarterly
Yearly
Day of use
Weekly
Monthly
Day of use
Monthly
Day of use
Periodically
Day of use
Day of use
Yearly
Yearly
Monthly/quarterly
Dependent on source type
Twice yearly
Each irradiator use
Yearly
Twice yearly
As specified by manufacturer

Appendices

847

Appendix 10. Suggested Quality Control Performance Intervals (cont’d)
Equipment and Reagents

Frequency

Thermometers (vs NIST-certified or traceable)
1. Liquid-in-glass
2. Electronic
G. Timers/clocks
H. Pipette recalibration
I. Sterile connecting device
1. Weld check
2. Function
J. Blood warmers
1. Effluent temperature
2. Heater temperature
3. Alarm activation
III. Blood Collection Equipment
A. Whole blood equipment
1. Agitators
2. Balances/scales
3. Gram weight (vs NIST-certified)
B. Microhematocrit centrifuge
1. Centrifuge timer check
2. Calibration
C. Cell counters/hemoglobinometers
D. Blood pressure cuffs
E. Apheresis equipment
Checklist requirements
IV. Reagents
A. Red cells
B. Antisera
C. Antiglobulin serum
D. Transfusion-transmissible disease marker testing
V. Miscellaneous
A. Copper sulfate specific gravity
B. Shipping containers for blood transport
(usually at temperature extremes)

Yearly
Monthly
Yearly
Yearly
Each use
Yearly
Quarterly
Quarterly
Quarterly

Day of use
Day of use
Yearly
Quarterly
Yearly
Day of use
Periodically
As specified by the
manufacturer
Day of use
Day of use
Day of use
Each test run
Day of use
Twice yearly

Note: The frequencies listed above are suggested intervals, not requirements. For any new piece of equipment,
installation, operational, and process qualification must be performed. After the equipment has been suitably
qualified for use, ongoing quality control (QC) testing should be performed. Depending upon the operational
and process qualification methodology, the ongoing QC may initially be performed at a greater frequency than
one ultimately wishes to use. Once a track record of appropriate in-range QC results has been established (either during equipment qualification or the ongoing QC), the frequency of testing can be reduced, but, at a minimum, the frequency must comply with the manufacturer’s suggested intervals. If no such guidance is provided
by the manufacturer, the intervals given in this table would be appropriate to use.

848

AABB Technical Manual

Appendix 11. Directory of Organizations
AABB
8101 Glenbrook Road
Bethesda, MD 20814-2749
(301) 907-6977
FAX: (301) 907-6895
www.aabb.org

American Society of Hematology (ASH)
1900 M Street, NW, Suite 200
Washington, DC 20036
(202) 776-0544
FAX: (202) 776-0545
www.hematology.org

American Association of Tissue Banks (AATB)
1320 Old Chain Bridge Road, Suite 450
McLean, VA 22101
(703) 827-9582
FAX: (703) 356-2198
www.aatb.org

America’s Blood Centers (ABC)
725 15th Street, NW
Suite 700, The Folger Building
Washington, DC 20005
(202) 393-5725
FAX: (202) 393-1282
www.americasblood.org

American Medical Association (AMA)
515 N. State Street
Chicago, IL 60610
(800) 621-8335
www.ama-assn.org
American Red Cross National Headquarters (ARC)
2025 E Street, NW
Washington, DC 20006
(202) 303-4498
Disaster Assistance: (866) 438-4636
www.redcross.org
American Society for Apheresis (ASFA)
570 West 7th Avenue, Suite 402
Vancouver, BC, Canada V5Z 1B3
(604) 484-2851
FAX: (604) 874-4378
www.apheresis.org
American Society for Clinical Pathology (ASCP)
2100 West Harrison Street
Chicago, IL 60612-3798
(312) 738-1336
Outside Illinois: (800) 621-4142
FAX: (312) 738-1619
www.ascp.org
American Society for Histocompatibility
and Immunogenetics (ASHI)
15000 Commerce Parkway, Suite C
Mount Laurel, NJ 08054
(856) 638-0428
FAX: (856) 439-0525
www.ashi-hla.org

Armed Services Blood Program Office (ASBPO)
5109 Leesburg Pike, Suite 698
Falls Church, VA 22041-3258
(703) 681-8024
FAX: (703) 681-7541
www.tricare.osd.mil/asbpo
Association of Donor Recruitment Professionals
(ADRP)
P.O. Box 540524
Grand Prairie, TX 75054-0524
www.adrp.org
College of American Pathologists (CAP)
325 Waukegan Road
Northfield, IL 60093-2750
(800) 323-4040
FAX: (847) 832-8000
www.cap.org
Foundation for the Accreditation of Cellular Therapy
(FACT)
University of Nebraska Medical Center
986065 Nebraska Medical Center
Omaha, NE 68198-6065
(402) 559-1950
FAX: (402) 559-1951
www.factwebsite.org
ICCBBA, Inc.
204 St. Charles Way, Unit 179E
York, PA 17402
(717) 845-4790
FAX: (717) 845-9727
www.iccbba.com

American Society of Anesthesiologists (ASA)
520 N. Northwest Highway
Park Ridge, IL 60068-2573
(847) 825-5586
FAX: (847) 825-1692
www.asahq.org

850

AABB Technical Manual

Appendix 12. Resources for Safety Information
Centers for Disease Control and Prevention (CDC)
Office of Health and Safety, Biosafety Branch
Mail Stop F-05
1600 Clifton Road
Atlanta, GA 30333
(404) 639-2453
FAX: (404) 639-2294
www.cdc.gov
Clinical and Laboratory Standards Institute (CLSI)
940 West Valley Road, Suite 1400
Wayne, PA 19087-1898
(610) 688-0100
FAX: (610) 688-0700
www.clsi.org
Department of Transportation (DOT)
Office of Hazardous Materials Standards
Research and Special Programs Administration
DHM-10
400 7th Street, SW
Washington, DC 20590-0001
(202) 366-8553
FAX: (202) 366-3012
www.dot.gov
Environmental Protection Agency (EPA)
Chemical Emergency Preparedness and Prevention
Office (5104A)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
(800) 424-9346
In Washington, DC metropolitan area: (703) 412-9810
www.epa.gov
Environmental Protection Agency (EPA)
Office of Solid Waste (5305W)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
(800) 424-9346
www.epa.gov/osw
Food and Drug Administration (FDA)
Center for Biologics Evaluation and Research
Division of Blood Applications, HFM-370
1401 Rockville Pike
Rockville, MD 20852-1448
(301) 827-3524
FAX: (301) 827-3535
www.fda.gov/cber

International Air Transport Association (IATA)
1776 K Street, NW, Suite 400
Washington, DC 20006
(202) 293-9292
FAX: (202) 293-8448
www.iata.org
International Civil Aviation Organization (ICAO)
999 University Street
Montreal, Quebec
Canada H3C 5H7
(514) 954-8220
FAX: (514) 954-6376
www.icao.int
National Fire Protection Association (NFPA)
1 Batterymarch Park
Quincy, MA 02169-7471
(617) 770-3000
FAX: (617) 770-0700
www.nfpa.org
National Institute for Occupational Safety and
Health (NIOSH)
Education and Information Division
4676 Columbia Parkway
Cincinnati, OH 45226-1998
(800) 356-4674
Outside the US: (513) 533-8328
Clinicians’ Post-Exposure Prophylaxis Hotline:
(888) 448-4911
FAX: (513) 533-8588
www.cdc.gov/niosh
National Institutes of Health (NIH)
Division of Safety
Building 13, Room 3K04
Bethesda, MD 20892
(301) 496-2346
FAX: (301) 402-0313
www.nih.gov
Nuclear Regulatory Commission (NRC)
Office of Public Affairs
Washington, DC 20555
(800) 368-5642
In Washington, DC metropolitan area: (301) 415-8200
FAX: (301) 415-2234
www.nrc.gov

Appendices

849

Appendix 11. Directory of Organizations (cont'd)
International Society for Cellular Therapy (ISCT)
570 West 7th Avenue, Suite 402
Vancouver, BC, Canada V5Z 1B3
(604) 874-4366
FAX: (604) 874-4378
www.celltherapy.org

National Marrow Donor Program (NMDP)
3001 Broadway Street NE, Suite 500
Minneapolis, MN 55413-1753
(800) 627-7692
Outside the US: (612) 627-5800
www.marrow.org

International Society of Blood Transfusion (ISBT)
Central Office
C/O Jan van Goyenkade 11
1075 HP Amsterdam
The Netherlands
+ 31 (0) 20 679 3411
FAX: + 31 (0) 20 673 7306
www.isbt-web.org

Plasma Protein Therapeutics Association (PPTA)
147 Old Solomons Island Road, Suite 100
Annapolis, MD 21401
(410) 263-8296
FAX: (410) 263-2298
www.plasmainfo.org

Joint Commission on Accreditation of Healthcare
Organizations (JCAHO)
One Renaissance Boulevard
Oakbrook Terrace, IL 60181
(630) 792-5000
FAX: (630) 792-5005
www.jcaho.org

United Network for Organ Sharing (UNOS)
700 North 4th Street
P.O. Box 2484
Richmond, VA 23218
(804) 782-4800
FAX: (804) 782-4817
www.unos.org

National Hemophilia Foundation (NHF)
116 West 32nd Street, 11th Floor
New York, NY 10001
(212) 328-3700
FAX: (212) 328-3777
www.hemophilia.org

Note: Contact information changes rapidly. Therefore, the data listed above may not be current for the entire life of this publication.

Appendices

851

Appendix 12. Resources for Safety Information (cont'd)
Occupational Safety and Health Administration (OSHA)
Office of Communications
Room N-3647
200 Constitution Avenue, NW
Washington, DC 20210
(202) 693-1999
Workplace safety and health-related questions:
(800) 321-6742
www.osha.gov

US Postal Service (USPS)
Headquarters, Room 9301
475 L’Enfant Plaza, SW
Washington, DC 20260
(800) 275-8777
www.usps.com

Note: Contact information changes rapidly. Therefore, the data listed above may not be current for the entire life of this publication.

Index

Index
Page numbers in italics refer to tabular or illustrative material.

A
A, B, H substances, 290-291, 298, 301, 445,
736-739
ABO compatibility
of bone grafts, 623-624
of Cryoprecipitated AHF, 411, 500-501
of Granulocytes Pheresis, 411
of HPC transplants, 598-599, 600, 601-602
of kidney transplants, 401
of liver transplants, 402, 628
of plasma products, 411, 496, 498
of platelet components, 361-362, 411,
489-490, 569
of Red Blood Cells, 411, 418, 486
of solid organ transplants, 402, 627, 629-630
of Whole Blood, 411, 486
ABO hemolytic disease of the fetus and newborn, 536, 537, 538, 545
ABO system (ISBT 001), 289-303
acquired B phenotype, 298, 301-302
antibodies, 294-296
age variations, 295, 298, 299
anti-A and anti-B, 294-296
anti-A1, 295-296, 302
anti-A,B, 295, 296
in HDFN, 536, 538, 545

passively acquired, 563
reactivity, 295
antigens, 293
in newborns, 293
nomenclature, 226, 840, 844
on platelets, 361-362, 397
weakly expressed, 297-298, 300-301
B(A) phenotype, 298, 301
discovery of, 289
genetics and biochemistry, 229-230, 235,
290-293, 844
phenotypes, 844, 845
soluble substances, 290-291, 298, 301, 445,
736-739
subgroups, 293-294
in ABO discrepancies, 302
confirmation of by adsorption/elution,
735-736
testing for, 296
ABO testing
of autologous blood, 122
of blood components, 164, 165
in children, 296, 415, 545, 563, 574
with cold autoagglutinins, 299, 302-303, 469
comparison with previous records, 413, 526
confirmation of weak A or B subgroup,
735-736
853

854

AABB Technical Manual

of cord blood, 545, 595
discrepancies, 296-303, 469
absence of expected antibodies, 301
absence of expected antigens, 300-301
causes, 297
mixed-field agglutination, 298, 299
resolving, 300-303
specimen-related problems, 297-299
technical errors, 299-300
unexpected red cell reactions, 301-302
unexpected serum reactions, 302-303
hemolysis in, 295
microplate tests, 733-735
for organ transplantation, 621, 629
of Platelets Pheresis, 142
in prenatal studies, 538
reagents, 296
of recipients, 122, 410, 411
routine, 290, 296
slide tests, 731-732
in transfusion reaction evaluation, 654
tube tests, 732-733
ABTI antigen, 355
Accidents, 45-46
ACE (angiotensin-converting enzyme) inhibition, 636, 645
Acid-elution stain (Kleihauer-Betke), 550-551,
794-796
Acid glycine/EDTA, 446, 469, 747-748
Acidosis, in neonates, 561
Acquired antigens, 298, 301-302
Activated partial thromboplastin time (aPTT)
in massive transfusion, 511
monitoring hemostasis with, 494, 496
normal values, 837
in vitamin K deficiency, 498
Active failures, 26
Acute lung injury (ALI), 647
Acute normovolemic hemodilution (ANH), 117,
126-130
clinical studies, 128, 129, 130
physiologic considerations, 127-128
practical considerations, 128, 130
selection of patients, 131
Acute transfusion reactions. See Transfusion reactions
Adaptive (acquired) immunity, 243-244
ADCC (antibody-dependent cellular
cytotoxicity), 255, 441

Additives
anticoagulants-preservatives for red cells,
176, 186, 564-565
in serologic testing, 276-277, 437-438, 443,
753-754
Adhesion molecules, 246, 269
Adsol (AS-1), 176, 186, 565
Adsorption
allogeneic, 471-472, 781-782
autologous cold, 438, 466, 472, 775-776
autologous warm, 470-471, 779-781
differential, 781-782
and elution, 300-301, 448, 735-736
methods, 768-769, 775-776, 779-784
polyethylene glycol, 783-784
purposes, 446-447
with rabbit red cells, 439, 466
Adsorption separation, 140
Adult T-cell lymphoma/leukemia (ATL), 682
Adverse reactions
to apheresis, 141, 150-153
to DMSO, 607
in donors, 19, 107-109
to G-CSF, 594-595
management, 19, 20, 21
to transfusions
action for, 531
autologous, 123
infectious, 667-703
noninfectious, 633-661
records, 660-661
reporting, 19, 20, 21 , 661, 702-703
AET (2-aminoethylisothiouronium bromide),
342, 446
Affinity constant (Ko), 273
Age
of blood samples, 410, 431, 433
of donor, 98
effect on ABO antigens/antibodies, 293, 295,
298, 299
Agglutination
defined, 271
factors affecting, 272-275
antigen-antibody proportions, 274275
chemical bonding, 273
equilibrium (affinity) constant, 273
incubation time, 274, 444
ionic strength, 274

Index

pH, 274, 444
temperature, 273-274, 336, 443
grading reactions, 412, 728-729
inhibition, 275-276, 444-445
mixed-field, 298, 299, 348, 357
stages, 272-275
tests for granulocyte antibodies, 380
Agreements, 10, 177
AHF. See Cryoprecipitated Antihemophilic Factor
AHG test. See Antiglobulin test
AIDS (acquired immunodeficiency syndrome),
675-676, 677. See also HIV
AIHA. See Autoimmune hemolytic anemia
Air embolism, 636, 651-652
AITP (autoimmune thrombocytopenic
purpura), 158, 373-374, 492, 553-554
Alanine aminotransferase (ALT), 164, 673, 835
Alarm systems, 185, 198-199, 823-826, 846
Albumin
antibodies to ingredients in, 437-438
as colloid replacement, 507-508
physiology, 507
recombinant, 219
as replacement fluid in apheresis, 150, 151
in serologic testing, 276, 427, 753
use in exchange transfusion, 567
Aldehyde dehydrogenase (ALDH), 603
ALI (acute lung injury), 647
Aliquoting components, 193-194, 564, 571
Allele-specific oligonucleotide (ASO) hybridization. See Sequence-specific oligonucleotide
probes
Alleles, 225, 227. See also specific blood groups
blank, 389
dosage effect, 225, 227
frequencies, 227-229
rare, 233, 234
Allelic, defined, 241
Allergic reactions
in apheresis, 152
to latex, 46-47
to transfusions, 522, 634, 644-647
Alloantibodies, red cell. See also Antibody detection; Antibody identification; specific blood
groups
in ABO discrepancies, 299, 303
with autoantibodies, 438-439, 470-472
in bone graft patients, 623

855

clinical significance, 411, 418, 423-424, 439, 441
dosage effect, 225, 227, 327, 345, 425, 431
to high-incidence antigens, 356-357,
435-436, 441-442, 547
in liver transplant patients, 629
to low-incidence antigens, 340, 358, 436-437,
546
multiple, 236, 434-435, 441-442, 449
in selection of units, 418, 439-443
serologic behavior, 336
in sickle cell disease, 576
temperature of reaction, 273-274, 336, 443
in thalassemia patients, 576
titration, 449, 539-540, 761-764, 796-798
Allogeneic adsorption, 471-472, 781-782
Allogeneic HPC transplantation
ABO incompatibilities, 598-599, 600, 601-602
cell processing, 596-597, 598, 601-604
collection of products, 591-596
defined, 582
donor evaluation, 589-591
evaluation and QC of products, 607-608
freezing and storage of products, 604-606
graft-vs-host disease in, 586-587
of HPC-A (apheresis), 587-588, 592, 594-595
of HPC-C (cord blood), 588-589, 595-596
of HPC-M (marrow), 583, 585-587, 591-592
indications for, 582
infectious disease testing, 590-591
matched, unrelated transplantation,
585-586, 589
mobilization of HPCs, 587, 594-595
nonmyeloablative, 582-583
positive DAT after, 455
regulations, 608
related transplantation, 587-588, 589
standards, 609
thawing and infusion of products, 607
transfusion support in, 591-592, 599, 600,
601
transportation and shipping of products,
606-607
Allografts. See Organ donation and transplantation; Tissue transplantation
Alloimmune hemolytic anemia. See Hemolytic
disease of the fetus and newborn; Hemolytic
transfusion reactions
Alloimmunization. See also Alloantibodies, red cell
direct/indirect allorecognition, 265

856

AABB Technical Manual

HLA, 265, 266, 362-366, 397-398, 637
in sickle cell disease, 575, 576
as transfusion complication, 265, 637,
656-657
Allotypic, defined, 269
α-methyldopa, 455, 475-476
ALT (alanine aminotransferase), 164, 673, 835
AMD 3100, 594
American Rare Donor Program (ARDP), 441-442,
769-770
American trypanosomiasis (Chagas’ disease),
697-698, 700
2-aminoethylisothiouronium bromide (AET),
342, 446
Amniocentesis, 539, 540, 541, 549
Amniotic fluid analysis, 540-541, 542
Ana (Gerbich) antigen, 352, 843
Anaphylactic transfusion reactions, 644-647
evaluation, 655
frequency, 645
management, 635
pathophysiology, 635, 644-645
prevention, 646-647
symptoms, 639, 644-645
treatment, 645-646
Anaphylactoid transfusion reactions, 644, 645
Anaphylatoxins, 262, 639
Anemia. See also Hemolytic disease of the fetus
and newborn
alloimmune hemolytic, 459
autoimmune hemolytic, 458-477, 509-510
cold agglutinin syndrome, 459, 460, 464466
DAT-negative AIHA, 459, 468
drug-induced hemolytic, 459, 460, 472-477,
481-482
IgM warm AIHA, 464
mixed-type AIHA, 459, 460, 466-467
in neonates, 558, 559, 563-564
oxygen compensation in, 484
paroxysmal cold hemoglobinuria, 459, 460,
467-468
screening donors for, 102, 799
sickle-cell disease, 119, 155-156, 508-509,
574-576
in thalassemia, 508
treatment, 487-488
warm autoimmune hemolytic anemia, 459,
460, 461-464

Angiotensin-converting enzyme (ACE) inhibition, 636, 645
ANH. See Acute normovolemic hemodilution
Anhydrous, defined, 724
Ankylosing spondylitis, 403
Anti-A and anti-B, 294-296
Anti-A1, 293, 295-296, 302
Anti-A,B, 295, 296
Anti-C3b, -C3d, 279, 280, 456
Anti-CMV, 170
Anti-D
active vs passive antibody, 548
antibody titrations, 539-540
in D+ individuals, 327
discovery of, 315-316
dosage effect, 327
in HDFN, 330, 535, 536-537, 539-540
in liver transplantation, 628-629
partial D, 322-323
reagents, 328-330
and weak D, 323
Anti-Delta, 670
Anti-HAV, 671
Anti-HBc (antibody to hepatitis B core antigen)
blood component testing, 164, 166, 675
look-back for, 675
marker of infection, 669, 670
reentry of donors with, 675
as surrogate marker, 672
testing transplant donors for, 621
Anti-HBe (antibody to hepatitis B e antigen),
669, 670
Anti-HBs (antibody to hepatitis B surface antigen), 669, 670
Anti-HCV (antibody to hepatitis C virus)
blood component testing, 164, 166, 170
marker of infection, 670-671, 672-673
reentry of donors with, 673, 674, 675
supplemental testing, 170
testing organ donors for, 620, 621, 623
Anti-HEV, 671
Anti-HIV-1, 2
reentry of donors with positive tests, 674,
681
supplemental testing, 169-170
testing blood donors for, 164, 166, 169-170,
676, 679-681
testing organ donors for, 620, 621,
623

Index

Anti-HTLV-I, II
blood component testing, 120, 164, 166, 170,
683
donors for organ transplants, 620
supplemental testing, 170
Anti-IgA, 645, 646, 655
Anti-IgG, 279, 280, 427, 438, 456
Anti-K1, 536, 538, 540
Anti-Ku, 343
Anti-Pr, 777, 778
Antibodies. See also Alloantibodies; Antigenantibody reactions; Autoantibodies; other
Antibody entries; specific blood groups
complement-binding, 280
concomitant, 327-328, 441
defined, 269
distinguishing IgG and IgM, 764-765
drug-induced, 374-377, 472-477, 481-482,
786-791
equilibrium constant (Ko), 273
high-titer, low avidity, 449, 762, 765766
HLA
in antibody identifications, 433
and Bg antigens, 358
detection, 365-366, 397
in platelet refractoriness, 362-365,
397-398, 491
in transfusion reactions, 398-400, 637,
647, 656
monoclonal
in ABO testing, 296
anti-D, 328, 329-330
assays utilizing, 284, 373, 380
in autologous tumor purging, 597-598
for reagent use, 266-267
in neonates, 560
platelet
autoantibodies, 373-374
clinical importance, 370-373
detecting, 370-373, 374, 375, 377, 552
drug-induced, 374-377
HLA, 266, 362-366, 397-398
platelet-specific, 366, 367-368, 370-373,
552
production, 249
reagent, 266-267
to reagent components, 298, 437-438
temperature of reaction, 273-274, 443

