AABB Technical Manual 15th Ed. 2005

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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

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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
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Efforts are made to have publications of the AABB consistent in regard to acceptable
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The publication of this book does not constitute an endorsement by the AABB of any
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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 . . . . . . . . . . . . . . . .

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Copyright © 2005 by the AABB. All rights reserved.

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

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89
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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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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117
118
126
130
133
135

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

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139
139
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158

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

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163
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174
174

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

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184

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Contents

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

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xiii

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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 .

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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

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225
232
236
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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 .

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243
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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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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276
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283
286

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

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

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

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335
335
337
340
343
345
346
355

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357
358
358
360

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

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361
361
377
380

16.

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

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

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

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

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453
454
458
469
472
477
479

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

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

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

.
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.

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

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557
557
558
562
562
568
572
572
573
574
577

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

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

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

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

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667
667
675
682
683
686
689
690
695
695

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xviii

AABB Technical Manual

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

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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 . . . . . . .

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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

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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
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755
756
760
761

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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

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765
766
767
768
769

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772
772
773
774
775

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775
776
778
779

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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.

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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.

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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

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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

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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

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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.

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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

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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-

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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

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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

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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

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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

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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

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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)

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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
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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
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5-2).

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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

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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)

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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

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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-

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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)

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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-

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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

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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-

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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

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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

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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.

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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.

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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

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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

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68.

using DALI apheresis. J Clin Apheresis 2002;
17:161-9.
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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

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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

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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

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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

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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

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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

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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

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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

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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.

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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)

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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,

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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)

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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-

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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

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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
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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

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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.

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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.

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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
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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

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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.

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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.

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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.

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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

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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-

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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

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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.

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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

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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-

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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-

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38.

39.

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41.

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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.

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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.
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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

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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

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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,

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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

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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

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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;

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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).

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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-

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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

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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.

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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

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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 )

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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

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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

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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-

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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-

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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.

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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

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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
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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-

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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

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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

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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.

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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.

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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

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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,

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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

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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.)

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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

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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

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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

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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

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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

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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.

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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

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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.

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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.

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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.

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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.

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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

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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

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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-

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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-

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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 frequenc