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

TransfusionServicesonCD-ROM

Transfusion Therapy: Clinical Principles and Practice, 2nd Edition

Edited by Paul D. Mintz, MD

Transfusion Medicine Self-Assessment and Review

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and Darrell J. Triulzi, MD

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Edited by Jerry Gottschall, MD

Practical Guide to Transfusion Medicine

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Transfusion Medicine Interactive: A Case Study Approach CD-ROM

By Marian Petrides, MD; Roby Rogers, MD; and Nora Ratcliffe, MD

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AABB ISBN No. 1-56395-196-7

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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

MichaelC.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,

Linda Hahn,

MT(ASCP)SBB, MPM

Heather Hume, MD

Mark A. Janzen, PhD

SusanT.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

ArellS.Shapiro,MD

R. Sue Shirey, MS,

MT(ASCP)SBB

Bruce Spiess, MD, FAHA

JerryE.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 com-

mittees who reviewed

manuscriptsaspartof

committee resource

charges

The staff of the Armed

Services Blood Program

Office

The staff of the US Food

and Drug Administra-

tion, Center for

Biologics Evaluation

and Research

The staff of the Transplan-

tation and Transfusion

Service, McClendon

Clinical Laboratories,

UNC Hospitals

Special thanks are due to Laurie Munk, Janet McGrath, Nina Hutchinson, Jay Penning-

ton, 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

The 15th edition of the AABB Tech-

nical Manual is the first in the

second half century of this publica-

tion. The original Technical Manual (then

called Technical Methods and Procedures)

was published in 1953 and the 14th edi-

tion 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 pro-

fessionals (technologists, nurses, and phy-

sicians) around the world. Selected editions

or excerpts have been translated into

French, Hungarian, Italian, Japanese, Span-

ish, 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 activi-

ties.

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 infor-

mation to allow both students and experi-

enced individuals to rapidly familiarize

themselves with the rationale and scientific

basis of the AABB standards and current

standards of practice. As in previous edi-

tions, 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 individu-

als consult the text, or at a minimum, to di-

rect 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 ex-

perts in specific subject areas, AABB com-

mittees, and regulatory bodies such as the

Food and Drug Administration). As such,

this text is relatively unique, and represents

a major effort on the part of the AABB to

provide an authoritative and balanced

reference source.

As in previous recent editions, the con-

tent is necessarily limited in order to retain

thesizeoftheTechnical Manual to that of a

textbook that can be easily handled. Never-

theless, readers will find extensive new and

updated information, including expanded

coverage of quality approaches, apheresis

indications, cellular nomenclature, molec-

ular 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 permissi-

ble way in which requirements of Stan-

dards can be met. Other methods, not in-

cluded, 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 ef-

forts of both the Program Unit and the ex-

tensive number of outside reviewers, errors

mayremaininthetext.Aswithprevious

editions, the Program Unit welcomes sug-

gestions, 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 Lin-

den, and Sue Roseff—who have provided

countless invaluable hours in the prepara-

tion of this edition. Finally I would like to

thank Laurie Munk, AABB Publications Di-

rector, 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 Techni-

cal 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 de-

cades to come.

Mark E. Brecher, MD

Chief Editor

Chapel Hill, NC

x AABB Technical Manual

Contents 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

DetermineAdequateSampleSizeandLevelofConfidencefor

Validation of Pass/Fail Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Appendix 1-4. Assessment Examples: Blood Utilization. . . . . . . . . . . . . . . . . 36

2. Facilities and Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Facilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Safety Program. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Fire Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Electrical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Biosafety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Chemical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Radiation Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Shipping Hazardous Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

Waste Management. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Disaster Planning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .70

Appendix 2-1. Safety Regulations and Recommendations Applicable to

Health-Care Settings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

Appendix 2-2. General Guidelines for Safe Work Practices, Personal Protective

Equipment, and Engineering Controls . . . . . . . . . . . . . . . . . . . . . . . . . 73

Appendix 2-3. Biosafety Level 2 Precautions . . . . . . . . . . . . . . . . . . . . . . . 77

Appendix 2-4. Sample Hazardous Chemical Data Sheet. . . . . . . . . . . . . . . . . 78