857

Antibody-dependent cellular cytotoxicity
(ADCC), 255, 441
Antibody detection
antiglobulin test, 277-281, 282, 283
with autoantibodies, 438-439, 463-464,
470-472
in autologous blood, 122
autologous control, 412, 427, 438-439
automated testing, 285
in blood components, 165
in children, 415, 563, 574
column agglutination for, 278, 284-285
in cord blood, 595
ELISA for, 286
enhancement, 276-277, 427, 443-444,
753-754
enzyme-linked immunosorbent assay, 286
frequency of testing, 442-443, 463-464
of granulocyte autoantibodies, 380
of HLA antibodies, 365-366, 397
immunofluorescence, 285-286
indirect antiglobulin test, 277, 278, 752-754
interpreting reactions, 415-416, 417
MAIEA assay, 284, 286
in organ transplantation, 629
of platelet antibodies, 370-373, 374, 375, 377
in Platelets Pheresis, 142
in positive DAT evaluation, 456
practical considerations, 412
in prenatal evaluations, 539
in pretransfusion testing, 411-413, 417
prewarming technique, 308, 438, 754-755
reagents, 278-280, 425, 427, 468
solid-phase red cell adherence tests, 283-284
specimen requirements, 424
in transfusion reaction evaluation, 654
Antibody identification, 427-450
ABO type of red cells tested, 434
adsorption, 446-447, 448, 470-472
anomalous reactions, 437-438
antibodies to high-incidence antigens,
356-357, 435-436, 441-442, 547
antibodies to low-incidence antigens, 340,
358, 436-437, 546
with antibodies to reagent components,
437-438
with autoantibodies, 438-439, 463-464,
470-472
autologous control, 427, 429-430, 438-439

858

AABB Technical Manual

elution, 447-448, 456-458
enhancement techniques, 276-277, 427,
443-444, 753-754
flowchart for, 432
frequency of testing, 442-443, 463-464
inactivation of antigens, 445-446, 766-767
inhibition tests, 444-445
interpreting results, 428-429
medical history in, 424
multiple antibodies, 236, 434-435, 441-442,
449
no discernible specificity, 433-434
phenotyping autologous red cells, 429-430,
439
with positive DAT, 427, 436, 438-439, 456
in prenatal evaluations, 539
prewarming technique, 308, 438, 754-755
probability values, 429, 430
reagents, 278-280, 425, 426, 427, 468
and selection of blood, 418, 439-443, 463
specimen requirements, 424
sulfhydryl reagents in, 446, 448-449, 744-745,
766-767
titration studies, 449, 539-540, 761-764,
796-798
in transfusion reaction evaluation, 654
using immunohematology reference labs,
439
variations in antigen expression, 431, 433,
468
Antibody screens. See Antibody detection
Antibody specificity prediction (ASP) method,
365
Anticoagulant-preservative solutions, 178-179
CPD, CP2D, CPDA-1, 176, 178, 186, 564,
565
red cell changes during storage, 185, 186,
187, 431, 433
shelf life of components, 178, 188, 189-190
Antifibrinolytic agents, 513-514
Antigen-antibody reactions. See also Antibody
detection; Antibody identification
agglutination, 271, 272-276
hemolysis, 271-272
precipitation, 272
prozone, 272, 275
Antigen-presenting cells (APCs), 269
Antigens. See also specific blood groups
acquired, 298, 301-302

blood group nomenclature, 238-239,
318-319, 840-844
CD, 246, 247-248
collections, 335, 355-356
defined, 269
depressed, 341-342, 468
distribution in population, 335, 337
granulocyte/neutrophil, 377-380
high-incidence
antibodies to, 356-357, 435-436, 441-442,
547
of Cromer system, 353, 354
defined, 335
of Gerbich system, 352
GIL, 355
of Knops system, 354
of Lutheran system, 347
not assigned to a system or collection,
356-357
with positive DAT, 436
selecting blood negative for, 441-442
of Vel collection, 355-356
HLA, 362, 385, 388-389, 390-391
inactivation, 342, 445-446, 766-767
low-incidence, 357-358, 436-437, 546
antibodies to, 340, 358, 436-437, 546
of Cromer system, 353, 354
defined, 335
of Gerbich system, 352
of Lutheran system, 347
of MNS system, 227, 228, 337-338, 340
not assigned to a system or collection,
357
of Scianna system, 350
platelet, 361-363, 366, 367-368, 397, 552
public, 392
typing donor units for, 418, 440-441, 657,
745-748
variations in expression of, 431, 433, 468
Antiglobulin (AHG) test. See also Direct
antiglobulin test
in antibody detection/identification,
277-281, 282, 283
complement in, 280-281, 453
in crossmatching, 413, 414
direct, 278, 454-458, 760-761
indirect, 278, 752-754
principles, 277-278
reagents, 278-280, 427, 454, 468

Index

sources of error, 282, 283
use of IgG-coated cells, 281, 412
Antiprotease concentrates, 505-506
Antipyretics, 522
Antithrombin, 500, 505-506
AnWj antigen, 356-357
APCs (antigen-presenting cells), 269
Apheresis
complications, 141, 150-153
for component collection, 140-144
donation intervals, 141, 143
equipment, 139-140, 152, 847
HPCs collected by, 581, 587-588, 592-595
monitoring cell counts, 832
records, 142
separation techniques, 139-140
therapeutic, 144-158
indications for, 146-148, 153-158
photopheresis, 158
plasma volumes exchanged, 145, 149
removal of normal plasma constituents,
149-150
removal of pathologic substances, 145,
148-149
replacement fluids, 150, 151
SPA immunoadsorption, 158
vascular access, 145, 150
Aprotinin, 502, 513-514
aPTT. See Activated partial thromboplastin time
Arachis hypogaea (anti-T), 743
ARDP (American Rare Donor Program), 441-442,
769-770
Arrhythmias, 650
Arterial transplants, 624
Articular cartridge transplants, 624
AS (additive solutions), 176, 186, 564-565
ASP (antibody specificity prediction) method,
365
Assessments, 22-24
blood utilization, 23, 36-38, 514
competency, 8
external, 23-24
internal, 22
proficiency testing, 24
quality indicators, 22-23, 31
transfusion oversight, 514
Ata antigen, 356
ATL (adult T-cell lymphoma/leukemia), 682
Audits, transfusion, 23

859

Auto controls. See Autologous controls
Autoadsorption. See Adsorption
Autoantibodies
cold
ABO discrepancies with, 299, 302-303,
469
adsorption, 438, 466, 472, 775-776
with alloantibodies, 438-439, 472
anti-I/i, 306, 307-308, 438, 465-466
anti-IH, 308, 434, 777, 778
in cold agglutinin syndrome, 281, 307,
465-466
and complement, 281
determining specificity, 776-778
in mixed AIHA, 466, 467
in paroxysmal cold hemoglobinuria,
467
in Rh testing, 329, 469-470
titration, 458, 465, 778-779
use of sulfhydryl reagents with, 469-470,
744-745
in warm autoimmune hemolytic anemia,
461
in DAT-negative AIHA, 468
granulocyte, 380
platelet-specific, 373-374
posttransfusion, 657
warm
adsorption, 470-472, 779-781
with alloantibodies, 439, 470-472
drug-induced, 475-476
frequency of testing, 463-464
mimicking alloantibodies, 472
transfusion-stimulated, 462
transfusion with, 462-464
in warm autoimmune hemolytic anemia,
461-462
Autoclaving biohazardous waste, 57
Autoimmune hemolytic anemia (AIHA)
classification, 458, 459
cold agglutinin syndrome, 459, 460, 464-466
complement coating cells, 281, 460, 465
DAT-negative AIHA, 459, 468
IgM warm AIHA, 464
mixed-type AIHA, 459, 460, 466-467
paroxysmal cold hemoglobinuria, 459, 460,
467-468
serologic findings in, 460
transfusion in, 462-464, 467, 468, 509-510

860

AABB Technical Manual

warm autoimmune hemolytic anemia, 459,
460, 461-464
Autoimmune neutropenia, 379-380
Autoimmune platelet disorders, 373-374
Autoimmune thrombocytopenic purpura
(AITP), 158, 373-374, 492, 553-554
Autologous adsorption
cold, 438, 466, 472, 775-776
warm, 470-471, 779-781
Autologous blood
acute normovolemic hemodilution, 117,
126-130
clinical studies, 128, 129, 130
physiologic considerations, 127-128
practical considerations, 128, 130
selection of patients, 131
bacterial contamination of, 693
categories, 117
for HIV-positive donors, 117, 681
intraoperative blood collection, 117, 130-133
clinical studies, 131-132
controversies in, 132-133
direct reinfusion, 133
equipment for, 132-133
practical considerations, 132
processing, 132-133
requirements and recommendations, 133
inventory management, 94-95
for patients needing rare blood types, 442
postoperative blood collection, 117, 133-135
preoperative blood donation, 117, 118-126
advantages/disadvantages, 118
adverse reactions, 123
aggressive phlebotomy, 125
collection, 121, 126
compliance requirements, 119-120
continuous quality improvement,
123-124
contraindications, 119
cost-effectiveness, 125, 127
donor deferrals, 120
donor screening, 121-122, 124-125
erythropoietin use, 125
establishing program, 120-124
labeling, 120, 122-123
medical interview, 122
in pediatric patients, 118-119
physician responsibility, 120-121
records, 111, 123

shipping, 120
storage, 123
supplemental iron use, 121
testing, 119-120, 122
timing and red cell regeneration during,
122
transfusion of units, 123
transfusion trigger, 125
volume collected, 122
voluntary standards, 119
weak D in donor, 324
separation from transfused cells, 748-750
Autologous controls
in antibody identification, 427, 429-430,
438-439
positive, 417, 438-439
in pretransfusion testing, 412, 417, 454
Autologous HPC transplantation
autologous tumor purging, 597-598
cell processing, 596-604
collection of products, 591-596
defined, 582
donor evaluation, 589, 590-591
evaluation and QC of products, 607-608
freezing and storage of products, 604,
605-606
of HPC-A (apheresis), 587, 592-595
of HPC-C (cord blood), 589, 595-596
of HPC-M (marrow), 583, 591-592
infectious disease testing, 590-591
mobilization of HPCs, 592-594
regulations, 608
standards, 609
thawing and infusion of products, 607
transportation and shipping, 606-607
Autologous tumor purging, 597-598
Automated testing platforms, 285
Autosomal dominant and recessive traits, 233,
234

B
5b antigen, 378
B-cell receptor (BCR), 249
B lymphocytes, 249-250, 252-253
receptors/markers on, 249-253
stimulation of, 254-255
β2-microglobulin, 245
B(A) phenotype, 298, 301
Babesiosis, 695-696, 700

Index

Bacterial contamination, 690-695
clinical considerations, 692-693
fatalities associated with, 690-692
of HPCs, 602-603
organisms involved, 691, 692
platelet-associated, 690-692, 694-695
preventive measures for, 693-694
of reagents, 331, 332
of Red Blood Cells, 691
testing components for, 195, 655, 693, 694
in transfusion-associated sepsis, 635, 643,
655, 691-692
transfusion risk of, 700
Bacteriologic studies
of components, 195, 655, 693, 694
of hematopoietic products, 602-603
in transfusion reaction evaluation, 655
Bags, blood, 104-105
centrifugation of, 179-180
for frozen storage, 182
returning to lab, 531-532
Barcode labels, 171
Basophils, 255
BCR (B-cell receptor), 249
Bennett-Goodspeed (Bg) antigens, 358, 391
Bilirubin
in HDFN, 536, 540-541, 542, 547
hyperbilirubinemia, exchange transfusion
for, 566-567
total, normal values, 835, 836
Bioassays, 65
Biohazardous waste
defined, 55-56
disposal, 56-57
treating, 57
Biologic product deviations, 19, 21
Biological products, 717
Biological safety cabinets (BSCs), 50, 51, 52-53
Biosafety, 49-57
biosafety levels and precautions, 49-50,
54-55, 77
Bloodborne Pathogen Standard, 49
decontamination, 51, 54
emergency response plan, 55-57
engineering controls, 50-51
hazard identification and communication,
50
labeling, 719
personal protective equipment, 54

safe work practices, 54-55
shipping dangerous goods, 716-722
Standard Precautions, 49
storage, 54
training, 50
waste management, 55-57
Biphasic hemolysin of paroxysmal cold
hemoglobinuria, 467
Blank alleles, 364
Bleeding
indication for transfusion, 486
microvascular, 511, 651
with platelet defects, 490
transfusion-related, 639
Bleeding time, 488, 489, 837
Blocking phenomenon, 330
Blood administration
assessment, 37-38
blood warmers for, 522, 529, 560, 650, 847
compatibility testing, 524
component preparation in, 522, 523
delays in starting, 525
delivering blood to transfusion area, 524525
electromechanical pumps for, 522, 523,
529-530, 565
emergency release, 522-523
errors, 525-526, 527, 641, 642, 653, 654
filters for, 528-529, 565-566
identification procedures, 525-526
infusion sets for, 527-528, 565-566
IV solutions, 530
in neonates, 565-566
patient care during, 530-531
patient consent, 521-522
patient considerations in, 522
patient education, 521-522
post-administration events, 531-532
prescription for, 522-523
pressure devices for, 530
quality assurance, 532
reaction evaluation, 531, 652-656
special instructions for, 522-523
starting the transfusion, 526-527
venous access, 523-524, 565-566
Blood bags, 104-105
centrifugation of, 179-180
for frozen storage, 182
returning to lab, 531-532

861

862

AABB Technical Manual

Blood collection
adverse donor reactions, 19, 107-109
anticoagulants and preservatives, 178-179,
186
blood containers, 104-105
of blood samples, 105-106, 408-409, 801-804
care of donor, 106
of components by apheresis, 140-144
equipment quality control, 847
fatalities related to, 19
identification in, 98, 105
intraoperative, 117, 130-133
phlebotomy, 105-106, 178, 693, 801-804
postoperative, 117, 133-135
preoperative, 126
preoperative autologous, 118-126
preparing venipuncture site, 105, 800-801
records, 97-98, 165
safety, 41, 51
volume collected, 101, 122, 178-179
of whole blood, 178-179
Blood component selection
ABO/Rh compatibility in, 411, 418, 486,
489-490, 496, 498
after non-group-specific transfusions,
419-420
with alloantibodies, 418, 439-443
for exchange transfusions, 546-547, 567
for intrauterine transfusions, 544
for massive transfusions, 419, 510-511
platelets, 363-365, 489-490, 569
rare types, 441-442
red cell components, 411, 486
in urgent situations, 419, 510-511
in warm autoimmune hemolytic anemia,
463
Blood components. See also Blood collection;
Blood component selection; specific components
aliquoting, 193-194, 564, 571
appearance, 194, 525, 655
bacterial contamination, 195, 643, 655,
690-695
for children, 564-572, 574-577
CMV-negative, 95, 172
disposition, 196
distribution, handling and dispensing, 36-37
for exchange transfusion, 567
expiration and shelf life, 178, 188, 189-190

freezing, 180-183, 807-812
for hemostasis, 496, 497, 498-507, 570-572
identification, 105, 416, 524-526, 527
inspection, 93-94, 194-195, 416, 418, 525
irradiated, 183, 192-193, 493
issuing, 196-197, 416, 418, 524-525
labeling, 15, 170-172, 416, 440
leukocyte reduction, 180, 190, 492-493,
573-574
ordering, 36, 91-94, 522-523, 526
outdating, factors affecting, 90-91
pathogen inactivation, 702
phenotyping, 440-441
pooling, 183-184, 193, 815
preparation, 173, 179-184, 522, 523, 804-818
quality control, 197-199, 202
quarantine, 173-174, 194
records, 165, 172-173, 416, 418
reissuing, 197, 525
rejuvenation, 194, 806-807
returning, 525
storage, 93-94, 184-185, 186, 187
testing
ABO and D, 164, 165
alanine aminotransferase, 164
antibody screening, 165
cytomegalovirus, 170
equipment requirements, 164-165
general requirements, 163-164
hepatitis, 669-673
HIV, 164, 166, 169-170, 679-681
HTLV, 120, 164, 166, 170, 683
records, 165
syphilis, 165-166, 695
viral markers, 166-170
thawing, 191-192, 809, 815
transportation and shipping, 66, 179,
195-196, 717, 722
volume reducing, 193, 490, 569, 817-818
washed, 193
Blood containers, 104-105, 179-180, 182, 564
Blood donation. See Blood collection
Blood exposure, 45, 54-55
Blood Group Antigen Mutation Database, 236
Blood group sugars, 445
Blood group systems. See also specific blood
groups
chromosomal assignments, 225, 226,
235-236

Index

co-dominant traits in, 234-235
defined, 335
distribution of antigens, 335, 337
genetics
basic principles, 223-225
and heredity, 225-232
patterns of inheritance, 232-236
population genetics, 236-237
nomenclature, 238-239, 318-319, 840844
serologic behavior of antibodies, 336
Blood inspections
before release, 194-195, 416, 418, 525
of inventory, 93-94
Blood loss
dilutional coagulopathy in, 499-500, 651
due to platelet dysfunction, 490
in preterm neonates, 559
transfusion in, 486-487
Blood ordering practices
assessment, 36
improving, 91-94
physicians’ orders, 522-523, 526
for routine vs emergency orders, 93, 414
Blood pressure, 633
of donors, 102
hypertension, 633
hypotension
in donors, 107
in recipients, 585, 586, 591, 595
in therapeutic apheresis, 152
treatment, 592
Blood pressure cuffs, 847
Blood samples, 409-410
age of, 410, 431, 433
appearance, 410
frozen, 184-185
hemolyzed, 410
identification and collection, 105-106,
408-409, 801-804
incompletely clotted, 409, 722-723
labeling, 409, 719
lipemic, 410
packaging, 717-718, 720-721
requirements for testing, 424, 454
retention and storage, 410
transportation and shipment, 66, 717-721
Blood spills, 55, 56
Blood transfusions. See Transfusions

863

Blood utilization
assessments, 23, 36-38
blood ordering practices, 36, 91-94, 414
inventory levels, 89-90, 93
maximum surgical blood order schedules,
92-93, 414
outdating factors, 90-91
routine vs emergency orders, 93
of special components, 94-95
units processed, transfused and outdated in
2001, 91
Blood volume, 558-559, 839
Blood warmers, 522, 529, 560, 650, 847
Bloodborne Pathogen Standard, 49
Bombay (Oh) phenotype, 304
Bonds, chemical, 273
Bone banking, 623, 625
infectious disease transmission in, 619
preservation and dating periods for, 624
Bone marrow. See Marrow transplantation
Bovine aprotinin, 502, 513-514
Bovine spongiform encephalopathy (BSE), 690
Burkitt’s lymphoma, 687

C
C/c antigens and antibodies (Rh system), 316,
841
cis product antigens, 324
concomitant antibodies, 327-328, 441
dosage in, 317
expression of, 320
gene complexes, 319
in HDFN, 536
incidence of, 317, 321
phenotypes, 320
testing for, 330-331, 441
C1-esterase inhibitor, 500
Calcium, 151-152, 561, 636, 649-650
Calculations
for CRYO dose, 501
for Factor VIII dose, 503-504
for multiple alloantibodies, 236, 441
Calibration
of cell washers, 830-832
defined, 30
of platelet separation centrifuges, 179,
826-828
of serologic centrifuges, 828-830
of thermometers, 198, 821-823

864

AABB Technical Manual

CAP (College of American Pathologists), 514
Capture-P, 372
Cardiovascular system. See Heart and heart valves
Carriers, defined, 233
Cartwright (Yt) system (ISBT 011), 226, 336, 348,
349, 842
CAS. See Cold agglutinin syndrome
Catheters
for apheresis, 145
for HPC collection, 593, 594
for neonates, 565, 568
for transfusions, 523-524
Cause-and-effect diagrams, 26
CBER (FDA Center for Biologics Evaluation and
Research), 19, 702-703
CCE (counterflow centrifugal elutriation),
596-597
CCI (corrected platelet count increment), 362,
363, 489
CD (cluster of differentiation) markers, 246,
247-248, 250
CD34
analysis/enumeration, 603
collection targets, 593-594
defined, 592
selection of, 596-597
Cefotetan antibodies, 477
Cell adhesion molecules, 246, 269
Cell counters, 847
Cell counts
in apheresis components, 832
in HPC products, 607-608
Cell selection techniques, 596-598
Cell therapy. See Hematopoietic progenitor cell
transplantation
Cell washers, 830-832, 846
Center for Biologics Evaluation and Research
(CBER), 19, 702-703
Central venous catheters, 524, 593
Centrifugation
apheresis separation by, 139-140
in component preparation, 179-180
counterflow elutriation, 596-597
in separating autologous cells, 748-749
standardization of variables, 716
Centrifuges
calibrating, 179, 826-830
continuous-flow, 596-597
QC performance intervals, 846, 847

Centromere, 224, 241
Cephalosporins, 476
detection of antibodies to, 786-788
in positive DAT, 455, 474, 476, 477
Cephalothin (Keflin), 476
CFR (Code of Federal Regulations), 1, 32
CGD (chronic granulomatous disease), 343
Ch (Chido/Rodgers) antigens, 351-352, 842
Chagas’ disease, 697-698, 700
Change control, defined, 30
Charts, run and control, 23
Check cells, 281, 412
Chemical bonds, 273
Chemical Hygiene Plan (CHP), 57-58
Chemical safety, 57-63
chemical categories, 82-83
chemical data sheet, 78-79
chemical hygiene officer, 58
chemical spills, 61-62, 84-88
chemicals found in blood banks, 80-81
engineering controls, 61
general principles, 57-58
hazard communication, 59
hazardous vapors, 62, 63
health hazards categories, 58, 59
labeling and signs, 59-60
material data safety sheets, 59, 60-61, 78-79
personal protective equipment, 61
safe work practices, 61, 82-83
spills, 61-62, 84-88
training, 58-59
waste disposal, 63
Chemiluminescence assay, 380, 441
Chemokines, 594
Chido/Rodgers system (ISBT 017), 351-352
antibodies, 352
antigens, 351-352, 842
genes, 226
plasma inhibition studies, 445, 765-766
Children. See also Hemolytic disease of the fetus
and newborn
ABO antigens/antibodies in, 293, 295, 298,
420
ABO discrepancies in, 298
anemia in, 558, 559, 563-564
autologous collection from, 118-119
blood volume of, 558-559, 839
compatibility testing in, 415, 562-563, 574
cytomegalovirus in, 562

Index

DIC in, 567, 572
erythropoiesis in, 557-558, 559
extracorporeal membrane oxygenation in,
572-573, 574
graft-vs-host disease in, 560-561
hematopoietic transplantation in, 587, 593
hypothermia in, 560
immunologic status, 560-561
leukocyte reduction for, 573-574, 576
Lewis antigens in, 306
metabolic problems in, 561-562
normal laboratory values in, 836-837
paroxysmal cold hemoglobinuria in, 468
plasma volume of, 839
polycythemia in, 572
red cell volume of, 839
size of, 558-559
transfusions in
administration, 565-566
aliquoting for small volumes, 193-194,
564, 571
Cryoprecipitated AHF, 572
education and consent, 521
to enhance hemostasis, 570-572
exchange, 546-547, 560, 566-568
FFP, 571, 576-577
Granulocytes, 569-570
with hemoglobinopathies, 574-576
indications for, 563-564
older infants and children, 574-577
Platelets, 490, 552-553, 568-569, 576-577
Red Blood Cells, 564-568
with sickle cell disease, 574-576
with thalassemia, 575-576
volumes for, 558, 564, 568
variations in antigen expression, 431
Chills with transfusions, 633, 634, 643
Chimerism
blood group, 237, 299
hematopoietic, 298, 601
and posttransfusion GVHD, 399
transfusion, 298, 399
Chloroquine diphosphate, 446, 469, 746-747
Chorionic villus sampling, 539
CHP (chemical hygiene plan), 57-58
Christmas disease, 505
Chromatid, 241
Chromatin, 223, 241
Chromosomes. See also Genes

865

defined, 223-224, 241
DNA structure in, 203-204
locations of blood group genes, 225, 226,
235-236
Chronic granulomatous disease (CGD), 343
Chronic inflammatory demyelinating
polyneuropathy (CIDP), 156
Circulatory overload, 636, 648-649
Circulatory shock, 633
Cis product antigens, 319-320, 324, 342
Citrate toxicity, 151, 636, 649-650
CJD (Creutzfeldt-Jakob disease), 689-690
Class I, II, III antigens (HLA), 244-246, 386-394
biochemistry and structure, 390-391
biologic function, 393-394
defined, 269, 385
genetics, 386-387
nomenclature, 392-393
Clinical specimens
defined, 717
labeling, 718, 719
shipping, 717-718, 720-722
Clocks, 847
Clone, defined, 269
Cluster of differentiation (CD) markers, 246,
247-248
CMV. See Cytomegalovirus
Co (Colton) antigens and antibodies, 336, 349,
351
Co-dominant traits, 233, 234-235, 241
Coagulation
in hemolytic transfusion reactions, 640, 642
in liver transplants, 629
in massive transfusion, 511, 651
monitoring hemostasis, 494, 496
normal test values, 837, 838
physiologic principles, 493-494
role of platelets, 488-489
Coagulation factors
changes in red cell storage, 187-188
concentrates, 496, 497
in Cryoprecipitated AHF, 500-502
Factor VIIa concentrates, 497
Factor VIII concentrates, 497, 503-504
Factor IX concentrates, 497, 505
minimum levels needed for hemostasis, 495
in neonates, 571
normal values, 837
in plasma, 496, 498-500

866

AABB Technical Manual

in platelet concentrates, 838
replacement, 496, 497, 498-508
support during massive transfusion, 511
virus inactivation in concentrates, 701
for vitamin K deficiency, 497, 498-499
for warfarin reversal, 497, 498-499
Coagulopathy
dilutional, 499-500, 567, 651
in hemolytic transfusion reactions, 640,
642
in liver disease, 499
in liver transplantation, 629
in massive transfusions, 511, 651
in neonates, 571
treatment with heparin, 642
in vitamin K deficiency, 498-499
in warfarin reversal, 498-499
Code of Federal Regulations (CFR), 1, 32
Codons, 206-207
Cold-acid elutions, 457, 772
Cold agglutinin syndrome (CAS), 464-466
adsorption procedures, 466
classification, 459
complement in, 281
pretransfusion testing in, 466
serologic findings in, 460, 465
specificity of autoantibodies, 307, 465-466
Cold agglutinin titer, 458, 465, 778-779
Cold autoadsorption
in cold agglutinin syndrome, 308, 466
for detection of alloantibodies, 438, 472
method, 775-776
in resolving ABO discrepancies, 303
Cold autoantibodies
ABO discrepancies with, 299, 302-303, 469
adsorption, 438, 466, 472, 775-776
with alloantibodies, 438-439, 472
anti-I/i, 306, 307-308, 438, 465-466
anti-IH, 308, 434, 777, 778
in cold agglutinin syndrome, 281, 307,
465-466
and complement, 281
determining specificity, 776-778
in mixed AIHA, 466, 467
in paroxysmal cold hemoglobinuria, 467
in Rh testing, 329, 469-470
titration, 458, 465, 778-779
use of sulfhydryl reagents with, 469-470,
744-745

in warm autoimmune hemolytic anemia,
461
Cold hemagglutinin disease (CHD). See Cold agglutinin syndrome
Cold reactive alloantibodies, 273-274, 299, 303
Cold stress. See Hypothermia
Collection of blood. See Blood collection
Collections, antigen, 335, 355-356
College of American Pathologists (CAP), 514
Colloid solutions, 486, 507-508, 572
Colony-forming cell assays, 603-604
Colony-stimulating factors, 264
Colton system (ISBT 015), 226, 336, 349, 351, 842
Column agglutination technology, 278, 284-285
Compatibility testing. See Pretransfusion testing
Competency assessments, 8
Complement, 259-262
in acute transfusion reactions, 639-640
alternative pathway, 260, 261
in antiglobulin testing, 280-281, 453
in autoimmune hemolytic anemia, 281, 460,
465
C1-esterase inhibitor, 500
classical pathway, 259-261
complement receptors, 262, 354
membrane attack complex, 261-262, 263
physiologic effects of activation, 262
and positive DAT, 453
regulation of activation, 262, 263
Complications. See Adverse reactions
Computer crossmatch, 413, 414-415
Computer systems
alternative systems, 18-19
backups, 19
for record storage, 17-19
security, 18
validation, 13-14, 415
Confidential unit exclusion, 101
Confidentiality of records, 18
Consent
for apheresis, 140, 142
for blood donation, 103
for transfusion, 521-522
for transplantation, 620-621
Containers
for biological specimens, 717
for blood collection, 104-105, 179-180
damaged or leaking, 720
freezer storage bags, 182

Index

quality control, 847
for shipments, 195, 607, 717-718
Contamination. See Bacterial contamination
Continuous-flow centrifuges, 596-597
Continuous Quality Improvement, 123-124
Contracts, 10
Control charts, 23, 30
Controls
autologous, 412, 427, 438-439, 454
for high-protein reagents, 328-329
IgG-coated cells, 281, 412
for indirect antiglobulin tests, 754
for low-protein reagents, 330
in viral marker testing, 167-169
Convulsions in donors, 108
Copper sulfate
quality control, 819-821, 847
in screening donors, 102, 799-800
Cord blood
antigen expression on, 431, 433
HPC-C transplantation, 588-589
autologous, 589
collection, 588, 595-596
colony-forming cell assays, 603-604
cord blood banks, 589
diseases treated with, 583, 584
engraftment in, 588-589
evaluation and quality control, 607608
freezing and storage, 604, 605-606
infectious disease testing, 591
microbial cultures, 602-603
processing, 602
regulations, 608
related donors, 589
standards, 609
stem cell enumeration, 603
thawing and infusion, 607
transportation and shipping, 606-607
serologic testing, 544-546, 595
Cordocentesis, 539, 541-542, 552
Corneal transplants, 619, 621, 624
Corrected platelet count increment (CCI), 362,
363, 489
Corrective action, 24-25
Corticosteroids, 144
Cost blood group collection, 352, 355
Counterflow centrifugal elutriation (CCE),
596-597

867

CP2D (citrate-phosphate-dextrose-dextrose),
176, 178
CPD (citrate-phosphate-dextrose), 176, 178, 186
CPDA-1 (citrate-phosphate-dextrose-adenine),
176, 178, 564-565
CR (complement receptors), 262, 354
Cra (Cromer) antigen/antibody, 353, 354, 843
CREG (cross-reactive groups), 392
Creutzfeldt-Jakob disease (CJD), 689-690
Cromer system (ISBT 021), 226, 353, 354, 843
Cross-reactive groups (CREGs), 392
Crossmatch-to-transfusion (C:T) ratios, 92, 93
Crossmatching
antiglobulin test in, 413, 414
for children, 415, 546, 563
computer, 413, 414-415
HLA, 397, 398, 401, 402, 492
immediate-spin, 413, 414, 751-752
“in-vivo,” 509
interpretation of results, 415-416, 417
platelets, 364, 365, 398
in pretransfusion testing, 412, 413-415, 417
in transfusion reaction evaluation, 654, 655
Crossover, gene, 208, 209, 230-231, 232, 241, 389
CRYO. See Cryoprecipitated Antihemophilic Factor
Cryoglobulinemia, 156
Cryoprecipitate Reduced Plasma, 177, 190, 496
Cryoprecipitated Antihemophilic Factor (CRYO)
ABO compatibility, 411, 500-501
calculating dose, 501
for coagulation factor replacement, 496
description, 177
for disseminated intravascular coagulation, 502
expiration dates, 189
for fibrinogen abnormalities, 501-502
for hemophilia A, 502-503
indications for use, 499, 500-503
inspection, 194
inventory management, 94
for neonates, 558, 572
pooled, 183-184, 191, 193, 815
preparation, 814-815
quality control, 199, 202
room temperature storage, 185
thawing, 191, 815
topical use, 502
transportation and shipping, 196
for von Willebrand syndromes, 501

868

AABB Technical Manual

Cryopreservation
agents, 181, 604
computer-controlled, 605
effects of, 181
of hematopoietic progenitor cells, 604-606
passive controlled-rate, 605-606
of platelets, 183
of red cells, 181-183, 807-812
storage bags, 182
Crystalloids, 150, 151, 486, 572
Cs (Cost) antigens and antibodies, 352, 355
C:T (crossmatch-to-transfusion) ratios, 92, 93
Cultures, bacterial, 195, 602-603, 655, 693, 694
Customer relations, 9-10
CXCR4, 594
Cytokines
defined, 269
in immune response, 251, 254, 262-263, 264
in transfusion reactions, 640, 643
Cytomegalovirus (CMV), 686-687
associated with hepatitis, 667, 668
blood component testing for, 170
clinical manifestations, 686
and leukocyte reduction, 687
in neonates, 562
prevention, 687
transfusion-transmitted, 686-687
in transplantation, 590, 591, 621, 629, 630
Cytomegalovirus (CMV)-negative products, 95
granulocytes, 492, 570
for intrauterine transfusion, 544, 553
labeling, 172

D
D antigen. See also Rh system; Rh testing
clinical significance, 316
discovery of, 315-316
expression of, 319-320
in HDFN, 330, 536-537
ISBT classification, 841
partial D, 322-323, 538
testing, 165, 328-330, 414
weak D, 322-324
in autologous donations, 324
in donors, 165, 323
“microscopic,” 550
partial D, 322-323
as position effect, 302
in pregnancy, 538-539

quantitative, 322
in recipients, 323-324, 411
Dangerous goods, 716-722
DAT. See Direct antiglobulin test
DAT-negative autoimmune hemolytic anemia,
459, 468
DDAVP (desmopressin), 513
as fibrinolytic inhibitor, 514
in hemophilia A, 503, 513
indications for, 513
in liver disease, 499
in von Willebrand syndrome, 501, 513
Decay-accelerating factor (DAF), 353
Decontamination of biohazardous waste, 51, 54,
57
Deferrals, donor
of autologous donors, 120
for babesiosis, 696
for Creutzfeldt-Jakob disease, 689, 690
for drugs taken by donor, 100-101, 113
for hepatitis, 674, 703
for HIV, 674, 680, 681, 703
for HTLV, 683, 684, 685, 703
implicated in posttransfusion infections, 703
for Lyme disease, 696-697
for malaria, 697
records of, 98, 173
Deglycerolized Red Blood Cells
checking adequacy of deglycerolization,
812-813
expiration date, 189
preparation, 191-192, 809
refreezing, 183
of sickle cell trait, 192
storage, 192
Delayed hemolytic transfusion reactions
(DHTR), 346, 575, 637, 656-657
Delayed serologic transfusion reaction (DSTR),
656
Delayed transfusion reactions. See also Transfusion reactions
alloimmunization, 637, 656-657
graft-vs-host disease, 399, 560, 637, 657-659
hemolytic, 346, 575, 637, 656-657
immunomodulation, 638, 660
iron overload, 638, 660
Kidd antibodies in, 346, 656
posttransfusion purpura, 366, 370, 638, 659-660
Density gradient separation, 601

Index

Derivatives. See also specific derivatives
for coagulation factor replacement, 496
virus inactivation, 496, 699, 701
Desensitization therapy, 505
Design output, defined, 4, 30
Designated donations, 98, 103-104
Desmopressin. See DDAVP
Deviations, 19, 20, 21
Dextrose solution, 530
Dha (Gerbich) antigen, 352, 843
DHTR (delayed hemolytic transfusion reactions), 346, 575, 637, 656-657
Di (Diego) antigens and antibodies, 336, 348, 349
Diagnostic specimens
defined, 717
labeling, 718, 719
shipping, 717-718, 719, 720-722
DIC. See Disseminated intravascular coagulation
Dichloromethane/methylene chloride elutions,
457, 775
Diego system (ISBT 010), 348
antigens/antibodies, 336, 348, 842
chromosomal location of genes, 226
and En(a–), 338
in HDFN, 348
phenotype frequencies, 349
in transfusion reactions, 348
Differential centrifugation, 179-180
Differential warm adsorption, 781-782
Digitonin acid elutions, 457
Dilution
of % solutions, 726-727
of serum, 725-726
Dilutional coagulopathy, 499-500, 567, 651
Dimethylsulfoxide (DMSO), 604, 607
2,3-Diphosphoglycerate. See 2,3-DPG
Direct antiglobulin test (DAT)
in classifying AIHA, 458
complement in, 280-281
method, 760-761
positive test
in antibody identification, 427, 436,
438-439
causes, 453-454
in cold agglutinin syndrome, 460, 465
drug-induced, 455, 472-477, 481-482
elution with, 456-458, 745-746
evaluation, 454-458, 480

869

in HDFN, 545-546
in IgM warm AIHA, 464
medical history in, 454-456
in mixed-type AIHA, 460, 466
in paroxysmal cold hemoglobinuria, 460,
467
red cell testing with, 745-747
serologic studies, 456
in warm autoimmune hemolytic anemia,
459, 460, 461
in pretransfusion testing, 412, 454
principles, 278
reagents, 278-280 , 427, 454, 468
specimens, 454
in transfusion reaction evaluation, 653-654
in transplantation, 455, 629
vs autologous control, 427
Directed donations, 94-95, 537-538
Disaster planning, 67-68
Disinfectants, 51, 54
Disposal
of biohazardous waste, 56-57
of blood components, 196
of chemical waste, 63
of radioactive waste, 66
Disposition of blood components, 196
Disseminated intravascular coagulation (DIC)
in neonates, 567, 572
in transfusion reactions, 640, 642
treatment for, 502, 567
Dithiothreitol. See DTT
Diversion, 693
DMSO (dimethylsulfoxide), 604, 607
DNA (deoxyribonucleic acid)
libraries, 217
molecular techniques
cloning, 217, 222
isolation of nucleic acids, 209-211
microarrays, 218
polymerase chain reaction technique,
211, 212, 213-214, 222, 394-396
profiling (typing or fingerprinting), 216, 222
recombinant proteins, 218-219, 222
restriction endonucleases, 214, 215
restriction fragment length polymorphism analysis, 214, 215, 222
sequencing, 217-218, 222, 396
structure, 203-204
transcription, 204, 205

870

AABB Technical Manual

Do (Dombrock) antigens and antibodies, 336,
349, 350-351, 842
Documents, 14-17. See also Records
management, 14-15, 17
quality system, 14, 16
types, 15
Dolichos biflorus lectin, 293, 296, 302, 743-744
Dombrock system (ISBT 014)
antibodies, 336, 350-351
antigens, 350, 842
genes, 226
phenotype frequencies, 349
Dominant traits, 232-233, 234, 241
Donath-Landsteiner test, 458, 467, 784-785
Donation intervals for apheresis, 141, 143
Donation process. See Blood collection
Donor area, 55, 97
Donor deferrals
of autologous donors, 120
for babesiosis, 696
for Creutzfeldt-Jakob disease, 689, 690
for drugs taken by donor, 100-101, 113
for hepatitis, 674, 703
for HIV, 674, 680, 681, 703
for HTLV, 683, 684, 685, 703
implicated in posttransfusion infections, 703
for Lyme disease, 696-697
for malaria, 697
for positive infectious disease tests, 680, 683,
684, 685
records, 98, 173
Donor history questionnaire, 110-112, 114-118
Donors
adverse reactions in, 19, 107-109
autologous, 103, 121-122, 124-125, 131, 135,
693
care of, after phlebotomy, 106
confidential unit exclusion, 101
consent, 103, 620-621
designated donations, 98, 103-104
directed donations, 104, 537-538
drugs taken by, 100-101, 113, 115
eligibility, 99-100
family members as, 442
hematocrit, 102
for hematopoietic transplantation, 589-591
hemoglobin levels, 102, 799-800
for HLA-matched platelets, 398
hypotension in, 107

implicated in posttransfusion infection, 703
information provided to, 98-99, 114
medical history, 100-101, 110-112, 693
notification of abnormal tests, 99, 103, 703
for organ and tissue transplantation, 618,
620-623
physical examination of, 100, 101-103, 620
for plateletpheresis, 141
records, 165, 173
red cell density, 102
reentry with positive screening tests, 673,
674, 675, 681-682
registration, 97-98
report of illness after donation, 103
testing
ABO, 165, 290, 296, 413
alanine aminotransferase, 164
antibody detection, 165
for blood group antigens, 418, 440-441,
745-748
cytomegalovirus, 170
equipment requirements, 164-165
general requirements, 163-164
hepatitis, 120, 166-170, 669-673
HIV, 120, 164, 166-170, 679-681
HTLV, 120, 683
repeat, 413
Rh, 165, 328-332, 413
syphilis, 120, 165-166, 695
viral markers, 166-170
weak D, 165, 323, 328
weight, 101, 143
Doppler flow studies, 542
Dosage effect
in antibody identification, 425, 431
in Duffy system, 345
genetics, 225, 227
in Rh system, 327
Dosimeters, 64-65
Dot blot, 213, 395
2,3-DPG
in neonatal transfusions, 561-562
in red cell storage lesion, 185, 187
in tissue oxygenation, 484, 511-512
Dra (Cromer) antigen, 353, 354, 843
Drug-induced immune hemolytic anemia,
472-477
autoantibodies, 474, 475-476
classifications, 473-476

Index

drug adsorption mechanism, 473, 474, 475
drug-dependent antibodies, 473, 474-475
drugs associated with, 476, 481-482
laboratory investigation, 460, 476-477,
786-791
metabolites, 477
nonimmunologic protein adsorption, 476
theories of, 472-473
Drugs
administered for leukapheresis, 143-144
alteration of pharmacodynamics in
apheresis, 150-151
for autologous tumor purging, 597
as cause of thrombocytopenia, 374-377
for febrile transfusion reactions, 644
in positive DAT, 455, 472-477, 481-482
to prevent allergic transfusion reactions, 646
taken by donors, 100-101, 113, 115
Dry ice, 196, 719, 720-721
Dry shippers, 607
DSTR (delayed serologic transfusion reaction),
656
DTT (dithiothreitol)
applications for, 448-449
differentiating IgG from IgM, 764-765
to disperse autoagglutination, 302, 469, 470,
744-745
inactivating blood group antigens, 342, 445,
446, 766-767
Duffy system (ISBT 008), 343-345
antibodies, 336, 345, 844
antigens, 343-344, 841
biochemistry, 344-345
chromosomal locations of genes, 226
effect of enzymes on, 276, 336, 345
in HDFN, 345
and malaria, 337, 344
phenotypes and frequencies, 344, 844
in transfusion reactions, 345
Dura mater transplants, 619, 624
Dysfibrinogenemias, 501-502

E
E/e antigens and antibodies
cis product antigens, 324
concomitant antibodies, 327-328, 441
dosage in, 317
expression of, 320
gene complexes, 319

871

in HDFN, 536
incidence of, 317, 321
phenotypes, 320
testing for, 330-331, 441
E/e antigens and antibodies (Rh system), 316,
841
EACA (epsilon aminocaproic acid), 513-514
EBV (Epstein-Barr virus), 667, 668, 687-689
ECMO (extracorporeal membrane oxygenation),
572-573, 574
Education
donor, 98-99, 114, 117-118
patient, 521-522
EIA. See Enzyme-linked immunosorbent assay
ELBW (extremely low birthweight) neonates,
557, 568. See also Neonates
Elderly people, 295, 299
Electrical safety, 48-49
Electromechanical infusion devices, 522,
529-530, 565
ELISA. See Enzyme-linked immunosorbent assay
Elutions
with adsorption, 300-301, 448, 735-736
in antibody identification, 447-448
in cold agglutinin syndrome, 460, 465
in IgM warm AIHA, 464
methods, 457, 771-775
cold-acid, 457, 772
dichloromethane/methylene chloride,
457, 775
digitonin-acid, 457
gentle-heat, 745-746
glycine-HCl/EDTA, 772-773
heat, 457, 773-774
Lui freeze thaw, 457, 774
in mixed-type AIHA, 460, 467
in paroxysmal cold hemoglobinuria, 460, 467
in positive DAT evaluation, 456-458
technical factors in, 447-448
uses, 448
in warm antibody AIHA, 460, 461
Embolism, air, 636, 651-652
Emergency procedures
blood orders, 93
identification of patients, 408
issue of blood, 419, 510-511, 522-523
in pretransfusion testing, 419
Emergency release, 510-511, 522-523

872

AABB Technical Manual

Emergency response plans, 44
for biohazardous waste, 55-57
for chemicals, 61-62, 63
for electrical emergency, 49
for fires, 48
for radiation safety, 66
Emergency showers, 61
Emergency supplies for donor area, 108
Employees. See Personnel
En(a–) red cells, 338
Engineering controls
for biosafety, 50-51, 52-53, 54
for chemical safety, 61
for electrical safety, 48
for fire prevention, 47
general guidelines, 43-44, 73-76
for radiation safety, 65-66
Engraftment data, 608
Enhancement of antibodies
albumin additives, 276, 427, 753
alteration of pH, 274, 444
in antibody detection/identification,
276-277, 427, 443-444, 753-754
combined adsorption/elution, 300-301, 448,
735-736
effect of incubation times, 274, 444
enzymes, 276, 443, 445 , 446, 756-760
increased serum-to-cell ratio, 444
LISS and LISS additives, 274, 275, 277, 427,
443, 753, 754
polyethylene glycol, 276-277, 427, 443, 753
temperature reduction, 443
Environmental chambers, 185, 197-198, 846
Enzyme-linked immunosorbent assay
(EIA/ELISA)
in anti-HTLV testing, 683
in antibody detection, 286
in antigen-antibody detection, 286
for Chagas’ disease testing, 698
for heparin-dependent antibodies, 377
in hepatitis testing, 672-673
in HIV testing, 679
in viral marker testing, 166
Enzymes, proteolytic
in adsorption, 470, 471, 781-782
in antibody detection/identification, 276,
443, 445, 446
effect on blood group antigens, 276, 336,
339-340, 345

evaluating treated red cells, 758-759
ficin preparation, 756
one-stage technique, 759
papain preparation, 756-757
in resolving ABO discrepancies, 300, 301
standardization of procedures, 757-758
two-stage technique, 759-760
Eosinophils, 255
Epistasis, 236
Epitope, defined, 269
EPO. See Erythropoietin
Epsilon aminocaproic acid (EACA), 513-514
Epstein-Barr virus (EBV), 667, 668, 687-688
Equilibrium constant (Ko), 273
Equipment. See also specific equipment
for apheresis, 139-140, 152
calibration, 821-823, 826-830
for component testing, 164-165
critical, 10
management, 10-11
personal protective, 43-44, 73-75
quality control
automatic cell washers, 830-832
centrifuge calibration, 826-830
continuous temperature monitoring systems, 197-198
freezer alarms, 198-199, 824-826, 846
performance intervals, 846-847
refrigerator alarms, 198-199, 823-824, 846
thermometers, 198, 821-823, 847
for transfusions, 523-524, 527-530
validation, 11, 13
Er blood group collection, 355
Ergonomics, 41-42
Errors
in ABO typing, 299-300
in antiglobulin testing, 282, 283
identification, 525-526, 527, 641, 642, 653,
654
Erythroblastosis fetalis. See Hemolytic disease of
the fetus and newborn
Erythropoiesis, 557-558, 559
Erythropoietin (EPO)
in anemia treatment, 219, 488
in autologous donations, 125, 219
in newborns and infants, 558, 559
recombinant, 125, 219, 488, 512, 559
as transfusion alternative, 512
Esa (Cromer) antigen, 353, 354, 843