Appendix 2-5. Sample List of Hazardous Chemicals in the Blood Bank . . . . . . . . 80

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

These Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

Appendix 2-7. Incidental Spill Response . . . . . . . . . . . . . . . . . . . . . . . . . . 84

Appendix 2-8. Managing Hazardous Chemical Spills . . . . . . . . . . . . . . . . . . 87

3. Blood Utilization Management. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

Minimum and Ideal Inventory Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

Determining Inventory Levels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

Factors that Affect Outdating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

Improving Transfusion Service Blood Ordering Practices . . . . . . . . . . . . . . . . 91

Special Product Concerns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

Blood Donation and Collection

4. Allogeneic Donor Selection and Blood Collection . . . . . . . . . . . . . . . . . . . 97

Blood Donation Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Collection of Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Appendix 4-1. Full-Length Donor History Questionnaire . . . . . . . . . . . . . . . 110

Appendix 4-2. Medication Deferral List. . . . . . . . . . . . . . . . . . . . . . . . . . 113

Appendix 4-3. Blood Donor Education Materials . . . . . . . . . . . . . . . . . . . . 114

Appendix 4-4. Some Drugs Commonly Accepted in Blood Donors . . . . . . . . . 115

5. Autologous Blood Donation and Transfusion . . . . . . . . . . . . . . . . . . . . . 117

Preoperative Autologous Blood Collection . . . . . . . . . . . . . . . . . . . . . . . . 118

Acute Normovolemic Hemodilution . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

Intraoperative Blood Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

Postoperative Blood Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

6. Apheresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Separation Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Component Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

Therapeutic Apheresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

7. Blood Component Testing and Labeling . . . . . . . . . . . . . . . . . . . . . . . . 163

Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

Labeling, Records, and Quarantine . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

8. Collection, Preparation, Storage, and Distribution of Components from

Whole Blood Donations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

Blood Component Descriptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178

Prestorage Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

Storage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184

xii AABB Technical Manual

Inspection, Shipping, Disposition, and Issue . . . . . . . . . . . . . . . . . . . . . . 194

Blood Component Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199

Appendix 8-1. Component Quality Control . . . . . . . . . . . . . . . . . . . . . . . 202

Immunologic and Genetic Principles

9. Molecular Biology in Transfusion Medicine . . . . . . . . . . . . . . . . . . . . . . 203

From DNA to mRNA to Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203

Genetic Mechanisms that Create Polymorphism . . . . . . . . . . . . . . . . . . . . 207

Genetic Variability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

Molecular Techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220

Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

Appendix 9-1. Molecular Techniques in Transfusion Medicine . . . . . . . . . . . . 222

10. Blood Group Genetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

Basic Principles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

Genetics and Heredity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

Patterns of Inheritance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232

Population Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236

Blood Group Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239

Appendix 10-1. Glossary of Terms in Blood Group Genetics . . . . . . . . . . . . . 241

11. Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243

Immune Response. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243

Organs of the Immune System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

Cells of the Immune System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

Soluble Components of the Immune Response . . . . . . . . . . . . . . . . . . . . . 256

Immunology Relating to Transfusion Medicine . . . . . . . . . . . . . . . . . . . . . 263

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

Appendix 11-1. Definitions of Some Essential Terms in Immunology . . . . . . . . 269

12. Red Cell Antigen-Antibody Reactions and Their Detection . . . . . . . . . . . . 271

Factors Affecting Red Cell Agglutination . . . . . . . . . . . . . . . . . . . . . . . . . 272

Enhancement of Antibody Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . 276

The Antiglobulin Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277

Other Methods to Detect Antigen-Antibody Reactions. . . . . . . . . . . . . . . . . 283

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286

Contents xiii

Blood Groups

13. ABO, H, and Lewis Blood Groups and Structurally Related Antigens . . . . . . 289

The ABO System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289

The H System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303

The Lewis System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304

The I/i Antigens and Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306

The P Blood Group and Related Antigens . . . . . . . . . . . . . . . . . . . . . . . . 308

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311

Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312

14. The Rh System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315

The D Antigen and Its Historical Context. . . . . . . . . . . . . . . . . . . . . . . . . 315