Index

Ethnic groups, differences in
in ABO system, 845
in Cromer system, 353
in distribution of antigens, 335, 337
in Duffy system, 337, 344, 345
in HLA system, 585
in Kell system, 341, 343
in Kidd system, 337
in Lutheran system, 347
in Rh system, 316, 317, 320, 321, 325
Etiologic agents, 719
Exchange transfusions
with antibody against high-incidence antigen, 547
blood selection for, 546-547, 567
blood warming in, 560
in DIC, 567
in HDFN, 546-547
for hyperbilirubinemia, 566-567
methods used, 568
“partial,” 563
for removal of toxins, 567
in sickle cell disease, 574
vascular access, 568
volume and hematocrit, 568
Executive management, 6
Exons, 206
Expiration dates
checking before issue, 525, 526
of components, 188, 189-190
of tissues and organs, 624
Exposure control plan, 49, 50
External assessments, 23-24
External controls in viral marker testing, 167-169
Extracorporeal membrane oxygenation (ECMO),
572-573, 574
Extracorporeal photochemotherapy, 158
Eye washes, 50, 75

F
Face shields, 74
Facilities
design and workflow, 40
ergonomics, 41-42
housekeeping, 40-41
mobile sites, 41
organizational management, 6-7
restricted areas, 41
safety, 27, 39-42

873

FACT standards, 609
Factor V, 188
Factor VIIa, 497, 505
Factor VIII
calculating dose, 503-504
concentrates, 497, 503-504
deficiency, 503
for hemophilia A, 502-504
inhibitors to, 504-505
in stored blood, 188
virus inactivation, 710
for von Willebrand syndromes, 501
Factor IX, 497, 498, 504, 505
Failures, active and latent, 26-27
Fainting in donors, 107
False-positive/false-negative results
in antiglobulin testing, 282, 283
in Rh testing, 329, 330, 331-332
Fascia lata transplants, 624
Fatalities
during apheresis, 153
due to bacterial contamination, 690-692
due to transfusions, 641, 645, 661, 691,
702-703
employees, 46
reporting, 19, 46, 661, 702-703
FDA. See Food and Drug Administration
Febrile nonhemolytic transfusion reactions
(FNHTR), 643-644
granulocyte antibodies in, 379
HLA antibodies in, 398-399
management, 634
manifestations, 643-644
pathophysiology, 634, 643-644
preventing, 576, 644
treatment, 644
Fetal hemoglobin (hemoglobin F), 550, 558, 835
Fetomaternal hemorrhage (FMH)
as immunizing event, 536
Kleihauer-Betke acid-elution test, 550-551,
794-796
microscopic weak D test, 550
postpartum evaluation, 548, 549-551
rosette test, 550, 793-794
Fetus. See also Hemolytic disease of the fetus
and newborn
erythropoiesis in, 557-558, 559
hematocrit, 541
Rh typing, 539

874

AABB Technical Manual

Fever, transfusion-associated, 398-399, 633, 634,
643
FFP. See Fresh Frozen Plasma
Fibrin degradation products, 837
Fibrin sealant, 502
Fibrinogen
abnormalities, 501-502
CRYO for replacement of, 500, 501
in monitoring coagulation, 494, 511
normal values, 837
Fibrinolytic inhibitors, 513-514
Ficin, preparation of, 756. See also Enzymes,
proteolytic
Filgrastim. See Granulocyte colony-stimulating
factor
Filters
leukocyte-reduction, 180, 190, 528-529
microaggregate, 528, 565-566
standard in-line, 527-528
Fire safety, 47-48
Fish-bone diagrams, 26
Fisher/Race Rh terminology, 319
Flow cytometry
in CD34 analysis/enumeration, 603
in granuloctye autoantibody testing, 380
in HLA crossmatch, 397, 401
in platelet antibody detection, 372, 375, 376
to predict clinical significance of antibodies,
441
Flow PRA, 366
Fluids, replacement, 150, 151
Fluosol, 513
FMH. See Fetomaternal hemorrhage
FNHTR. See Febrile nonhemolytic transfusion
reactions
Food and Drug Administration (FDA)
cGMP training, 8
quality assurance regulations, 1, 2
regulations for autologous blood, 119-120
regulations for tissue transplantation, 626
reporting adverse events to, 19, 20, 21, 661,
702-703
Forensic testing, 402
Forms, 15, 17. See also Documents; Records
Foundation for the Accreditation of Cell Therapy
(FACT), 609
Freeze-thaw elutions, 457, 774
Freezers, 185
alarm systems for, 185, 198-199, 824-826

quality control, 197-199, 846
temperature monitoring systems for,
197-198
thermometers for, 198
Freezing
blood samples, 184-185
cryoprotective agents, 181, 604
freeze-thaw damage, 181
Fresh Frozen Plasma, 180-181, 814
hematopoietic progenitor cells, 604-606
Platelets, 183
Red Blood Cells, 181-183, 807-812
refreezing deglycerolized cells, 183
skin grafts, 624, 625
storage bags for, 182
Fresh Frozen Plasma (FFP)
ABO compatibility, 411, 496, 498
coagulation factors in, 496
collection by apheresis, 141, 142
description, 177
expiration dates, 189-190
indications for, 496, 498-500
inspection, 194
inventory management, 94
misuse, 500
for neonates, 558, 571
in older infants and children, 576
preparation, 180-181, 813-814
small-volume, 571
thawing, 191
transfusion thresholds, 494, 496
transportation and shipping, 196
virus inactivation, 701-702
Frozen components. See specific components
Fume hoods, 61
Fy (Duffy) antigens and antibodies, 336,
343-344, 345, 841

G
G antigen (Rh system), 324-325, 841
G-CSF. See Granulocyte colony-stimulating factor
GBV-C virus, 668
Ge (Gerbich) antigens and antibodies, 352-353,
843
GEIS antigen, 352, 843
Gel test, 285
Gene chips, 218
Gene therapy, 217, 219-220

Index

Genes
blood group terminology, 844
chromosomal assignment, 225, 226, 235-236
defined, 241
DNA structure, 203-204
frequencies, 227-229, 320-321
gene conversion, 209, 210
major histocompatibility complex, 244-246,
386-389
modifier, 235-236
nucleotide insertions and deletions, 208
nucleotide substitutions in, 207-208, 228
obligatory, 237
single crossover, 208, 209, 230-231, 232, 241, 389
suppressor, 235-236
variability, 208-209, 210
Genetics of blood groups
of ABO system, 229-230, 235, 290-293
alleles, 225, 227-229
basic principles, 223-225
definitions of terms, 241
of H system, 290-293
and heredity, 225-232
of HLA system, 386-389
Lewis system, 305
of MNS system, 338
nomenclature, 238-239, 318-319, 840-844
patterns of inheritance, 232-236
polymorphism in, 207-208
population genetics, 236-237
of Rh system, 316-318
Genotypes. See also specific blood groups
defined, 233
frequencies, 227-228
nomenclature for, 238-239
Gentle heat elutions, 745-746
Gerbich system (ISBT 020), 226, 352-353, 843
GIL system (ISBT 029), 226, 355, 843
Glanzmann’s thrombasthenia Type I, 366, 369
Gleevec (imatinib mesylate), 220
Globoside (GLOB) blood group, 226, 308, 843
Gloves, 46, 55, 73-74, 106
Glycerolization of red cells, 181-182, 808-809,
810-812
Glycine-HCl/EDTA
to disperse autoagglutination, 469
to dissociate IgG from red cells, 469, 747-748
elutions, 772-773
to inactivate red cell antigens, 446

875

Glycine max (anti-T, -Tn), 743
Glycophorins, 338, 339
GM-CSF (granulocyte-macrophage colony-stimulating factor), 264, 512, 592, 594
Goggles, safety, 74
Gov antigens, 370
Grading test results, 412, 728-729
Graft failure, 598
Graft-vs-host disease (GVHD)
acute/chronic, 586
and graft-vs-leukemia effect, 586-587
in hematopoietic transplantation, 585-588
HLA system in, 399, 400
in neonates, 560-561
transfusion-associated, 399, 400, 560-561,
637, 657-659
Graft-vs-leukemia (GVL) effect, 586-587
Gram-equivalent weight, 723
Gram-molecular weight, 723
Gram’s stain, 693
Granulocyte colony-stimulating factor (G-CSF)
and false-positive test results, 590
for HPC mobilization, 587, 592-595
in leukapheresis, 144
in neonatal sepsis, 570
production and function, 264
side effects of, 594-595
uses for, 218, 222, 512
Granulocyte-macrophage colony-stimulating
factor (GM-CSF), 264, 512, 592, 594
Granulocytes
alloantigens, 377-380, 647
antibodies to
in neonatal alloimmune neutropenia, 379
testing for, 380
in TRALI, 379-380, 647, 656
in immune system, 255-256
Granulocytes Pheresis
ABO compatibility, 411
collection, 143-144
description, 177-178
expiration dates, 189
indications/contraindications for use, 492,
570
irradiation, 144, 492
laboratory testing, 144
for neonates, 558, 569-570
quality control, 202
storage, 144

876

AABB Technical Manual

transfusion, 143, 492, 569-570
transportation and shipping, 196
validation, 35
Group O serum (anti-A,B), 295
Growth factors, recombinant
for HPC mobilization, 587, 592-595
in leukapheresis, 144
as transfusion alternative, 512, 559
uses for, 219, 222
GTI PAKAUTO, 374
Guillain-Barré syndrome, 156
GUTI (Cromer) antigen/antibody, 353, 354, 843
GVHD. See Graft-vs-host disease
GVL (graft-vs-leukemia) effect, 586-587
Gya (Dombrock) antigen, 350, 842

H
H system (ISBT 018), 303-304
anti-H, 304, 434
anti-IH, 308, 434, 777, 778
biochemistry and genetics, 290-293
chromosomal locations of genes, 226
H antigen, 290, 303-304, 361, 842
Oh phenotype (Bombay), 304
para-Bombay phenotypes, 304
saliva testing for substances, 301, 736-739
HAM (HTLV-associated myelopathy), 682
Hand-washing, 50, 75
Haplotypes
ancestral, 389
defined, 231
of HLA system, 387-388, 389, 400, 402
of Rh system, 318-319
Haptoglobin, 835
Hardy-Weinberg law, 227-229
HAV (hepatitis A virus), 667-668, 671, 700
Hazard Communication Standard, 59-60
Hazard identification and communication, 43
for biosafety, 50
for chemical safety, 59-61
for electrical safety, 48
for fire prevention, 47
Hazardous areas of facilities, 41
Hazardous materials
biohazards, 49-57
chemicals, 57-63
radioactive, 63-66
safety plan for, 42-47

transportation and shipping, 66, 716-722
waste management, 55-57, 67
HBeAg (hepatitis B e antigen), 669, 670
HBsAg. See Hepatitis B surface antigen
HBV. See Hepatitis B virus
HCV. See Hepatitis C virus
HDFN. See Hemolytic disease of the fetus and
newborn
HDV (hepatitis D virus), 668, 670
Heart and heart valves
arrhythmias in recipients, 650
cardiac arrest in donors, 108
cardiac output, 485-486
disease of, 119, 487-488
infectious disease transmission, 619
preservation, 624
transplantation, 402, 625, 629
Heat elutions, 457, 745-746, 773-774
Heating blocks, 846
Hemapheresis practitioner (HP), 139
Hematocrit
in autologous blood donors, 125, 126
in blood donors, 102
of blood in exchange transfusions, 568
fetal, 541
in neonates, 572
normal values, 835
of red cell components, 805
transfusion trigger, 487
Hematoma, in donors, 108
Hematopoietic growth factors
for HPC mobilization, 587, 592-595
for leukapheresis, 144
as transfusion alternative, 512, 559
uses for, 218-219, 222
Hematopoietic progenitor cell (HPC) transplantation, 581-609
ABO incompatibility, 598-599, 600, 601-602
ABO typing discrepancies in, 299
allogeneic, 582, 583, 585-588, 589-590,
594-595
autologous, 582, 583, 587, 589, 592-594, 597
autologous tumor purging, 597-598
cell processing, 596-604
chimerism in, 298, 601
collection of products, 144, 591-596
colony-forming cell assays, 603-604
diseases treated with, 583, 584
donor eligibility, 589-591

Index

evaluation and QC of products, 607-608
freezing and storage of cells, 604-606, 624
and graft-vs-host disease, 585, 586-587
HLA testing, 400-401, 585-587
of HPC-A (apheresis), 581, 587-588, 592-595
of HPC-C (cord blood), 588-589, 595-596,
602
of HPC-M (marrow), 583, 585-587, 591-592
indications for, 582
infectious disease testing, 590-591
matched, unrelated donors, 585-586, 589
microbial cultures, 602-603
mobilization of HPCs, 587, 592-595
nonmyeloablative, 582-583
positive DAT after, 455
processing, 596-598, 601-604
red cell depletion, 601
regulations, 608
related donors, 587-588, 589
selection of CD34+ cells, 596-597
sources of cells for, 583, 584-589
standards, 609
stem cell enumeration, 603
suitability criteria, 606
syngeneic, 582, 587
T-cell depletion, 586, 598
thawing and infusion of products, 607
transfusion support, 591-592, 599, 600, 601
transportation and shipping, 606-607
types, 581-582
xenogeneic, 582, 583
Hemoglobin
in autologous donors, 125
in blood donors, 102
bovine, 513
fetal (F), 550, 558, 835
in neonates, 558, 563, 572, 836
normal values, 835, 836
and oxygen, 185, 187, 483-485
recombinant, 219, 513
S, 544, 567, 574, 575
in transfusion reaction evaluation, 654-655
as transfusion trigger, 483, 486-488
Hemoglobin solutions, 512-513
Hemoglobinometers, 847
Hemoglobinopathies, 508-509, 574-576. See also
specific hemoglobinopathies
Hemolysis
in ABO testing, 295

877

in antibody detection, 412
defined, 271-272
immune-mediated
ABO/Rh typing problems with, 469-470
alloimmune hemolytic anemia, 459
antibodies associated with, 336
antibody detection with, 470-472
classification of anemias, 458, 459
cold agglutinin syndrome, 459, 460,
464-466
DAT-negative AIHA, 459, 468
defined, 458-459
drug-induced, 459, 460, 472-477, 481-482
in hemolytic transfusion reactions,
639-642
IgM warm AIHA, 464
intravascular/extravascular, 265-266, 267,
458
mixed-type AIHA, 459, 460, 466-467
paroxysmal cold hemoglobinuria, 459,
460, 467-468
warm antibody autoimmune hemolytic
anemia, 459, 460, 461-464
non-immune mediated, 152, 636, 642-643
passenger lymphocyte, 628, 629-630, 657
in patient samples, 410
and positive DAT evaluation, 455
in transfusion reaction evaluation, 653, 655
Hemolytic anemias. See also Hemolytic disease
of the fetus and newborn
alloimmune, 459
classification of, 458, 459
cold agglutinin syndrome, 459, 460, 464-466
DAT-negative AIHA, 459, 468
drug-induced, 459, 460, 472-477, 481-482
IgM warm AIHA, 426, 427, 428, 429-431
mixed-type AIHA, 459, 460, 466-467
paroxysmal cold hemoglobinuria, 459, 460,
467-468
serologic findings in, 460
warm autoimmune hemolytic anemia, 459,
460, 461-464
Hemolytic disease of the fetus and newborn
(HDFN), 535-551
ABO, 536, 537, 538, 545
ABO discrepancies in, 302
antibodies associated with, 336, 536-537,
545-546
elutions in, 456

878

AABB Technical Manual

exchange transfusion in, 546-547
intrauterine transfusion in, 541, 543-544
maternal immunization in, 536-538, 542-543
measuring severity of
amniotic fluid analysis, 540-541, 542
Doppler flow studies, 542
maternal antibody titer, 449, 539-540,
761-764, 796-798
percutaneous umbilical blood sampling,
541-542
pathophysiology, 535-536
postpartum evaluation, 544-546
prenatal evaluation, 538-540
Rh testing in, 330, 538-539, 545
use of Rh Immune Globulin, 547-551
Hemolytic transfusion reactions (HTR)
antibodies associated with, 336
coagulation activation in, 640, 642
complement activation in, 639-640
cytokines in, 640
delayed, 346, 575, 637, 656-657
due to ABO incompatibility, 639, 640, 641
elutions in, 456
frequency, 641
HLA antibodies in, 399-400
management, 634
non-immune, 642-643
pathophysiology, 639-641
prevention, 642
renal failure in, 640-641
shock in, 640
signs and symptoms of, 633, 639-641
treatment, 641-642
Hemolytic-uremic syndrome (HUS), 154-155
Hemophilia A, 502-505
calculating Factor VIII dose, 503-504
factor replacement for, 504
inheritance of, 233-234
inhibitors of Factor VIII, 504-505
Hemophilia B, 505
Hemorrhage, intraventricular, 569
Hemorrhagic disease of the newborn, 571
Hemostasis
in acute normovolemic hemodilution, 128
coagulation factors needed for, 495
components for, 496, 497, 498-508, 570-572
during massive transfusion, 511, 651
monitoring, 494, 496
in neonates, 570-571

normal test values, 837
physiologic principles, 488-489 , 493-494
Heparin, 375-377, 505, 642, 723
Heparin co-factor, 505-506
Heparin-induced thrombocytopenia (HIT),
375-377, 492
Hepatitis, 667-675
associated with CMV or EBV, 668
chronic carriers, 668-669
clinical manifestations, 668-669
donors implicated in, 703
markers, 669, 670-671, 672-673, 674
non A, non B (NANB), 673, 699
prophylaxis, 45, 668, 672, 703
quarantine and recipient tracing, 675
reentry of donors with, 673, 674, 675
reporting cases, 702
risk of, 673, 675, 700
surrogate markers, 673
vaccination for, 668
viruses, 667-668
Hepatitis A virus (HAV), 667-668, 671, 700
Hepatitis B core antigen, antibody to (anti-HBc)
blood component testing, 164, 166, 675
look-back for, 675
marker of infection, 669, 670
reentry of donors with, 675
as surrogate marker, 672
testing transplant donors for, 621
Hepatitis B e antigen (HBeAg), 669, 670
Hepatitis B immune globulin (HBIG), 45, 668,
703
Hepatitis B surface antigen (HBsAg)
antibody to (anti-HBs), 669, 670
blood component testing, 164, 166, 169
chronic carriers, 668-669
look-back for, 675
marker of infection, 669, 670, 672
reentry of donors with positive tests, 674, 675
testing organ and tissue donors for, 620, 621,
623
Hepatitis B virus (HBV)
chronic carriers, 668-669
clinical manifestations, 667, 668-669
employee exposure to, 45
HBV DNA, 669, 672
look-back for, 675
markers of infection, 120, 669, 670, 672, 674
NAT testing, 669, 672

Index

prophylaxis for, 45, 668, 672, 703
reentry of donors with, 674, 675
testing transplantation donors for, 590
transfusion risk of, 672, 679, 700
Hepatitis C virus, antibody to (anti-HCV)
blood component testing, 164, 166, 170
marker of infection, 670-671, 672-673
reentry of donors with, 673, 674, 675
supplemental tests, 170
testing organ donors for, 620, 621, 623
Hepatitis C virus (HCV)
autologous blood testing, 120
blood component testing, 164, 166-167, 170
chronic carriers, 669
clinical manifestations, 667, 668, 669
employee exposure to, 45
false-positive test results, 590
infections due to IGIV, 699
look-back for, 675
markers of infection, 669, 670-671, 672-673,
674
NAT testing, 164, 166, 170, 213, 590, 673, 675
reentry of donors with, 673, 674, 675
testing transplantation donors for, 590, 620,
621, 623
transfusion risk of, 673, 675, 699, 700, 701
Hepatitis D virus (HDV), 668, 670
Hepatitis E virus (HEV), 667-668, 671
Hepatitis G virus (HGV), 668
Hereditary angioneurotic edema, 500
Hereditary hemochromatosis (HH), 387
Heredity, genetics of
alleles, 225, 227-229
Hardy-Weinberg Law, 227-229
independent assortment, 229-230, 231
linkage, 230-231, 232
linkage disequilibrium, 231-232, 337, 389
segregation, 229, 230
Herpesviruses, 686-688. See also
Cytomegalovirus
HES (hydroxyethyl starch), 143-144
Heterozygous, defined, 225
HEV (hepatitis E virus), 667-668, 671
HGV (hepatitis G virus), 668
HH (hereditary hemochromatosis), 387
HHV-6/HHV-8 (human herpesviruses), 688
High-incidence antigens
antibodies to, 356-357, 435-436, 441-442,
547

879

of Cromer system, 353, 354
defined, 335
of Gerbich system, 352
GIL, 355
of Knops system, 354
of Lutheran system, 347
not assigned to a system or collection,
356-357
selecting blood negative for, 441-442
of Vel collection, 355-356
High-titer, low-avidity antibodies, 449, 762,
765-766
HIT (heparin-induced thrombocytopenia),
375-377, 492
HIV (human immunodeficiency virus), 675-682
in AIDS, 675-676
in autologous donors, 117, 681
clinical manifestations, 99, 676-677
confirmatory tests for, 169-170, 679-681
donors implicated in, 703
employee exposure to, 45
false-positive test results, 590
HIV-1, 675, 676, 678
HIV-2, and HIV-1, group O, 675, 677-679
information provided to donors about, 99
nucleic acid testing, 164, 166-167, 213, 590,
678-681
recipient tracing (look-back), 681-682
reentry of donors with positive screens, 674,
681
reporting cases, 702
risk factors for, 677
testing components for, 164, 166-167,
169-170, 213, 679-681
testing transplantation donors for, 590, 620,
621, 623
transfusion considerations, 678-679
transfusion risks of, 679, 700, 701
Hives, 634, 644-647
HLA-B27, 403
HLA Matchmaker, 365
HLA system, 385-404
alloimmunization to platelets, 265, 266,
362-366, 397-398, 637
antibody detection, 365-366, 397
antigens, 362, 385, 388-389, 390-391
and Bg antigens, 358
biochemistry, 390-391
biologic function, 393-394