Genetic and Biochemical Considerations . . . . . . . . . . . . . . . . . . . . . . . . 316

Rh Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318

Serologic Testing for Rh Antigen Expression . . . . . . . . . . . . . . . . . . . . . . . 319

Weak D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322

Other Rh Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324

Rhnull Syndrome and Other Deletion Types . . . . . . . . . . . . . . . . . . . . . . . . 325

Rh Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327

Rh Typing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332

Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

15. Other Blood Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335

Distribution of Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335

MNS System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337

Kell System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340

Duffy System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343

Kidd System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345

Other Blood Group Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346

Blood Group Collections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355

High-Incidence Red Cell Antigens Not Assigned to a Blood Group

System or Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356

Low-Incidence Red Cell Antigens Not Assigned to a Blood Group

System or Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357

Antibodies to Low-Incidence Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . 358

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358

Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360

16. Platelet and Granulocyte Antigens and Antibodies . . . . . . . . . . . . . . . . 361

Platelet Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361

Granulocyte Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380

xiv AABB Technical Manual

17. The HLA System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385

Genetics of the Major Histocompatibility Complex . . . . . . . . . . . . . . . . . . 386

Biochemistry, Tissue Distribution, and Structure . . . . . . . . . . . . . . . . . . . . 390

Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391

Biologic Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393

Detection of HLA Antigens and Alleles . . . . . . . . . . . . . . . . . . . . . . . . . . 394

The HLA System and Transfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397

HLA Testing and Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400

Parentage and Other Forensic Testing . . . . . . . . . . . . . . . . . . . . . . . . . . 402

HLA and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404

Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405

Serologic Principles and Transfusion Medicine

18. Pretransfusion Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407

Transfusion Requests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407

Blood Sample. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409

Serologic Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410

Crossmatching Tests. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413

Interpretation of Antibody Screening and Crossmatch Results . . . . . . . . . . . . 415

Labeling and Release of Crossmatched Blood at the Time of Issue. . . . . . . . . . 416

Selection of Units . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 420

Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 420

19. Initial Detection and Identification of Alloantibodies to Red Cell Antigens . . . . 423

Significance of Alloantibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423

General Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424

Basic Antibody Identification Techniques . . . . . . . . . . . . . . . . . . . . . . . . 427

Complex Antibody Problems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431

Selecting Blood for Transfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439

Selected Serologic Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450

20. The Positive Direct Antiglobulin Test and Immune-Mediated Red

Cell Destruction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453

The Direct Antiglobulin Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454

Immune-Mediated Hemolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458

Serologic Problems with Autoantibodies . . . . . . . . . . . . . . . . . . . . . . . . . 469

Drug-Induced Immune Hemolytic Anemia . . . . . . . . . . . . . . . . . . . . . . . 472

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477

Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479

Contents xv

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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483

Red Blood Cell Transfusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483

Platelet Transfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488

Granulocyte Transfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492

Special Cellular Blood Components. . . . . . . . . . . . . . . . . . . . . . . . . . . . 492

Replacement of Coagulation Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . 493

Cryoprecipitated AHF Transfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500

Special Transfusion Situations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508

Pharmacologic Alternatives to Transfusion . . . . . . . . . . . . . . . . . . . . . . . 512

Oversight of Transfusion Practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515

22. Administration of Blood and Components . . . . . . . . . . . . . . . . . . . . . . 521

Pre-Issue Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521

Blood Issue and Transportation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 524

Pre-Administration Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525

Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 527

Post-Administration Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531

Quality Assurance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 532

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 532

23. Perinatal Issues in Transfusion Practice . . . . . . . . . . . . . . . . . . . . . . . . 535

Hemolytic Disease of the Fetus and Newborn . . . . . . . . . . . . . . . . . . . . . 535

Neonatal Immune Thrombocytopenia . . . . . . . . . . . . . . . . . . . . . . . . . . 551

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 554

24. Neonatal and Pediatric Transfusion Practice . . . . . . . . . . . . . . . . . . . . . 557

Fetal and Neonatal Erythropoiesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557

Unique Aspects of Neonatal Physiology . . . . . . . . . . . . . . . . . . . . . . . . . 558