880

AABB Technical Manual

and chimerism, 399
crossmatching, 397, 398, 401, 402, 492
disease associations, 402-404
in forensic testing, 402
genetics, 386-389
in graft-vs-host disease, 399, 400
major histocompatibility complex, 244-246,
386-389
matching
granulocytes, 492
in HPC transplantation, 388, 400-401,
585-586
in organ transplantation, 387-388,
401-402
of platelets, 363-365, 398
nomenclature, 391-393
in parentage testing, 402
in platelet refractoriness, 362-365, 397-398,
491
structure of molecules, 244-246, 390-391
tissue distribution, 390-391
and transfusion, 397-400
in transfusion reactions, 398-400, 637, 643,
656
in transfusion-related acute lung injury, 399,
647
and transplantation, 388, 400-402
typing, 394-397, 595, 621
HMIWI (hospital/medical/infectious waste incinerators), 57
HNA antigens, 378
Homozygous, defined, 225
Homozygous type II familial hypercholesterolemia, 140, 157
Housekeeping, 40-41
HPA antigens, 366, 367-368, 369-370, 552
HPCs. See Hematopoietic progenitor cell transplantation
HTLA (high-titer, low-avidity antibodies), 449,
762, 765-766
HTLV-associated myelopathy (HAM), 682
HTLV (human T-cell lymphotropic virus),
682-683
autologous blood testing, 120
blood component testing, 164, 166, 170, 674,
683
clinical manifestations, 682
donors implicated in, 703
quarantine and look-back, 683

testing transplantation donors for, 590, 620
transfusion risk, 700
transmission, 682
Type I, II, 682
HTR. See Hemolytic transfusion reactions
Human albumin (5% and 25%), 507
Human Genome Project, 217
Human granulocytic erlichiosis, 696
Human herpesviruses, 688
Human immunodeficiency virus. See HIV
Human monocytic erlichiosis, 696
Human resources, 7-9
Human T-cell lymphotropic virus. See HTLV
Human thrombin, 502
Hy (Dombrock) antigen, 350, 842
Hydatid cyst fluid, 310, 445
Hydroxyethyl starch (HES), 143-144
Hydroxyurea, 575
Hyper-IgM immune deficiency, 255
Hyperbilirubinemia, 536, 566-567
Hypercholesterolemia, familial, 140, 157
Hyperhemolytic syndrome, 575
Hyperkalemia, 650
Hyperleukocytosis, 154
Hypertension, 633
Hypertransfusion programs, 575-576
Hyperventilation, in donors, 107
Hyperviscosity, serum, 153-154
Hypoalbuminemia, 507
Hypocalcemia, 561, 636, 649-650
Hypofibrinogenemia, 500, 501
Hypokalemia, 650
Hypotension
associated with ACE inhibition, 636, 645
in donors, 107
in recipients, 633, 640, 641, 645
in therapeutic apheresis, 152
treatment, 646
Hypothermia, 511, 560, 637, 650
Hypovolemia, 141, 152

I
I/i antigens and antibodies, 306-308
in ABO discrepancies, 302
chromosomal locations of genes, 226
in cold agglutinin syndrome, 307, 465-466
complex reactivity, 308
ISBT nomenclature, 843

Index

in pregnancy, 539
serologic behavior, 307-308, 777, 778
IAT. See Indirect antiglobulin test
ID-NAT (individual donor NAT), 669, 678, 680, 685
Identification
of blood samples, 409
of components, 105, 416, 524-526, 527
of donors, 98, 105
of equipment, 11
errors in, 525-526, 527, 641, 642, 653, 654
of persons issuing blood, 416, 525
of phlebotomists, 409
of recipients, 407, 408-409, 418, 524-526, 527
traceability of components, 173
Idiopathic thrombocytopenic purpura (ITP),
158, 373-374, 492, 553-554
Idiotypes, 256, 269
IFC (Cromer) antigen, 353, 354, 843
IgA, 259
antibodies to, 645, 646, 655
normal values, 835, 837
polymers, 258, 259
properties, 253
secretory, 258, 259
IgD, 253, 259, 835
IgE, 259
in allergic transfusion reactions, 644-645
normal values, 835
properties, 253
IgG, 258
anti-A and -B, 295
in autoimmune hemolytic anemias,
458-459, 460
complement activation by, 260
dissociation of, by chloroquine, 469, 746-747
distinguishing from IgM, 764-765
in neonates, 560
normal values, 835, 837
in positive DAT, 280, 453, 461
properties, 253
subclasses, 258, 458-459
temperature of reaction, 274
IgG-coated cells (check cells), 281, 412
IGIV. See Immunoglobulin, Intravenous
IgM, 258
anti-A and -B, 295
in antiglobulin testing, 281
cold-reactive autoagglutinins, 302-303, 465,
466, 467, 469, 776-778

881

complement activation by, 260
dispersing autoagglutination, 302, 469-470,
744-745
distinguishing from IgG, 764-765
normal values, 835, 837
polymers, 257, 258
properties, 253
temperature of reactions, 274
IgM warm AIHA, 464
IgSF (immunoglobulin superfamily), 244, 245,
246
IL (interleukins), 264, 512, 640
Imatinib mesylate (Gleevec), 220
Immediate-spin crossmatch, 413, 414, 751-752
Immune complexes
in antigen-antibody reactions, 280, 281
demonstration of, 788-789
mechanism of drug-induced antibodies,
473, 474-475
Immune-mediated hemolysis. See also
Hemolytic disease of the fetus and newborn
ABO/Rh typing problems with, 469-470
alloimmune hemolytic anemia, 459
classification of anemias, 458, 459
cold agglutinin syndrome, 459, 460, 464-466
DAT-negative AIHA, 459, 468
defined, 458-459
drug-induced, 459, 460, 472-477, 481-482
in hemolytic transfusion reactions, 639-642
intravascular/extravascular, 265-266, 267,
458
mixed-type AIHA, 459, 460, 466-467
paroxysmal cold hemoglobinuria, 459, 460,
467-468
warm autoimmune hemolytic anemia, 459,
460, 461-464
Immune serum globulin (ISG), 668
Immune system
cell adhesion molecules in, 246
cells of, 249-250, 251, 252-256
cluster of differentiation molecules in, 246,
247-248
complement in, 259-262, 263
cytokines in, 262-263, 264
defined, 269
and drug-dependent antibodies, 472-473
in HLA alloimmunization to platelets, 265,
266
immune response, 243-246, 247-248

882

AABB Technical Manual

immunoglobulin superfamily in, 244, 245
immunoglobulins in, 253, 256-259
innate and adaptive immunity, 243-244
lymphocytes in, 249-250, 251, 252-255
major histocompatibility complex in,
244-246
of neonates, 560-561
organs of, 249
phagocytic cells in, 255-256
in production of reagent antibodies, 266-267
receptors/markers, 250
in red cell alloimmunization, 265
in red cell destruction, 265-266, 267
signal transduction in, 246
soluble components, 256-263
Immune thrombocytopenic purpura (ITP), 158,
373-374, 492, 553-554
Immune tolerance, 237
Immunity. See Immune system
Immunization, maternal
antibody titers, 449, 539-540, 761-764,
796-798
mechanisms, 536-538
prenatal evaluation, 538-542
suppression, 542-543
Immunocompromised patients, 298-299
Immunofluorescence tests
in antigen-antibody detection, 285-286
in confirming HIV tests, 169
for granulocyte antibodies, 380
for platelet antibody detection, 372
Immunogen, defined, 269
Immunoglobulin, Intravenous (IGIV), 505
ABO discrepancies with, 299
for idiopathic thrombocytopenic purpura, 554
indications for, 505, 506
for neonatal immune thrombocytopenia,
552-553
positive DAT with, 456
for posttransfusion purpura, 659-660
to suppress maternal alloimmunization, 543
virus inactivation in, 699
Immunoglobulin products
hepatitis B immune globulin (HBIG), 45,
668, 703
immune serum globulin (ISG), 668
immunoglobulin, intravenous (IGIV), 505,
506, 543
virus inactivation in, 699, 701

Immunoglobulin superfamily (IgSF), 244, 245,
246
Immunoglobulins, 256-259. See also specific
immunoglobulins
classes, 258-259
Fab and Fc fragments, 256-257
functions, 256
interchain bonds, 256
J chains, 258
normal values, 835, 837
polymers, 257-258
properties, 253
secretory component, 258
structure, 256, 257
Immunohematology reference laboratories, 439
Immunomagnetic cell separation, 596, 598
Immunomodulation, 638, 660
In (Indian) antigens and antibodies, 349, 354,
843
“In-vivo” crossmatch, 441, 509
Incinerators, hospital/medical/infectious waste
(HMIWI), 57
Incompletely clotted specimens, 409, 722-723
Incubation
temperatures, 443, 715
times, 274, 444
Independent assortment, 229-230, 231
Indian system (ISBT 023), 226, 349, 354, 843
Indirect antiglobulin test (IAT)
false-positive/false-negative results, 282, 283
methods, 752-754
principles, 278
reagents, 278-280, 425, 427, 468
role of complement, 280-281
use of additives, 276-277, 443, 753-754
use of IgG-coated cells, 281, 412
Infants. See Children; Neonates
Infectious disease testing
of autologous blood, 119-120
of blood components, 166-170
external controls in, 167-169
of granulocytes, 144
for hepatitis, 669, 670-671, 672-673, 674
for HIV, 164, 166-167, 169-170, 213, 679-681
of HPC donors, 590-591
for HTLV, 120, 164, 166, 170, 683
invalidation of results, 167-169
neutralization, 169
notification of abnormal tests, 703

Index

in organ and tissue transplantation, 621-623
of Platelets Pheresis, 142
records of, 173
supplemental tests, 169-170
surrogate markers, 673
for syphilis, 120, 165-166, 695
Infectious diseases. See also Transfusion-transmitted diseases
as apheresis complication, 152
in blood donors, 99
safety precautions for, 49-57
transfusion-transmitted, 667-699, 700,
701-703
transmitted by allografts, 617, 619-620
Infectious mononucleosis, 687
Infectious substances
defined, 716-717
labeling, 718, 719
shipping, 717-718, 720-722
waste, 55-57
Inflammation, 262
Informed consent
for apheresis, 140, 142
of blood donors, 103
for transfusion, 521-522
for transplantation, 620-621
Infusions. See also Transfusions
of hematopoietic components, 607
infusion pumps, 522, 523, 529-530, 565
infusion sets, 527-529, 565-566
rates for, 531, 566
Inheritance patterns
autosomal dominant, 233, 234
autosomal recessive, 233, 234
blood group co-dominant, 234-235
chromosomal assignment, 226, 235-236
dominant and recessive, 232-233
of major histocompatibility complex,
387-389
sex-linked dominant or co-dominant, 233,
234
sex-linked recessive, 233-234
Inhibition tests
agglutination, 275-276
in antibody identification, 444-445
for Chido/Rodgers, 445, 765-766
for secretor status, 737-739
Inhibitors
of complement activation, 262, 263

883

to Factor VIII, 504-505
fibrinolytic, 513-514
Injuries, 45-46, 51
Innate immunity, 243-244
Inspections
of components before release, 194-195, 416,
418, 525
external assessments, 23-24
of incoming supplies, 10
of inventory, 93-94
Integrins, 246, 369
Interferon γ (IFNγ ), 264
Interleukins, 264, 512, 640
Internal assessments, 22
Internal controls, 167
Internal event report, 19, 20
International Organization for Standardization
(ISO), 1-2, 3
Intervening sequences, 206
Intraoperative blood collection, 117, 130-133
clinical studies, 131-132
controversies in, 132-133
direct reinfusion, 133
equipment for, 132-133
practical considerations, 132
processing, 132-133
requirements and recommendations, 133
Intrauterine transfusions, 541, 543-544
Intraventricular hemorrhage, 569
Introns, 206
Inventory
counts and inspection, 93-94
determining levels, 89-90
minimum and ideal levels, 89
and outdating, 90-91
routine vs emergency orders, 93
of special products, 94-95
Ionic strength, 274
Iron, supplemental, 121
Iron overload, 638, 660
Irradiated blood components, 192-193
expiration dates, 183, 189, 192
Granulocytes, 144, 492
HPC products, 592
indications for, 183, 193, 493, 658-659
for intrauterine transfusion, 544
inventory management, 95
labeling, 172
platelet products, 144, 189, 192-193, 492, 553

884

AABB Technical Manual

potassium leak in, 561
to prevent TA-GVHD, 183, 192, 493, 658-659
quality control, 202
Red Blood Cells, 189, 192, 193, 493
to reduce platelet alloimmunization, 365, 398
ultraviolet B, 365, 398
washing, 561
Irradiators, blood, 65-66, 192, 846
ISBT (International Society of Blood Transfusion)
128 barcode labeling, 171
nomenclature
for platelet antigens, 366, 367-368
for red cell antigens, 226, 238-239,
840-843
ISG (immune serum globulin), 668
Ishikawa diagrams, 26
ISO 9001 standards, 1-2, 3
Isotype switching, 252
Issuing blood
delivering blood to patient area, 524-525
identification of recipient and component,
418, 524-526
policies and procedures of, 416, 418, 524-525
reissue, 197, 525
from transfusion service, 196-197
in urgent situations, 419, 510-511, 522-523
ITP (idiopathic thrombocytopenic purpura),
158, 373-374, 492, 553-554
IV solutions, 530

J
J chains, 258
Jaundice, 566, 639
JCAHO (Joint Commission on Accreditation of
Healthcare Organizations), 25, 514
Jk (Kidd) antigens and antibodies, 336, 345-346,
841
JMH antigen/antibodies, 355, 843
Joa (Dombrock) antigen, 350, 842
Job descriptions, 7
John Milton Hagen (JMH) system (ISBT 026),
226, 355, 843
Joint Commission on Accreditation of
Healthcare Organizations (JCAHO), 25, 514
Jra antigen, 356
Js (Kell) antigens and antibodies, 336, 341, 343,
841
Juran’s Quality Trilogy, 2, 4

K
K/k (Kell) antigens and antibodies, 336, 340-342,
343, 841
Kaposi’s sarcoma-associated herpesvirus, 688
Kell system (ISBT 006), 340-343
anti-Ku, 343
antibodies, 336, 343, 844
antigens, 340-342, 468, 841, 844
biochemistry, 342
genes, 226, 844
in HDFN, 343, 536, 540
inheritance, 229-230
Kx antigen, 226, 342, 842
McLeod phenotype, 342-343
phenotypes and frequencies, 341, 844
in transfusion reactions, 343
Kernicterus, 536, 566
Kidd system (ISBT 009), 345-346
allele frequencies in, 227-229
antibodies, 336, 346
antigens, 345-346, 841, 844
chromosomal locations of genes, 226
genes, 844
in HDFN, 346
inheritance patterns of, 235
phenotypes and frequencies, 345, 346, 844
in transfusion reactions, 346, 656
Kidney
diseases treated with apheresis, 156-157
failure, 640-641
transplantation, 401-402, 619, 624, 627
Kinetic PCR, 213
Kleihauer-Betke acid-elution test, 550-551,
794-796
Kn (Knops) antigens, 354, 843
Knops system (ISBT 022), 226, 352, 354, 843
Kp (Kell) antigens and antibodies, 336, 341, 343,
841
Kx antigen (ISBT 019), 226, 342, 842

L
Labeling
aliquots, 564
for antigen typing, 440
autologous blood, 120, 122-123
biohazardous materials, 50, 719
blood components, 15, 170-172, 416, 440
blood samples, 409

Index

dry ice, 719
hazardous chemicals, 59-60
ISBT 128 system, 171
reagents, 715
shipments, 718, 719
Laboratory coats, 73
Lactated Ringer’s solution, 530
Lan antigen, 356
Landsteiner-Wiener blood group (ISBT 016),
226, 327, 351, 842
Latent failures, 26-27
Latex allergies, 46-47
Law of mass action, 273
LCR (ligase chain reaction), 213, 214
LDL apheresis, 140, 157
Le (Lewis) antigens/antibodies, 304-306, 336,
844
Leach phenotype, 352
Lectins
Dolichos biflorus (anti-A1), 293, 296, 302,
743-744
preparation and use, 743-744
Ulex europaeus, 304, 743-744
Leishmania sp., 699
Leukapheresis, 143-144, 154, 587, 593
Leukocyte-reduction filters, 180, 190, 528-529
Leukocytes-reduced components
for children, 573-574, 576
expiration dates, 189
indications for, 493
labeling, 172
leukocyte content in, 492-493
Platelets, 180, 184, 189, 199, 202, 365, 493,
817
Platelets Pheresis, 35, 202, 493
poststorage, 190
prestorage, 180, 184, 199, 805-806, 817
to prevent bacterial contamination, 693
to prevent CMV infection, 687
quality control, 202, 832-834
Red Blood Cells, 180, 189, 199, 202, 493,
805-806
to reduce platelet alloimmunization, 265,
365
Whole Blood, 180
Lewis blood group (ISBT 007), 304-306
antibodies, 305-306, 336, 434, 539
antigens, 304-305, 841
in children, 306

885

genes, 226, 305
Lewis substance, 445
phenotypes, 305
saliva testing for, 445, 736-739
transfusion practices, 306
Ligands, 269
Ligase chain reaction (LCR), 213, 214
Liley graphs, 540, 541
Linkage, 230-231, 232
Linkage disequilibrium, 231-232, 337, 389
Lipemic samples, 410
Liquid-in-glass thermometers, 821-822
Liquid nitrogen
shipping, 718, 719
storage, 185, 189, 606
Liquid Plasma, 177, 189
LISS (low-ionic-strength saline)
antibodies to ingredients in, 437-438
and antigen-antibody proportions, 275, 277
to enhance antigen-antibody reactions, 277,
427, 443, 753, 754
and incubation time, 274
Liver
disease, 499
transplantation, 402, 619, 624, 627-629
LKE (Luke) antigen, 308, 310, 311
Locus, defined, 241
Long distance PCR, 213, 214
Look-back
for hepatitis, 675
for HIV, 681-682
for HTLV, 683
Low-incidence antigens
antibodies to, 340, 358, 436-437, 546
of Cromer system, 353, 354
defined, 335
of Diego system, 348
of Gerbich system, 352
of Lutheran system, 347
of MNS system, 227, 228, 337-338, 340
not assigned to a system or collection, 357
of Scianna system, 350
Low-ionic-strength saline. See LISS
Low-volume units, 101, 122, 179
Lsa (Gerbich) antigen, 352, 843
Lu (Lutheran) antigens and antibodies, 336,
347-348, 841
Lui freeze-thaw elution, 457, 774
Luke (LKE) antigen, 308, 310, 311

886

AABB Technical Manual

Lung transplants, 402
Lutheran system (ISBT 005), 347-348
antibodies, 336, 347-348
antigens, 347, 841
biochemistry, 347
chromosomal location of genes, 226
in HDFN, 348
phenotypes and frequencies, 347
LW system (ISBT 016), 226, 327, 351, 842
Lyme disease, 696-697
Lymphocytes
B cells, 249-250, 251, 252-253
NK (natural killer) cells, 255
passenger, 628, 629-630, 657
T cells, 253-254
Lymphocytotoxicity assays, 365-366, 395, 396-397
Lyonization, 224, 241

M
Macrophage chemoattractant protein (MCP),
640
Macrophage colony-stimulating factor (M-CSF),
264
Macrophages, 250, 255, 256
Magnetic cell separation, 596
MAIEA (monoclonal antibody-specific immobilization of erythrocyte antigens) assay, 284,
286
MAIGA (monoclonal antibody-specific immobilization of granulocyte antigens), 380
MAIPA (monoclonal antibody-specific immobilization of platelet antigen), 373
Major histocompatibility complex (MHC). See
also HLA system
Class I, II, III molecules, 244-246, 390-391
defined, 269
organization of regions, 386-387
patterns of inheritance, 387-389
restriction, 393
Malaria, 697, 700
Management
assessment, 38
of critical supplies and services, 9-10
of documents and records, 14-19
of equipment, 10-11
of facilities, 6-7
of organization, 6-7
of personnel, 7-9
of safety program, 45

Manual, safety, 45
Marrow transplantation
ABO incompatibilities, 598-599, 600, 601602
ABO typing discrepancies, 299
allogeneic, 582, 583, 585-587
autologous, 583
bacterial contamination, 602-603
collection of marrow, 591-592
diseases treated with, 583, 584
donor databases, 585
donor evaluation, 589-591
engraftment in, 588-589
evaluation and quality control, 607-608
freezing and storage of product, 604-606
and graft-vs-host disease, 585-587
HLA testing, 400-401, 585-586
infectious disease testing, 590-591
matched, unrelated donor, 585-586
nonmyeloablative, 582-583
positive DAT after, 455
processing, 596-598, 601-604
red cell depletion, 601
regulations, 608
standards, 609
thawing and infusion, 607
transportation and shipping product,
606-607
MART antigen, 378, 379
Masks, 75
Massive transfusions
2,3-DPG levels in, 511-512
changing blood types, 511, 628-629
citrate toxicity in, 649-650
coagulopathy in, 511, 651
emergency issue, 510-511
hyper- and hypokalemia in, 650
hypothermia in, 511, 650
selection of blood, 419, 510-511
tissue oxygenation in, 511
Material data safety sheets (MSDS), 59, 60-61,
78-79
Materials, critical, 9
Maternal immunization
antibody titers, 449, 539-540, 761-764,
796-798
mechanisms, 536-538
prenatal evaluation, 538-542
suppression, 542-543

Index

Maximum surgical blood order schedules
(MSBOS), 92-93, 414
McCoy (McC) antigens, 352, 354
McLeod phenotype, 342-343
MCP (macrophage chemoattractant protein),
640
2-ME (2-mercaptoethanol)
applications for, 448-449
to disperse autoagglutination, 469, 470,
744-745
inactivation of Kell antigens, 342
Mechanical barrier systems, 527
Mechanical hemolysis, 152, 642
Medical history
in allogeneic donor selection, 100-101,
110-112
in antibody identification, 424
in autologous donations, 122
in cord blood collection, 595
in evaluation of positive DAT, 454-456
in prenatal evaluations, 538
in tissue and organ transplantation, 618,
620
Medical waste, 55, 56, 57
Medications. See Drugs
Meiosis, 224-225, 241
Membrane attack complex (MAC), 261-262, 263
MER2 (Raph) antigen, 354-355, 843
2-mercaptoethanol. See 2-ME
Meryman method of red cell cryopreservation,
807-810
Messenger RNA (mRNA)
isolation of, 210-211
processing, 204-206
translation, 206-207
Metabolic abnormalities, in neonates, 561-562
Methemoglobin, 835
Methylene chloride (dichloromethane) elutions,
457, 775
MHC. See Major histocompatibility complex
MHO4 clone, 298, 301
Microaggregate filters, 528, 565-566
Microangiopathic hemolysis, 502
Microarrays, 218
Microbead array assay, 395-396
Microfilariasis, 699
Microhematocrit centrifuges, 847
Microlymphocytotoxicity tests, 365-366, 395,
396-397

887

Microplate tests
for ABO group, 733-735
in antigen-antibody detection, 283-284
for Rh typing, 328, 741
Microscopic weak D test, 550
Microvascular bleeding (MVB), 511, 651
Miltenberger system, 338
Missense responses (mutations), 208, 227
Mitosis, 224, 225, 241
Mixed-field agglutination, 298, 299, 348, 357
Mixed lymphocyte culture (reaction)
(MLC/MLR), 397
Mixed type AIHA, 466-467
classification, 459
serologic findings in, 460, 466-467
specificity of autoantibodies, 467
transfusion in, 467
MLC (mixed lymphocyte culture), 397
MLR (mixed lymphocyte reaction), 397
MNS system (ISBT 002), 337-340
antibodies, 303, 336, 340, 444
antigens
linkage disequilibrium in, 231, 232, 337
linkage in, 232
low-incidence, 227, 228, 337-338, 340
M, N, S, s, U, 337
nomenclature, 840, 844
phenotypes and frequencies, 337, 844
biochemistry, 338-339
effect of enzymes on, 276, 339-340
genes, 226, 227, 228, 338, 844
in HDFN, 340
hybrid molecules, 338
in transfusion reactions, 340
MoAb. See Monoclonal antibodies
Mobile blood collection, 41, 179
Mobilization of hematopoietic progenitor cells,
587, 592-595
Modifier genes, 235-236
Molar solutions, 723
Mole, defined, 723
Molecular biology, 203-220
DNA and mRNA, 203-207
genetic variability, 208-209, 210
polymorphism, genetic mechanisms in,
207-208
techniques
DNA cloning, 217, 222
DNA microarrays, 218

888

AABB Technical Manual

DNA profiling, 216-217, 222
DNA sequencing, 217-218, 222, 396
gene therapy, 219-220
isolation of nucleic acids, 209-211
phage display/repertoire cloning, 222
polymerase chain reaction, 211, 212,
213-214, 215, 222, 394-396
protein and RNA targeted inactivation, 220
recombinant proteins, 218-219, 222
restriction endonucleases, 214, 216
restriction fragment length polymorphism analysis, 214, 216, 222
Monoclonal antibodies
in ABO testing, 296
anti-D, 328, 329-330
assays utilizing, 284, 373, 380
in autologous tumor purging, 597-598
for reagent use, 266-267
Monoclonal antibody-specific immobilization
of granulocyte antigens (MAIGA), 380
Monoclonal antibody-specific immobilization
of platelet antigens assay (MAIPA), 373
Monoclonal antibody-specific immobilization
of erythrocyte antigens (MAIEA) assay, 284,
286
Monocyte monolayer assay, 441
Monocytes, 250, 255, 256
Mononuclear phagocytic system, 255
Monospecific AHG reagents, 280
MP-NAT (minipooled NAT)
for HCV, 669
for HIV, 678, 679
for parvovirus, 689
for West Nile virus, 685
MPHA (mixed passive hemagglutination assay),
371-372
mRNA. See Messenger RNA
MSBOS (maximum surgical blood order schedules), 92-93, 414
MSDS (material data safety sheets), 59, 60-61,
78-79
Multiplex PCR, 213
Muscular spasms, in donors, 108
Mutations, genetic, 208, 227, 228
Myasthenia gravis, 156

N
N antigen/antibody, 336, 337, 340, 840
NA/NB neutrophil antigens, 378

NAIT. See Neonatal alloimmune
thrombocytopenia
NAN (neonatal alloimmune neutropenia), 379
Nasopharyngeal carcinoma, 687
NAT. See Nucleic acid amplification test
National Marrow Donor Program (NMDP), 585,
589
Natural killer (NK) cells, 255
Nausea, 107-108, 639
Needles, 51, 523, 565
Negative selection, 597-598
Neonatal alloimmune neutropenia (NAN), 379
Neonatal alloimmune thrombocytopenia
(NAIT), 551-553
management after delivery, 553
platelet-specific antigens in, 366, 552
prenatal considerations for, 552-553
scheduling therapy, 553
serologic testing in, 552
sources of platelets for, 553
Neonatal immune thrombocytopenia (NIT)
alloimmune (NAIT), 366, 551-553
secondary to maternal ITP, 553-554
Neonatal polycythemia, 572
Neonates. See also Hemolytic disease of the fetus
and newborn
ABO antigens/antibodies in, 293, 295, 298
ABO discrepancies in, 299
ABO/Rh typing in, 415, 545
anemia in, 558, 559, 563-564
antigen variations in, 431
blood volume, 558-559, 839
compatibility testing in, 415, 562-563
cytomegalovirus infection in, 562
DIC in, 567, 572
direct antiglobulin testing in, 545-546
erythropoiesis in, 557-558, 559
extracorporeal membrane oxygenation in,
572-573, 574
graft-vs-host disease in, 560-561
hypothermia in, 560
immune thrombocytopenia in, 551-554
immunologic status, 560-561
leukocyte reduction for, 573-574
Lewis antigens in, 306
low birthweight, 557
metabolic problems in, 561-562
neonatal alloimmune neutropenia, 379
normal laboratory values in, 836-837

Index

plasma volume in, 839
polycythemia in, 572
red cell volume in, 839
size of, 558-559
transfusions in
administration, 565-566
Cryoprecipitated AHF, 558, 572
effect of additive/preservative solutions,
564-565
to enhance hemostasis, 570-572
exchange, 546-547, 560, 566-568
of Fresh Frozen Plasma, 558, 571
Granulocytes, 558, 569-570
indications for, 563-564
Platelets, 490, 552-553, 558, 568-569
Red Blood Cells, 558, 564-568
volumes for, 558, 564, 568, 571
Neutralization
in antibody identification, 445
of Sda antigen, 357, 445, 767-768
in viral marker testing, 169
Neutropenia, 379-380
Neutrophils
alloantigens, 377-380, 647
antibodies to
in neonatal alloimmune neutropenia, 379
testing for, 380
in TRALI, 379-380, 647, 656
in immune system, 255
NIT. See Neonatal immune thrombocytopenia
NK (natural killer) cells, 255
Nomenclature
of blood group systems, 238-239, 318-319,
840-844
CD (clusters of differentiation), 246, 247-248
of HLA system, 391-393
ISBT, 226, 238-239, 840-843
of platelet antigens, 366, 367-368
of Rh system, 315, 317, 318-319
Non-A, non-B hepatitis (NANB), 673, 699
Non-immune hemolysis, 636, 642-643
Nonconforming products or services
management, 19, 20, 21
quarantine, 173-174, 194
Nonimmunologic protein adsorption, 476
Nonsense response (mutations), 208, 227
Normal solutions, 723
Nuclear Regulatory Commission (NRC), 41, 64,
65, 66

889

Nucleic acid amplification test (NAT)
in HBV testing, 669, 672
in HCV testing, 164, 166, 213, 590, 672, 673,
675, 701
in HIV testing, 164, 166-167, 213, 590,
678-681
in HTLV testing, 683
in parvovirus testing, 688-689
in viral maker testing, 166-167
in West Nile virus testing, 213
Nucleic acids
isolation of, 209-211
sequencing, 217-218, 222
Nucleotides
defined, 203
insertion and deletion, 208
sequences, 204, 205
substitution, 207-208, 228
Nutricel (AS-3), 176, 186, 565

O
Obligatory gene, 237
Occupational Safety and Health Administration
(OSHA), 41
Officers
chemical hygiene, 58
safety, 42-43
Oh phenotype (Bombay), 304
Ok system (ISBT 024), 226, 354, 843
Oligonucleotide probes, sequence-specific
(SSOP), 214, 373, 395-396
OND antigen, 378, 379
“Open” system, 189, 192
Opsonization, 262
Optisol (AS-5), 176, 186, 565
Oral thermometers, electronic, 822-823
Ordering policies, 36, 91-94, 414
Organ donation and transplantation
ABO compatibility in, 401, 402, 627, 629-630
consent for, 620-621
cytomegalovirus in, 629, 630
disease transmission in, 617, 619-620
donor eligibility, 620-621
of heart, 629
HLA testing, 401-402
of kidney, 401-402, 619, 624, 627
of liver, 402, 627-629
of pancreas, 401-402, 629
positive DAT after, 455

890

AABB Technical Manual

preservation conditions and dating periods,
624
records, 626
recovery of tissue, 621
regulations, 626
risk reduction in, 618, 620
serologic testing for, 621-623, 629
skills and experience appropriate for, 618
transfusion support, 627, 629-630
types of donors for, 622
Organisms
in bacterial contamination, 691, 692
genetically modified, 717
Organizational management, 6-7
Organizations, 848-850. See also specific organizations
regulating quality systems, 1-2
regulating safety, 39-40, 71-72
Organs of immune system, 249
Orientation program, 8
OSHA (Occupational Safety and Health Administration), 41
Outdating, 90-91
Outpatients, 408-409
Oxygen
compensation for anemia, 484
delivery of, in ANH, 127-128
dissociation in red cell storage, 185, 187
in massive transfusion, 511
measuring adequacy of supply, 484-485
supply and demand, 483-484
treating inadequate supply of, 485
Oxygen therapeutics, 512-513

P
P blood group (ISBT 003), 308-311
anti-P1, 303, 310-311
antibodies, 311, 468
antigens, 308-309, 310, 311, 840, 844
association with PCH, 468
biochemistry and genetics, 309-310, 844
chromosomal locations of genes, 226
hydatid cyst fluid/P1 substance, 310, 445
phenotypes, 309, 844
P1A1 and P1A2 (HPA-1) antigens, 366, 367, 369,
552
p24 antigen, 679
Packaging biological materials, 717-718, 720-721
PAD. See Preoperative blood donation

Pain at infusion site, 633
Pancreatic transplantation, 401-402, 620, 624,
629
Panel-reactive antibody (PRA), 362, 397, 401
Panels, red cell, 425, 426
Papain, preparation of, 756-757. See also Enzymes, proteolytic
Para-Bombay phenotype, 304
Parasitic worms, 698-699
Parentage testing
genetics, 236-237
HLA testing in, 402
linkage disequilibrium in, 389
Paresthesias, 141, 151
Pareto analysis, 27
Paroxysmal cold hemoglobinuria (PCH),
467-468
classification, 459
serologic findings in, 460, 467
specificity of autoantibodies, 469
transfusion in, 467
Paroxysmal nocturnal hemoglobinuria, 378
Partial D, 322-323
Parvovirus B19, 688-689, 700
Passenger lymphocyte hemolysis, 628, 629-630,
657
Paternity testing. See Parentage testing
Pathogen inactivation, 702
Patients. See Recipients
PCH. See Paroxysmal cold hemoglobinuria
PCR. See Polymerase chain reaction
PCR-SSO/PCR-SSOP. See Sequence-specific
oligonucleotide probes
Pedi packs, 564
Pediatric patients. See Children; Neonates
Peer review, 23, 514
PEG. See Polyethylene glycol
Penicillin, 455, 474, 477, 786-788
Percentage solutions, 723, 726-727
Percutaneous umbilical blood sampling (PUBS),
541-542
Performance improvement standards, 25
Pericardium transplants, 619
Personal protective equipment (PPE), 43-44
for biosafety, 54
for chemical safety, 61
face shields, 74
gloves, 46, 55, 73-74, 106
laboratory coats, 73

Index

masks, 75
safety goggles, 74
uniforms, 73
Personnel
accidents and injuries in, 45-46
competency assessment, 8
hepatitis prophylaxis for, 45, 703
job descriptions for, 7
latex allergies in, 46-47
medical first aid and follow-up, 45
orientation program, 8
protective equipment for, 43-45, 73-75
records, 19
safety monitoring programs, 45
selection of, 7
staffing levels, 8-9
training
biosafety, 50
chemical safety, 58-59
computer systems, 32
electrical safety, 48
FDA cGMP, 8
fire safety, 47
general safety, 43, 44
new employee, 8
radiation safety, 65
PF4 ELISA, 377
pH, 274, 431, 444
pH meters, 846
Phage display/repertoire cloning, 222, 267
Phagocytic cells, 255-256, 262
Phagocytosis, 262, 269
Pharmacologic alternatives to transfusion,
512-514
Pharmacologic purging, 597
Phenotypes. See also specific blood groups
of autologous red cells, 429-430, 439
calculations for combined phenotypes, 236,
441
defined, 233
of donor units, 440-441
frequencies, 236
nomenclature for, 238-239, 844
Phlebotomy
adverse reactions to, 19, 107-109
aggressive, in autologous donations, 125
care of donors after, 106
collection of blood samples, 105-106,
408-409, 801-804

891

of donors, 105-106, 800-804
prevention of contamination in, 693
Phosphate buffer, 728
Photochemical inactivation, 702
Photopheresis, 158
Physical examination of donors, 100, 101-103, 620
Physicians’ orders, 522-523, 526
Physician’s responsibilities, in autologous program, 120-121
Physiologic anemia of infancy, 558, 559, 563-564
Physiologic jaundice, 566
Phytanic acid disease, 157-158
Pipettes, recalibration, 847
PlA antigens, 366, 552
Plasma
ABO compatibility, 411, 496, 498
avoiding use, 701
coagulation factor replacement with, 496,
498-500
collection by apheresis, 141, 142-143
components
derivatives/substitutes, 496, 503-508, 699,
701
Fresh Frozen Plasma, 142, 177, 496,
498-500
inventory management, 94
Liquid Plasma, 177, 189
Plasma, 177
Plasma, Cryoprecipitate Reduced, 177,
190, 496, 498
Plasma Frozen within 24 Hours after
Phlebotomy, 181, 189
Pooled Plasma, Solvent/detergent-treated, 496, 702
Recovered Plasma (for manufacture), 177
Source Plasma, 142
Thawed Plasma, 189, 191
constituents removed in apheresis, 149-150
description, 177
expiration dates, 189-190
for pretransfusion testing, 409, 424
as replacement fluid in apheresis, 150, 151
use in children, 571
virus inactivation, 701-702
volume, normal value, 839
Plasma D-dimers, 837
Plasma exchange. See Therapeutic apheresis
Plasma inhibition of anti-Ch and-Rg, 445,
765-766

892

AABB Technical Manual

Plasma protein fraction (PPF), 507
Plasmapheresis, 142-143, 500
Plasmids, 217
Platelet chambers, 185, 197-198, 846
Platelet concentrates (Platelets)
aliquoting, 193-194
bacterial growth in, 690-692, 694-695
biochemical changes during storage, 188
coagulation factors in, 838
description, 176-177, 489
expiration dates, 188, 189
freezing, 183
infectious risks of, 700
inspection, 194-195
inventory management, 94, 95
irradiated, 189, 192-193, 553
leukocyte-reduced, 184, 189, 190, 199, 202,
365, 493, 817
pooled, 184, 189, 193
preparation, 176-177, 815-817, 827-828
quality control, 199, 202
storage, 185, 188, 694-695
transfusions, 488-492
ABO/Rh matching, 361-362, 489-490, 569
in children, 490, 552-553, 558, 568-569,
576-577
contraindications to, 491-492
HLA alloimmunization to, 265, 266,
362-366, 397-398, 637
indications for, 490-491, 569
physiologic principles, 488-489
prophylactic, 490-491
refractoriness to, 362-365, 397-398, 491
therapeutic, 490
transportation and shipping, 196
volume reduction, 193, 490, 569, 817-818
washed, 193, 646
Platelet counts
in assessing hemostasis, 488
corrected platelet count increment, 362, 363,
489
in idiopathic thrombocytopenic purpura, 554
in massive transfusion, 511, 651
in NAIT, 552, 553
in neonates, 568-569
normal values, 835, 836
predicted platelet count increment, 362, 363,
489
transfusion triggers, 490-491, 569, 576

Platelet disorders
DDAVP in, 513
in liver disease, 499
thrombocytopenia
in blood loss, 499
drug-induced, 374-377
heparin-induced, 375-377, 492
idiopathic thrombocytopenic purpura,
158, 373-374, 492, 553-554
neonatal alloimmune thrombocytopenia,
511-513, 551-553
posttransfusion purpura, 366, 370, 638,
659-660
secondary to maternal ITP, 553-554
thrombotic thrombocytopenic purpura,
154-155, 158, 492, 500
treatment, 158, 490-491
Platelet incubators, 185, 197-198, 846
Platelet-rich plasma (PRP), 176, 177
Plateletpheresis, 140-142, 154
Platelets. See also Platelet concentrates; Platelet
disorders; Platelets Pheresis
antigens/antibodies
ABH, 361-362, 397
autoantibodies, 373-374
clinical importance, 370-373
detecting, 370-373, 374, 375, 377, 552
drug-induced, 374-377
HLA, 265, 362-366, 397-398
platelet-specific, 366, 367-368, 369-373, 552
assessing function, 488-489
corrected count increment, 362, 363, 489
life span and kinetics, 489
membrane glycoproteins, 369-370
normal values, 835, 836
predicted platelet count increment, 362, 363,
489
refractoriness, 362-365
antibody specificity prediction method
for, 365
causes, 362-363, 364, 397-398, 491
defined, 362
finding compatible donors, 398
HLA-matched platelets for, 363-365, 398
preventing alloimmunization, 365-366
selecting platelets with, 363-365
Platelets Pheresis
ABO/Rh matching, 361-362, 411, 489-490, 590
bacterial growth in, 690-692, 694-695

Index

collection, 140-141
crossmatching, 364, 365, 398
described, 489
donor selection and monitoring, 141
HLA matched, 95, 363-365, 398
infectious risks of, 700
inspection, 194-195
inventory management, 94, 95
laboratory testing, 142
leukocytes reduced, 142, 202, 493
product validation, 35
quality control, 202
records, 141
storage, 185, 694-695
transfusions, 488-492
ABO/Rh matching, 361-362, 411, 489-490
contraindications to, 491-492
HLA alloimmunization to, 265, 266,
362-366, 397-398, 637
physiologic principles, 488-489
prophylactic, 490-491
refractoriness to, 362-365, 397-398, 491
therapeutic, 490
transportation and shipping, 196
Pluripotent hematopoietic stem cell, 249, 251
Policies and procedures, 7, 11, 15
Polyagglutination, 298, 331, 743
Polycythemia, 572
Polyethylene glycol (PEG)
in antibody detection/identification,
276-277, 427, 443, 753-754
effect on incubation times, 274
use in adsorption, 783-784
Polymerase chain reaction (PCR)
applications, 213-214, 215, 222
in HLA typing, 394-396
kinetic, 213
ligase chain reaction, 213, 214
long distance, 213, 214
multiplex, 213
oligonucleotide probes, 214, 373, 395-396
in platelet typing, 373
reaction procedure, 211, 212, 213
reverse transcriptase, 214, 217
sequence-specific primers, 214, 373, 395, 396
in testing fetus for D antigen, 538
variations, 213
Polymorphisms, 207-209
Polymorphonuclear granulocytes, 255

893

Polyspecific AHG, 279-280, 427
Pooled components
Cryoprecipitated AHF, 183-184, 191, 193, 815
expiration, 183, 189
labeling, 172, 183
Platelets, 184, 189, 193
preparation, 183
Pooled plasma, solvent-detergent-treated, 496, 702
Population genetics, 236-237
Position effect, 319-320, 324
Postoperative blood collection, 117, 133-135
Posttransfusion platelet recovery (PPR), 362,
363, 489
Posttransfusion purpura (PTP), 366, 370, 638,
659-660
Potassium, 187, 561, 650
PPE. See Personal protective equipment
PPF (plasma protein fraction), 507
PPR (posttransfusion platelet recovery), 362,
363, 489
PRA (panel-reactive antibody), 362, 397, 401
Preadmission testing, 409
Precipitation, 272
Pregnancy. See also Hemolytic disease of the fetus and newborn; Prenatal studies
autologous collection in, 119
as immunizing stimulus, 536-537, 552
Lewis antibodies in, 305
Premedication in transfusions, 522, 646-647
Prenatal studies
amniotic fluid analysis, 540-541, 542
antibody titration, 449, 539-540, 761-764,
796-798
Doppler flow studies, 542
maternal history, 538
in neonatal immune thrombocytopenia, 552
percutaneous umbilical blood sampling,
541-542
serologic studies, 538-539
typing the fetus, 539
Preoperative blood donation (PAD), 117,
118-126
advantages/disadvantages, 118
adverse reactions, 123
aggressive phlebotomy, 125
collection, 121
compliance requirements, 119-120
component collection, 126
continuous quality improvement, 123-124

894

AABB Technical Manual

contraindications, 119
cost-effectiveness, 125, 127
donor deferrals, 120
donor screening, 121-122, 124-125
erythropoietin use, 125
establishing program, 120-124
labeling, 120, 122-123
medical interview, 122
in pediatric patients, 118-119
physician responsibility, 120-121
records, 123
shipping, 120
storage, 123
supplemental iron in, 121
testing, 119-120, 122
timing and red cell regeneration during, 122
transfusion, 123
transfusion trigger, 125
volume collected, 122
voluntary standards, 119
weak D in donor, 324
Prescriptions for blood orders, 522-523, 526
Preservatives
anticoagulant-preservative solutions,
178-179
CPD, CP2D, CPDA-1, 178-179, 186, 564, 565
red cell changes during storage, 185, 186,
187, 431, 432
shelf life of components, 178, 188,
189-190
reagent, antibodies to, 437
Pressure devices, 530
Pretransfusion testing, 407-420
after non-group-specific transfusions, 419-420
with autoantibodies, 469-472
of autologous blood, 122
blood labeling and release, 416, 418
blood samples, 409-410, 424
in children, 415, 562-563, 574
in cold agglutinin syndrome, 466
comparison with previous records, 413, 526
crossmatching, 413-415, 417
interpretation of results, 415-416, 417
in massive transfusions, 419, 510-511
patient identification, 407, 408-409
procedures included, 524
selection of units, 418-420
serologic testing, 410-413, 417
surgical blood orders, 91-93, 414

transfusion requests, 407-408, 522-523
type and screen, 91-93, 414
in urgent situations, 419, 510-511
Preventive action, 24-25
Prewarming technique, 308, 438, 754-755
Probability values in antibody identification,
429, 430
Problem identification and resolution, 25-27
Procainamide, 455
Procedures and policies, 7, 11, 15
Process capability, defined, 30
Process control, 30
Process flowcharting, 26, 27
Process improvement, 24-27
Process management, 11-14
computer system validation, 13-14
concepts, 4-5, 12
equipment validation, 13
process validation, 11-12
quality control, 14
validation plan, 12-13
Processes, 7, 11-12, 15
Production, principles of, 5
Products, nonconforming, 19, 20, 21
Proficiency testing (PT), 24
Promoter sequence, 204, 205
Protein C, 500, 507, 571, 837
Protein inactivation, 220
Protein S, 500, 507, 837
Protein synthesis, 204-207
Proteolytic enzymes. See Enzymes, proteolytic
Prothrombin time (PT)
in liver disease, 499
in massive transfusion, 511
in monitoring hemostasis, 494, 496
normal value of, 837
in vitamin K deficiency, 498
Prozone, 272, 275
PRP (platelet-rich plasma), 176, 177
Psoralen (S59), 702
PT. See Prothrombin time
PTP (posttransfusion purpura), 366, 370, 638,
659-660
Public antigens, 392
PUBS (percutaneous umbilical blood sampling),
541-542
Pulmonary edema, 648-649
Pulse, of donor, 102
Pumps, infusion, 522, 523, 529-530, 565

Index

Q
QSE (Quality System Essentials), 3
Quad packs, 564
Qualification, defined, 30
Quality assurance (QA)
of blood administration, 527, 532
defined, 2, 30
Quality control (QC)
of blood components, 197-199, 202
apheresis, 832
Cryoprecipitated AHF, 199
leukocyte-reduced components, 199,
832-834
Platelets, 199, 694
Red Blood Cells, 199
Red Blood Cells, Deglycerolized, 812-813
of copper sulfate solution, 819-821, 847
defined, 2, 31
of equipment
automatic cell washers, 830-832
centrifuge calibration, 826-830
continuous temperature monitoring systems, 197-198
freezer alarms, 198-199, 824-826, 846
frequency of, 14, 846-847
refrigerator alarms, 198-199, 823-824, 846
thermometers, 198, 821-823, 847
of hematopoietic products, 607-608
in process control, 14
records, 14
unacceptable results for, 14
Quality improvement, 4
Quality indicators, 22-23, 31
Quality management, 2, 4-6, 31
Quality oversight, 6-7, 8
Quality System Essentials (QSEs), 3
Quality systems, 1-38
application of principles, 6-28
Code of Federal Regulations references, 32
common terms, 30-31
customer and supplier relations, 9-10
defined, 1-2
deviations and nonconforming products or
services, 19, 20, 21
documents and records, 14-15, 16, 17-19
equipment management, 10-11
human resources, 7-9
monitoring and assessment, 22-24, 36-38
organizational management, 6-7

895

process improvement, 24-27
process management, 11-14
quality assurance, 2
quality concepts, 2, 4-6
quality control, 2
quality management, 2
work environment, 27-28
Quarantine
of nonconforming components, 173-174
of repeatedly reactive units, 673, 674, 675,
683
of unusable products, 93-94

R
Race of donor, 98
Radiation safety, 63-66
blood irradiators, 65-66
effects of radiation, 63-64
emergency response plan, 66
exposure limits, 64
monitoring radiation, 64-65
radiation measurement units, 63
regulations, 64
safe work practices, 66
training, 65
waste management, 66
Raph system (ISBT 025), 226, 354, 843
Rapidly progressive glomerulonephritis (RPGN),
156-157
Rare Donor Program, 441-442, 769-770
Rd (Scianna) antigen, 349, 350, 842
Reagents
in ABO testing, 296
albumin additives, 276, 427, 753
antibodies to components of, 298, 437-438
antiglobulin, 278-280 , 427, 454, 468
chloroquine diphosphate, 446, 469, 746747
contamination of, 331, 332
for elutions, 457, 772-773, 775
enzymes, 276, 443, 445, 446, 756-760
glycine-HCl/EDTA, 446, 469, 747-748
labeling, 715
LISS and LISS additives, 274, 275, 277, 427,
443, 753, 754
monoclonal, 266-267, 296, 328, 329-330
for phenotyping, 440-441
polyethylene glycol (PEG), 276-277, 427, 443,
753-754

896

AABB Technical Manual

preparation of, 715, 743-744, 756-757
quality control intervals, 847
red cells, 425, 426, 438
in Rh testing, 328-331
sulfhydryl, 448-449, 744-745, 764-765,
766-767
use of manufacturer’s directions, 11
ZZAP, 445, 446, 781-783
Receptors
on B cells, 249-253
complement, 262, 354
defined, 269
immunoglobulin superfamily, 244, 245
on macrophages and monocytes, 250,
255-256
for pathogens, P antigens as, 311
on phagocytic cells, 255-256
on T cells, 245, 254, 393, 394
Recessive traits, 233-234, 241
Recipients
ABO and Rh testing in, 122, 410, 411
care during transfusions, 530-531
education and consent, 521-522
identification, 407, 408-409, 416, 524-526,
527
phenotyping, 429-430, 439
records, 413, 416, 660-661
tracing (look-back), 675, 681-682, 683
weak D in, 323-324, 411
Recombinant human erythropoietin (rHuEPO),
219
in anemia treatment, 488
in autologous donations, 125
in neonates, 559
Recombinant immunoblot assay (RIBA), 170,
670-671, 672
Recombinant interleukin-11, 512
Recombinant proteins
for HPC mobilization, 587, 592-595
in leukapheresis, 144
as transfusion alternative, 512, 559
uses for, 218-219, 222
Records
archiving, 17-18
autologous donation/transfusion, 123
blood component, 165, 172-173, 416, 418
changing, 18
checking before blood release, 416, 526
confidentiality, 18

donor, 97-98, 103, 165, 173
electronic, 17-18
infectious disease testing, 173
linking personnel to, 19
management, 17-19
of patients with special needs, 661
plateletpheresis, 142
pretransfusion testing, 413
storage, 18
stored tissue allograft, 626
transfer, 173
transfusion, 416
transfusion complication, 660-661
Recovered Plasma, 177
Red blood cells
abnormalities in Rhnull, 326
alloimmunization to, 265, 637, 656-657
antigen nomenclature, 238-239, 318-319,
840-844
changes in storage, 185, 186, 187-188, 431,
433
immune-mediated destruction, 265-266
membrane components of, 290-291
normal values, 835
preparation of 3% suspension, 727
reagents, 425, 426, 438
removal from marrow, 601
separating autologous and transfused,
748-750
survival studies, 441, 509, 655
volume, normal value, 839
Red Blood Cells, Deglycerolized
adequacy of deglycerolization, 812-813
expiration date, 189
preparation, 191-192, 809
refreezing, 183
storage, 192
Red Blood Cells, Frozen
cryoprotective agents for, 181
expiration dates, 184, 189
freeze-thaw damage in, 181
preparation, 181-183, 807-812
storage, 184-185, 809
thawing and deglycerolizing, 191-192, 809
transportation and shipping, 196
Red Blood Cells, Pheresis, 35, 144
Red Blood Cells (RBCs)
ABO/Rh compatibility, 411, 418, 486
antigen-matched, 95

Index

bacterial contamination, 691
collection by apheresis, 35, 143
description, 176
expiration dates, 189
freezing, 181-183, 807-812
infectious risks of, 700
irradiated, 189, 192, 193, 493
leukocytes-reduced, 189, 190, 199, 202, 493,
805
low volume, 101, 122, 179
open system, 189
preparation, 176, 804-807
quality control, 199, 202
rejuvenated, 189, 194, 806-807
storage, 185, 186, 187-188
substitutes, 512-513
transfusion, 483-488
in HPC transplantation, 591-592, 599,
600, 601
indications for, 485-488, 563-564
in neonates, 562-568, 588
physiologic principles, 483-485
selection of components, 411, 486
transportation and shipping, 195-196
washed, 189, 193
Red cell depletion, 601
Reentry protocols, 673, 674, 675, 681-682
Reference laboratories, 439
Refractoriness to platelets, 362-365
antibody specificity prediction method for,
365
causes, 362-363, 364, 397-398, 491
defined, 362
finding compatible donors, 398
HLA-matched platelets for, 363-365, 398
platelet crossmatching for, 365, 398
preventing alloimmunization, 365-366
selecting platelets with, 363-365
Refrigerators, 184
alarm systems for, 198-199, 823-824, 846
quality control, 197-199, 846
temperature monitoring systems for,
197-198
thermometers for, 198
Refsum’s disease, 157-158
Registration of donors, 97-98
Regulations
for autologous donations, 119-120
for hematopoietic transplantation, 608

897

quality-related CFR regulations, 32
for radiation safety, 64
for safety, 28, 39-40, 64, 71-72
for tissue transplantation, 626
for transport and shipping dangerous goods,
716-721
Reissuing blood products, 197, 525
Rejuvenated RBCs, 189, 194, 806-807
Relative risk (RR), 403-404
Release of blood
identification of recipient and component,
524-526
policies of, 416, 418
reissue, 197, 525
in urgent situations, 419, 510-511, 522-523
Remedial action, 25
Renal system
diseases treated with apheresis, 156-157
failure in transfusion reactions, 640-641
kidney transplantation, 401-402, 619, 624,
627
Reports
of deviations, nonconformances and complications, 19, 20, 21
of fatalities, 19, 46, 661, 702-703
of injuries, 45-46
internal event, 19, 20
Requests
for autologous blood collection, 121
for transfusion, 407-408, 522-523
Requirement, defined, 31
Respiratory distress, 152, 639, 647-648
Restricted work areas, 41
Restriction endonucleases, 214, 215
Restriction fragment length polymorphism
analysis (RFLP), 214, 215, 222, 373
Reticulocyte counts, normal value, 835
Reverse line technique, 395
Reverse transcriptase PCR (RT-PCR), 214, 217
RFLP (restriction fragment length polymorphism analysis), 214, 215, 222, 373
Rg (Chido/Rodgers) antigens/antibodies,
351-352, 842
Rh Immune Globulin (RhIG), 547-551
in amniocentesis, 541, 549
antepartum administration of, 538, 541,
548
contraindications for, 549
dosage for, 547, 549, 550-551

898

AABB Technical Manual

for fetomaternal hemorrhage, 549-551
in platelet transfusions, 490
in positive DAT, 456
postpartum administration of, 548-549
Rh system (ISBT 004), 315-331
antibodies, 327-329
anti-D in D+ individuals, 327
concomitant, 327-328, 441
directed at cis products, 324
dosage effect, 327
antigens
C, c, E, e, 316, 319, 320, 330-331
cis product, 324
D antigen, 315-316, 319-320
deleted phenotypes, 326
G antigen, 324-325
incidence, 317
nomenclature, 841, 844
partial D, 322-323
Rhmod, 326
Rhnull, 325-326
variants, 325
weak D, 322-324
association with LW, 351
in component selection, 418, 441, 486, 490,
511
ethnic differences in, 316, 317, 320, 321,
325
genes, 226, 320-321
genetics and biochemistry, 316-318
genotypes, 321-322, 844
in HDFN, 536
phenotypes and haplotypes, 319, 320, 844
position effect in, 319-320, 324
red cell abnormalities in, 326
RhAG (Rh-associated glycoprotein), 318
terminology, 315, 317, 318-319
in transplantation, 599, 628-629
Rh testing, 328-332
with autoagglutinins, 329, 331, 469-470
of autologous blood, 122
of blood components, 164, 165
of C, c, E, e, 320, 330-331
in children, 330, 415, 545, 563, 574
comparison with previous records, 413,
526
controls, 328-329, 330
of cord blood, 545
for D, 319-320, 328-330

of donors and recipients, 328-332, 410, 411,
413, 441
false-positive/false-negative results, 329,
330, 331-332
of Granulocytes, 144
in HDFN, 330, 539, 545
high-protein reagents in, 328-329, 330
low-protein reagents in, 329-330
microplate test, 328, 741
of Platelets Pheresis, 142
in prenatal evaluation, 538-539
slide test, 328, 739-740
in transfusion reaction evaluation, 654
for transplantation, 629
tube test, 740-741
of umbilical cord blood, 595
for weak D, 165, 323, 328, 411, 538-539,
741-744
RIBA (recombinant immunoblot assay), 170,
670-671, 672
Rigors, 633, 643
Risks of disease transmission
HIV infection, 677
with plasma derivatives, 699, 701
posttransfusion hepatitis, 673, 675
reducing, 618, 620, 699, 701-703
with tissue transplantation, 617-618,
619-620, 620
with transfusions, 700
RNA interference, 220
RNA (ribonucleic acid)
isolation of, 209-211
mRNA processing, 204-206
mRNA translation, 206-207
splicing, 206
transfer, 207
Rocky Mountain spotted fever, 696
Rodgers blood group. See Chido/Rodgers
Room temperature antibodies, 302-303
Room temperature storage, 185
Rosette test, 550, 793-794
Rouleaux
in ABO discrepancies, 298, 299, 303
in antibody detection, 412
in Rh typing, 329, 330
saline replacement technique, 303, 755756
RT-PCR. See Reverse transcriptase PCR
Run charts, 23

Index

S
S/s antigens and antibodies, 336, 337, 340, 840
Safe work practices
for biosafety, 54-55
for electrical safety, 48-49
for fire prevention, 47-48
general guidelines, 44, 73-76
for radiation safety, 66
Safety goggles, 74
Safety program
accidents and injuries, 45-46
biosafety, 49-57, 77
chemical safety, 57-63, 78-88
disaster planning, 67-68
electrical safety, 48-49
emergency response plans, 44
employee monitoring programs, 45
engineering controls, 43
fire prevention, 47-48
first aid and follow-up, 45
general elements, 42-47
hazard identification and communication,
43
hepatitis prophylaxis, 45, 668, 672, 703
latex allergies, 46-47
management controls, 45
personal protective equipment, 43-44
policy manual, 45
radiation safety, 63-66
regulations and recommendations, 71-72
resources for information, 850
safe work practices, 44
safety officer, 42-43
shipping hazardous materials, 66
training, 43, 44
waste management, 67
in work environment, 27-28
Saline replacement technique, 303, 755-756
Saliva
ABH substances in, 290-291, 301, 736-739
Lewis substance in, 445, 736-739
Salvia lectins, 743
Samples. See Blood samples
SBO (standard blood orders), 92-93
Scianna system (ISBT 013), 226, 336, 349-350,
842
Sda antigen, 357
agglutination pattern of antibody, 357, 412
neutralization of, 357, 445, 767-768

899

SDF-1 (stromal-derived growth factor-1), 594
Secretor gene (Se), 290, 291, 304-305, 737-739
Secretory component, 258
Segregation, 229, 230
Selectins, 246
Semen, 620, 621, 624
SEN-V virus, 668
Sensitization, 272-275
Separation techniques
in apheresis, 139-140
immunomagnetic, 596, 598
of multiple antibodies, 449
for transfused and autologous cells, 748-750
Sepsis
neonatal, 569-570
and positive DAT, 456
prevention, 693-694
transfusion-associated, 635, 643, 655,
691-692
Sequence-specific oligonucleotide probes
(SSOP), 214, 373, 395-396
Sequence-specific primers (SSPs), 214, 373, 395,
396
SERF (Cromer) antigen, 353, 354, 843
Serious Hazards of Transfusion (SHOT) initiative, 641
Serologic centrifuges, calibration, 828-830
Serologic testing
additives in, 276-277, 443, 753-754
of autologous blood, 120, 122
of blood components, 165-166
in cold agglutinin syndrome, 460, 465
comparison with previous results, 413
of cord blood, 595
of Granulocytes, 144
in HDFN, 538-539, 544-546
for HLA antigens, 396-397
in IgM warm AIHA, 464
incubation temperatures, 443, 715
in mixed-type AIHA, 460, 466-467
in neonatal immune thrombocytopenia, 552
for organ and tissue transplantation, 620,
621-623, 629
in paroxysmal cold hemoglobinuria, 460,
467
for platelet antibodies, 375
of Platelets Pheresis, 142
with positive DAT, 456
in pretransfusion testing, 410-413, 562-563

900

AABB Technical Manual

records of, 165
for syphilis, 165-166, 695
in transfusion reaction evaluations, 653-654
in warm autoimmune hemolytic anemia,
460, 461
in warm IgM AIHA, 464
Serotonin release assay, 377
Serum
dilution, 725-726
for pretransfusion testing, 409, 424
Serum hyperviscosity syndrome, 153-154
Serum proteins in typing discrepancies, 298,
299, 303, 331
Serum-to-cell ratio, 444
Services
critical, 6-7
nonconforming, 19, 20, 21
quality principles, 5
Severe acute respiratory syndrome (SARS), 590
Sex-linked traits, 233-234, 241
SH antigen, 378
Shelf life of components, 178, 188, 189-190
Shipments
of autologous units, 120
of blood components, 66, 179, 195-196
cargo aircraft only, 719
of clinical specimens, 66, 717-718, 720-722
containers for, 195, 847
of dangerous goods, 66, 716-722
of hematopoietic components, 606-607
labeling, 718, 719
monitoring temperature during, 722
packaging with dry ice, 196, 719, 720-721
packaging with liquid nitrogen, 718, 719
Shock, 633, 640
Short supply agreement, 177
SHOT (Serious Hazards of Transfusion) initiative, 641
Showers, emergency, 61
Sickle cell disease (SCD)
alloantibody production in, 576
autologous donation with, 119
in children, 574-576
delayed transfusion reactions in, 656-657
separation of transfused from autologous
cells, 749-750
transfusion in, 508-509, 574-576
treatment with apheresis, 155-156
Signal transduction, 246

Signs, safety, 47, 51, 60
Silent mutations, 207
Single-donor platelets. See Platelets Pheresis
Skin appearance
in donors, 102-103
in transfusion reactions, 639, 644-646
Skin banking, 619, 624, 625
Sl (Knops) antigens, 354, 843
Slide tests
for ABO group, 731-732
for Rh typing, 328, 739-740
Sodium, 187
Software, computer, 17
Solid organ transplantation. See Organ donation
and transplantation
Solid-phase red cell adherence assay (SPRCA)
in detecting antigen-antibody reactions,
283-284
in platelet antibody detection, 371, 372, 375
in platelet crossmatching, 364
Soluble antigens
ABH, 290-291, 298, 301, 445, 736-739
Chido/Rodgers, 445, 765-766
HLA, 391
Lewis, 445, 736-739
P1, 310, 445
Sda, 357, 445, 767-768
testing, 301, 445, 736-739
Solutions
additive, 176, 186, 564-565
colloid, 486, 507-508, 572
dilution, 726-727
hemoglobin, 512-513
IV, 530
phosphate buffer, 728
preparation, 723-725
Solvent/detergent-treated pooled plasma, 496,
702
Somatic cells, 223, 241
Source Plasma, 142
Southern blotting, 214, 216
SPA immunoadsorption, 158
Specification, defined, 31
Spills
blood, 55, 56
chemical, 61-62, 84-88
radioactive, 66
SPRCA. See Solid-phase red cell adherence assay
SRA (C-serotonin release assay), 377

Index

SSOP (sequence-specific oligonucleotide
probes), 214, 373, 395-396
SSP (sequence-specific primers), 214, 373, 395,
396
Staffing, 8-9
Standard blood orders (SBO), 92-93
Standard Precautions, 49, 54
Standards
for autologous donations, 119
for hematopoietic transplantation, 609
ISO 9001, 1- 2, 3
performance improvement, 25
STAR (Scianna) antigen, 349, 350, 842
Statistical tables for binomial distribution,
33-35
Stem cells, pluripotent, 249, 251, 581
Sterile connection devices, 847
Sterility testing, 195
Storage
acceptable temperatures for, 184, 185, 715
antigen deterioration with, 431, 433
of autologous blood, 94-95, 123
biochemical changes with, 185, 186, 187
of biohazardous material, 57
of blood components, 93-94, 184-185, 186,
187-188
of blood samples, 184-185, 410
of directed donor units, 94-95
frozen, 184-185, 606
of Granulocytes, 144
of hazardous chemicals, 61
of hematopoietic progenitor cells, 604-606
liquid, 185, 186, 187-188
liquid nitrogen, 185, 606
of organs and tissue, 624
of Platelets, 185, 188, 694-695
of records, 18-19
of Red Blood Cells, 185, 186, 187-188
of Red Blood Cells, Deglycerolized, 192
of Red Blood Cells, Frozen, 184-185, 809
refrigerated, 184
room temperature, 185
of untested or infectious products, 606
vapor-phase, 606
Storage lesion
of platelets, 188
of red cells, 185, 186, 187-188
Stroma-free hemoglobin solution, 512-513
Stromal-derived growth factor-1 (SDF-1), 594

901

STS (serologic testing for syphilis), 165-166,
695
Subgroups of ABO system, 293-294
in ABO discrepancies, 302
confirmation of by adsorption/elution,
735-736
testing for, 296
Sulfhydryl reagents, 448-449
to alter blood group antigens, 342, 445, 446,
766-767
applications for, 448-449
in dispersing autoagglutination, 469-470,
744-745
to distinguish IgM from IgG, 764-765
effect on Kell antigens, 342
Supertransfusion programs, 575-576
Supplier relations, 9-10
Supplies, critical, 9, 10, 11
Suppressor genes, 235-236
Surgical blood order systems, 91-93, 414
Survey meters, 65
Survival studies of red cells, 441, 509, 655
Syncope, in donors, 107
Syngeneic HPC transplantation, 582, 587
Syphilis, 695
testing autologous blood, 120
testing blood components, 164, 165-166, 695
testing transplantation donors for, 590, 620,
621

T
T-cell receptor (TCR), 245, 254, 393, 394
T-cell reduction (depletion), 586, 598
T lymphocytes, 249, 253-255
cytotoxic T cells, 249, 253-254
depletion methods, 598
helper T cells, 249, 253-254
receptors (TCR), 245, 254, 393, 394
recognition by T cytotoxic cells, 254
stimulation of B cells, 254-255
subpopulations of, 249, 253
TA-GVHD. See Transfusion-associated
graft-vs-host disease
Tc (Cromer) antigens/antibodies, 353, 354, 843
TCR (T-cell receptor), 245, 254, 393, 394
Temperature
of donor, 101-102
effect on agglutination, 273-274, 336, 443
incubation, 443, 715

902

AABB Technical Manual

monitoring systems, 197-198, 823-826
during shipments, 195-196, 722
storage, 184, 185, 715
Tendon transplants, 624
Test results, grading, 412, 728-729
Thalassemia, 508, 575-576
Thawed Plasma, 189, 191
Thawing
Cryoprecipitated AHF, 191, 815
devices for, 846
effects of, 181
Fresh Frozen Plasma, 191
hematopoietic components, 607
Red Blood Cells, Frozen, 191-192, 809
Therapeutic apheresis, 144-158
complications, 150-153
in HDFN, 145, 149
indications for, 146-148, 153-158, 500
photopheresis, 158
plasma volumes exchanged, 145, 149
removal of normal plasma constituents,
149-150
removal of pathologic substances, 145,
148-149
replacement fluids in, 150, 151
SPA immunoadsorption, 158
vascular access in, 145, 150
Therapeutic cells, 581. See also Hematopoietic
progenitor cell transplantation
Therapeutic plasma exchange (TPE), 145. See
also Therapeutic apheresis
Thermal amplitude studies, 441, 458, 465
Thermometers, 198
electronic oral, 822-823
liquid-in-glass, 821-822
standardization and calibration, 198,
821-823, 847
Thrombin, 502
Thrombin time, normal value, 837
Thrombocythemia, 154
Thrombocytopenia
in blood loss, 499
drug-induced, 374-377
and exchange transfusion, 567
heparin-induced, 375-377, 492
idiopathic thrombocytopenic purpura, 158,
373-374, 492, 553-554
neonatal alloimmune thrombocytopenia,
551-553, 568-569

posttransfusion purpura, 366, 370, 638,
659-660
secondary to maternal ITP, 553-554
thrombotic thrombocytopenic purpura,
154-155, 158, 492, 500
treatment, 158, 490-491
Thrombopoietin, recombinant human, 219
Thrombotic thrombocytopenic
purpura/hemolytic-uremic syndrome
(TTP/HUS), 154-155, 158, 492, 500
Tick-borne infections, 695-697
Timers/clocks, 847
Tissue antigens. See HLA system
Tissue oxygenation, 484-485, 511-512
Tissue transplantation
ABO compatibility in, 627
bone banking, 623, 625
consent for, 620-621
disease transmission in, 617, 619-620
donor eligibility, 620-621
heart valves, 625
preservation conditions and dating periods,
624
records, 626
recovery of tissue, 621
regulation, 626
risk reduction in, 618, 620
serologic testing for, 621-623
skills and experience appropriate for, 618
skin banking, 625
types of donors for, 622
Titration of antibodies
applications, 449
cold agglutinins, 458, 465, 778-779
maternal antibodies, 449, 539-540, 730-731,
796-798
methods for, 761-764, 796-798
TNF (tumor necrosis factor), 264, 640
Toxoplasmosis, 698
TPE (therapeutic plasma exchange), 145. See
also Therapeutic apheresis
Training
biosafety, 50
chemical safety, 58-59
for dry ice shipments, 721
electrical safety, 48
FDA cGMP, 8
fire safety, 47
general safety, 43, 44

Index

new employee, 8
radiation safety, 65
Traits, 232-236
TRALI. See Transfusion-related acute lung injury
Tranexamic acid, 513-514
Trans effect, 319, 320
Transforming growth factor-β (TNF-β ), 264
Transfusion-associated graft-vs-host disease
(TA-GVHD), 657-659
HLA system in, 399, 400
management, 637
manifestations, 658
in neonates, 560-561
pathophysiology, 637, 658
treatment and prevention, 658-659
Transfusion-associated sepsis, 635, 643, 655,
691-692
Transfusion Committee, 514
Transfusion reactions
acute
air embolism, 636, 651-652
allergic (urticarial), 522, 634, 644-647
anaphylactic, 635, 644-647
circulatory overload, 636, 648-649
citrate toxicity, 649-650
coagulopathy, 651
evaluation, 531, 652-656
febrile nonhemolytic, 379, 398-399, 576,
634, 643-644
hemolytic, 336, 399-400, 634, 639-642
hyper- and hypokalemia, 650
hypocalcemia, 561, 636, 649-650
hypotension associated with ACE inhibition, 636, 645
hypothermia, 511, 637, 650
nonimmune hemolytic, 636, 642-643
sepsis, 635, 643, 655, 691-692
signs and symptoms, 633, 639
transfusion-related acute lung injury,
379-380, 399, 635, 647-648
classification, 633, 634-638
delayed
alloimmunization, 637, 656-657
graft-vs-host disease, 399, 400, 560-561,
637, 657-659
hemolytic, 346, 575, 637, 656-657
immunomodulation, 638, 660
iron overload, 638, 660
Kidd antibodies in, 346, 656

903

posttransfusion purpura, 366, 370, 638,
659-660
management, 634-639
records, 660-661
reporting, 19, 661
Transfusion-related acute lung injury (TRALI),
647-648
evaluation, 656
and granulocyte transfusions, 648
HLA antibodies in, 399, 647
management, 635
neutrophil antibodies in, 379-380, 647
pathophysiology, 635, 647
prevention, 648
symptoms, 634, 647-648
treatment, 648
Transfusion-transmitted diseases
babesiosis, 695-696
bacterial sepsis, 635, 643, 655, 691-692
Chagas’ disease, 697-698
cytomegalovirus, 686-687
Epstein-Barr virus, 687
erlichiosis, 696
fatalities from, 702-703
hepatitis, 667-675
herpesviruses, 686-689
human immunodeficiency viruses, 675-682
human T-cell lymphotropic viruses, 682-683,
684
Lyme disease, 696-697
malaria, 697
management, 703
parasitic worms, 698-699
parvovirus, 688-689
reducing risks of, 699, 700, 701-702
reporting, 702
Rocky Mountain spotted fever, 696
syphilis, 695
tick-borne infections, 695-697
toxoplasmosis, 698
transmissible spongiform encephalopathies,
689-690
West Nile virus, 683, 685-686
Transfusion triggers
for autologous transfusions, 125
for platelet transfusions, 490-491, 569, 576
for red cell transfusions, 483, 486-488
Transfusions
administration procedures, 521-532, 565-566

904

AABB Technical Manual

alternatives to, 512-514
of antiprotease concentrates, 505-506
assessments of, 23, 38, 514
in autoimmune hemolytic anemias,
462-464, 467, 468, 509-510
of autologous blood, 123
cause of ABO discrepancies, 298
cause of positive DAT, 455, 462
in children, 562-572, 574-577
of coagulation factors, 493-494, 495, 496,
497, 498-505
of colloid solutions, 486, 507-508, 572
consent for, 521-522
of Cryoprecipitated AHF, 500-502, 572
delays in starting, 525
equipment for, 523-524, 527-530
events following, 531-532
exchange, 546-547, 560, 566-568
fatalities from, 19, 641, 645, 661, 690-691,
702-703
of Fresh Frozen Plasma, 496, 498-500, 571
of Granulocytes, 144, 492, 569-570
and HLA system, 397-400
in HPC transplantation, 591-592, 599, 600,
601
identification of recipient and components,
524-526, 527
of Immune Globulin, Intravenous,
505, 506
as immunizing stimulus in HDFN,
537-538
of incompatible blood, 509, 641
indications for, 486-488, 563-564
infusion rates, 531, 566
infusion sets for, 527-529, 565-566
intrauterine, 541, 543-544
of irradiated components, 493
IV solutions for, 530
of leukocyte-reduced components, 492-493
massive, 419, 510-512, 628-629, 649-651
in neonates, 562-572
non-group-specific, 419-420, 510-511
in older infants and children, 574-577
patient care during, 530
perioperative, 486-487
of plasma derivatives and substitutes, 496,
503-508
of Platelets, 488-492, 553, 568-569
premedication in, 522

of protein C and protein S, 507
quality assurance, 514, 527, 532
of RBCs, 483-488, 558, 562-568
of recombinant proteins, 512
requests for, 407-408, 522-523
selection of components, 411, 486
in sickle cell disease, 508-509, 574-576
starting, 526-527
in thalassemia, 508, 575-576
in transplantation, 627, 629-630
in urgent situations, 419, 510-511,
522-523
of Whole Blood, 485, 486
Transmissible spongiform encephalopathies
(TSEs), 689-690
Transplantation. See Hematopoietic progenitor
cell transplantation; Organ donation and
transplantation; Tissue transplantation
Transplantation antigens. See HLA system
Transportation
of autologous units, 120
of blood components, 66, 179, 195-196
of blood to patient area, 524-525
cargo aircraft only, 719
of clinical specimens, 66, 717-718, 720-722
containers for, 195, 847
of dangerous goods, 66, 716-722
of hematopoietic components, 606-607
labeling, 718, 719
monitoring temperatures during, 722
packaging with dry ice, 196, 719, 720-721
packaging with liquid nitrogen, 718, 719
Trial to Reduce Alloimmunization to Platelets
(TRAP) Study Group, 365
Tropical spastic paraparesis (TSP), 682-683
Trypanosoma cruzi, 698, 700
TSEs (transmissible spongiform
encephalopathies), 689-690
TTP/HUS. See Thrombotic thrombocytopenic
purpura/hemolytic-uremic syndrome
TTV virus, 668
Tube tests
for ABO group, 732-733
for Rh type, 740-741
Tumor cell detection, 608
Tumor necrosis factor (TNF), 264, 640
Tumor purging, 597-598
Twitching, in donors, 108
Type and screen (T/S), 91-93, 414

Index

U
U antigen/antibody (MNS system), 336, 337,
340, 840
Ulex europaeus (anti-H), 304, 743-744
Ultraviolet B (UVB) irradiation, 365
Umbilical cord blood. See Cord blood
UMC (Cromer) antigen, 353, 354, 843
Unasyn, 455
Uniforms, 73
Urgent release of blood, 419, 510-511, 522-523
Urine
examinations in transfusion reactions, 639,
653
neutralization of Sda, 357, 445, 767-768
Urticaria (hives), 634, 644-647
Utilization of blood. See Blood utilization
UVB (Ultraviolet B) irradiation, 365, 398

V
Vaccines, hepatitis, 45, 668, 672, 703
Valeri method for red cell cryopreservation,
810-812
Validation
of computer systems, 13-14, 415
defined, 31
of equipment, 13
of processes, 11-12
statistical tables for, 33-35
validation plans, 12-13
variances to, 11
Vapors, hazardous, 62, 63
Variant Creutzfeldt-Jakob disease (vCJD), 590,
689-690
Vascular access
in apheresis, 145, 150
in HPC collection, 593, 594
in neonates, 565-566, 568
for transfusions, 523-524
Vasovagal syndrome, 107, 141
vCJD (variant Creutzfeldt-Jakob disease), 590,
689-690
Vel collection, 355-356
Venipuncture, 105-106, 800-804
Venous access. See Vascular access
Venous transplants, 624
Verification, defined, 31
Vicia graminea (anti-N), 743
View boxes, 846

905

Vil antigen, 354
Viral marker testing. See Infectious disease testing
Viruses
inactivation of, 699, 701-702
transmitted by transfusion
cytomegalovirus, 686-687
Epstein-Barr, 687
hepatitis, 667-675
HIV, 675-682
HTLV, 682-683, 684
human herpesviruses, 688
parvovirus, 688-689
transmissible spongiform
encephalopathies, 689-690
West Nile, 683, 685-686
Viscosity, relative, 835
Vital signs, 526-527, 531
Vitamin K, 498-499, 571
VLBW (very low birthweight) neonates, 557, 568.
See also Neonates
Volume of blood
administered in intrauterine transfusions,
544
administered in neonatal transfusions, 558,
564, 568, 571
collections, 101, 122, 178-179
normal values, 558-559, 839
Volume of marrow collections, 591
Volume of plasma
exchanged in TPE, 145, 149
normal values, 839
Volume of red cells, 839
Volume reduction of platelets, 193, 490, 569,
817-818
Vomiting, 107-108, 639
von Willebrand syndromes, 501

W
WAIHA. See Warm autoimmune hemolytic anemia
Warfarin, 376, 498-499
Warm autoantibodies
adsorption, 470-472, 779-781
alloantibodies with, 439, 470-472
drug-induced, 475-476
frequency of testing, 463-464
mimicking alloantibodies, 472
transfusion-stimulated, 462

906

AABB Technical Manual

transfusion with, 462-464
in warm autoimmune hemolytic anemia,
461-462
Warm autoimmune hemolytic anemia (WAIHA),
459, 461-464
antiglobulin testing in, 281
chronic, 462-463
classification, 459
serologic findings in, 459, 460, 461
specificity of autoantibody, 461-462
transfusion in, 462-464
Warm-reactive alloantibodies, 273-274
Washed blood components, 189, 193, 561, 646
Wastage of autologous blood, 123-124
Waste
biohazardous, 55-57
chemical, 63
defined, 717
radioactive, 63
reduction program, 67
treating, 57
Water of crystallization, 724
Water of hydration, 724
Waterbaths, 693, 846
Wb (Gerbich) antigen, 352, 843
Weak D, 322-324
in autologous donations, 324
in donors, 165, 323
“microscopic,” 550
partial D, 322-323
in pregnancy, 538-539
quantitative, 322
in recipients, 323-324, 411
testing for, 165, 323, 328, 741-743
Weight, donor, 101, 143
WES (Cromer) antigens/antibodies, 353, 354,
843
West Nile virus (WNV), 683, 685-686
fatalities due to, 691
preventive measures, 590, 685
testing for, 166, 213, 595-596, 685
Western immunoblot, 169-170, 679-681, 698
WH (Chido/Rodgers) antigen, 352, 842
White cells
normal values, 835, 836
residual, in leukocyte-reduced components,
493, 832-834

Whole blood
ABO/Rh compatibility, 411, 486
biochemical changes during storage, 185,
186, 187-188
collection, 178-179
description, 175
equipment quality control, 847
expiration date, 189
leukocyte reduction, 180
processing, 179-180
transfusion, 485, 486
transportation and shipping, 179,
195-196
Wipe tests, 65
Work environment
design and workflow, 40
housekeeping, 40-41
restricted areas, 41
safety, 27-28, 54-55
Wr (Diego) antigens/antigens, 348, 842
Wristbands, patient, 408, 409, 526

X
X-linked traits, 233-234, 241
Xenogeneic HPC transplantation, 582
Xg system (ISBT 012), 226, 336, 348-349, 350,
842

Y
York (Yk) antigens, 352, 354, 843
Yt (Cartwright) system (ISBT 011), 226, 336, 348,
349, 842

Z
ZENA (Cromer) antigen, 353, 354, 843
Zygosity, 431. See also Dosage effect
ZZAP
in allogeneic adsorption, 471-472, 781-783
to alter antigens, 342, 445, 446
in autologous adsorption, 469, 470, 775-776,
779-781
in dispersing autoagglutination, 469

Glossary of Abbreviations
AATB
ACD
ACE
AChE
ACOG
ADCC
AET
AHF
AHG
AHTR
AICC
AIDS
AIHA
ALG
ALT
ANCA
ANH
APC
aPTT
AS
ASO
ATG
ATL
ATP
BFU
BPO
BSA
BSC
BUN
C:T
CAD
CAP
CAS
CBER
CCE

American Association of Tissue
Banks
acid-citrate-dextrose
angiotensin-converting enzyme
acetylcholinesterase
American College of Obstetricians
and Gynecologists
antibody-dependent cellular
cytotoxicity
2-aminoethylisothiouronium
antihemophilic factor
antihuman globulin
acute hemolytic transfusion reaction
anti-inhibitor coagulation complex
acquired immune deficiency
syndrome
autoimmune hemolytic anemia
antilymphocyte globulin
alanine aminotransferase
antineutrophil cytoplasmic
antibodies
acute normovolemic hemodilution
antigen-presenting cell
activated partial thromboplastin
time
additive solution
allele-specific oligonucleotide
antithymocyte globulin
adult T-cell lymphoma/leukemia
adenosine triphosphate
burst-forming unit
benzyl-penicilloyl
bovine serum albumin or body
surface area
biological safety cabinet
blood urea nitrogen
crossmatch-to-transfusion
cold antibody (agglutinin) disease
College of American Pathologists
cold antibody (agglutinin) syndrome
Center for Biologics Evaluation and
Research
counterflow centrifugal elutriation

CCI
CD
CDC

corrected count increment
clusters of differentiation
Centers for Disease Control and
Prevention
CFR
Code of Federal Regulations
CFU
colony-forming unit
CGD
chronic granulomatous disease
cGMP
current good manufacturing
practice
cGy
centiGray
CHD
cold hemagglutinin disease
CI
continuous improvement or
confidence interval
CIDP
chronic inflammatory demyelinating
polyneuropathy
CJD
Creutzfeldt-Jakob disease
CLIA
Clinical Laboratory Improvement
Amendments
CLSI
Clinical and Laboratory Standards
Institute
CML
cell-mediated lympholysis
CMS
Centers for Medicare and Medicaid
Services
CMV
cytomegalovirus
CNS
central nervous system
CPD
citrate-phosphate-dextrose
CPDA-1 citrate-phosphate-dextrose-adenine-1
CR
complement receptor
CREG
cross-reactive group
CRYO
cryoprecipitated AHF
CUE
confidential unit exclusion
DAF
decay-accelerating factor
DAT
direct antiglobulin test
DDAVP 1-deamino-8-d-arginine
vasopressin
DDR
donor deferral registry
DHTR
delayed hemolytic transfusion
reaction
DIC
disseminated intravascular
coagulation
DMSO dimethylsulfoxide
DNA
deoxyribonucleic acid
2,3-DPG 2,3-diphosphoglycerate
DRG
diagnosis-related group

DTT
EACA
EBAA
EBV
ECMO
EDTA
EIA
ELAT
ELBW
ELISA
EPO
ESR
FACT
FDA
FFP
FMH
FNHTR
FTA-ABS
5-FU
G-CSF
GalNAc
GM-CSF
GMP
Gp
GPA
GPB
GPC
GPD
GVHD
Gy
HAM
HAV
HAZMAT
Hb
HBc
HBIG
HBsAg

dithiothreitol
epsilon aminocaproic acid
Eye Bank Association of America
Epstein-Barr virus
extracorporeal membrane
oxygenation
ethylenediaminetetraacetic acid
enzyme immunoassay
enzyme-linked antiglobulin test
extremely low birthweight
enzyme-linked immunosorbent
assay
erythropoietin
erythrocyte sedimentation rate
Foundation for the Accreditation of
Cellular Therapy
Food and Drug Administration
Fresh Frozen Plasma
fetomaternal hemorrhage
febrile nonhemolytic transfusion
reaction
fluorescent treponemal antibody
absorption test
5-fluorouracil
granulocyte colony-stimulating
factors
N-acetylgalactosamine
granulocyte macrophage
colony-stimulating factors
good manufacturing practice
glycoprotein
glycophorin A
glycophorin B
glycophorin C
glycophorin D
graft-vs-host disease
Gray
HTLV-associated myelopathy
hepatitis A virus
hazardous material
hemoglobin
hepatitis B core antigen
hepatitis B immunoglobulin
hepatitis B surface antigen

HBV
Hct
HCV
HDFN
HDV
HES
HEV
HIV
HPC
HTLV-I
HTR
HUS
IAT
Ig
IGIV
IHA
IL-1α
IL-1ß
IL-2
IPT
IS
ISBT
ISCT
ITP
IUT
IVT
JCAHO
L/S
LDH
LDL
LISS
LT-CIC
MAC
2-ME
MF
MHC
MLC

hepatitis B virus
hematocrit
hepatitis C virus
hemolytic disease of the fetus and
newborn
hepatitis D virus
hydroxyethyl starch
hepatitis E virus
human immunodeficiency virus
hematopoietic progenitor cell
human T-cell lymphotropic virus
type I
hemolytic transfusion reaction
hemolytic uremic syndrome
indirect antiglobulin test
immunoglobulin
Immunoglobulin Intravenous
immune hemolytic anemia
interleukin 1 alpha
interleukin 1 beta
interleukin 2
intraperitoneal transfusion
immediate spin
International Society of Blood
Transfusion
International Society for Cellular
Therapy
idiopathic thrombocytopenic
purpura
intrauterine transfusion
intravascular transfusion
Joint Commission on Accreditation
of Healthcare Organizations
lecithin to sphingomyelin
lactate dehydrogenase
low-density lipoproteins
low ionic strength saline
long-term culture-initiating cells
membrane attack complex
2-mercaptoethanol
mixed field
major histocompatibility complex
mixed lymphocyte (leukocyte)
culture
(cont’d)

MLR

mixed lymphocyte (leukocyte)
reaction
MoAb
monoclonal antibody
mRNA messenger ribonucleic acid
MSBOS maximum surgical blood order
schedule
MSDS material safety data sheets
NAIT
neonatal alloimmune
thrombocytopenia
NAT
nucleic acid testing
NIH
National Institutes of Health
NK
natural killer
NMDP National Marrow Donor Program
NRC
Nuclear Regulatory Commission
NT
not tested
OSHA
Occupational Safety and Health
Administration
p
probability
PAD
preoperative autologous (blood)
donation
PBPC
peripheral blood progenitor cell
PBS
phosphate-buffered saline
PCH
paroxysmal cold hemoglobinuria
PCR
polymerase chain reaction
PEG
polyethylene glycol
PHA
phytohemagglutinin
PI
paternity index
PPE
personal protective equipment
PPF
plasma protein fraction
PPTA
Plasma Protein Therapeutics
Association
PRA
panel reactive antibody
PT
prothrombin time or proficiency test
PTP
posttransfusion purpura
PUBS
percutaneous umbilical blood
sampling
PVC
polyvinyl chloride
QA
quality assessment or quality
assurance
QC
quality control
QSE
quality system essential

RBCs
RCA
RES
RFLP
Rh
RhIG
RIBA
RNA
RPGN
RPR
RR
RT
SBO
SCF
SGP
SOP
SPA
SSO
STS
TA
TCR
TNF-α
TPE
TRALI
tRNA
TTP
UNOS
VLBW
vWD
vWF
WAIHA
WB
XM

Red Blood Cells (blood donor unit)
regulators of complement activation
reticuloendothelial system
restriction fragment length
polymorphism
Rhesus factor
Rh Immune Globulin
recombinant immunoblot assay
ribonucleic acid
rapidly progressive
glomerulonephritis
rapid plasma reagin (serologic test
for syphilis)
repeatedly reactive or relative risk
room temperature or reverse
transcriptase
standard blood order
stem cell factor
sialoglycoprotein
standard operating procedure
staphylococcal protein A
sequence-specific oligonucleotide
serologic test for syphilis
transfusion-associated
T-cell receptor
tumor necrosis factor alpha
therapeutic plasma exchange
transfusion-related acute lung
injury
transfer ribonucleic acid
thrombotic thrombocytopenic
purpura
United Network for Organ Sharing
very low birthweight
von Willebrand disease
von Willebrand factor
warm autoimmune hemolytic
anemia
Whole Blood or Western blot
crossmatch

Source Exif Data:

File Type                       : PDF
File Type Extension             : pdf
MIME Type                       : application/pdf
PDF Version                     : 1.4
Linearized                      : No
Modify Date                     : 2008:05:17 15:48:17+02:00
Create Date                     : 2002:06:26 10:53:24-04:00
Page Count                      : 931
Page Mode                       : UseOutlines
Mod Date                        : 2008:05:17 15:48:17+02:00
Creation Date                   : 2002:06:26 10:53:24-04:00
Metadata Date                   : 2008:05:17 15:48:17+02:00
Title                           : TM12 - Introduction/Index
Page Layout                     : OneColumn
Has XFA                         : No

EXIF Metadata provided by EXIF.tools

AABB Technical Manual 15th Ed. 2005

AABB%20Technical%20Manual%2015TH

Navigation menu

Versions of this User Manual:

Views

Navigation