Cytomegalovirus Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562

Red Cell Transfusions in Infants Less than 4 Months of Age . . . . . . . . . . . . . . 562

Transfusion of Other Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568

Neonatal Polycythemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572

Extracorporeal Membrane Oxygenation . . . . . . . . . . . . . . . . . . . . . . . . . 572

Leukocyte Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573

Transfusion Practices in Older Infants and Children . . . . . . . . . . . . . . . . . . 574

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577

xvi AABB Technical Manual

25. Cell Therapy and Cellular Product Transplantation . . . . . . . . . . . . . . . . 581

Diseases Treated with Hematopoietic Cell Transplantation . . . . . . . . . . . . . . 583

Sources of Hematopoietic Progenitor Cells . . . . . . . . . . . . . . . . . . . . . . . 583

Donor Eligibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589

Collection of Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591

Processing of Hematopoietic Progenitor Cells. . . . . . . . . . . . . . . . . . . . . . 596

Freezing and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604

Transportation and Shipping. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606

Thawing and Infusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607

Evaluation and Quality Control of Hematopoietic Products. . . . . . . . . . . . . . 607

Regulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608

Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609

26. Tissue and Organ Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . 617

Transplant-Transmitted Diseases and Preventive Measures. . . . . . . . . . . . . . 617

Bone Banking. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623

Skin Banking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625

Heart Valves. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625

Records of Stored Tissue Allografts . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626

FDA Regulation of Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626

The Importance of ABO Compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . 627

The Role of Transfusion in Kidney Transplants . . . . . . . . . . . . . . . . . . . . . 627

Liver Transplants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 627

Other Organ Transplants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 629

Transfusion Service Support for Organ Transplantation . . . . . . . . . . . . . . . . 629

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 630

27. Noninfectious Complications of Blood Transfusion . . . . . . . . . . . . . . . . 633

Manifestations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633

Acute Transfusion Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 639

Evaluation of a Suspected Acute Transfusion Reaction. . . . . . . . . . . . . . . . . 652

Delayed Consequences of Transfusion . . . . . . . . . . . . . . . . . . . . . . . . . . 656

Records of Transfusion Complications . . . . . . . . . . . . . . . . . . . . . . . . . . 660

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 661

28. Transfusion-Transmitted Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . 667

Hepatitis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667

Human Immunodeficiency Viruses. . . . . . . . . . . . . . . . . . . . . . . . . . . . 675

Human T-Cell Lymphotropic Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . 682

West Nile Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 683

Herpesviruses and Parvovirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 686

Transmissible Spongiform Encephalopathies . . . . . . . . . . . . . . . . . . . . . . 689

Bacterial Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 690

Syphilis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 695

Tick-Borne Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 695

Contents xvii

Other Nonviral Infectious Complications of Blood Transfusion . . . . . . . . . . . 697

Reducing the Risk of Infectious Disease Transmission . . . . . . . . . . . . . . . . . 699

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 703

Suggested Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 711

Methods

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

1. General Laboratory Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 715

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 715

Method 1.1. Transportation and Shipment of Dangerous Goods . . . . . . . . . . . 716

Method 1.2. Treatment of Incompletely Clotted Specimens . . . . . . . . . . . . . . . 722

Method 1.3. Solution Preparation—Instructions . . . . . . . . . . . . . . . . . . . . 723

Method 1.4. Serum Dilution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 725

Method 1.5. Dilution of % Solutions. . . . . . . . . . . . . . . . . . . . . . . . . . . . 726

Method 1.6. Preparation of a 3% Red Cell Suspension . . . . . . . . . . . . . . . . . 727

Method 1.7. Preparation and Use of Phosphate Buffer . . . . . . . . . . . . . . . . . 728

Method 1.8. Reading and Grading Tube Agglutination . . . . . . . . . . . . . . . . . 728

2. Red Cell Typing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 731

Method 2.1. Slide Test for Determination of ABO Type of Red Cells . . . . . . . . . 731

Method 2.2. Tube Tests for Determination of ABO Group of Red Cells and

Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 732

Method 2.3. Microplate Test for Determination of ABO Group of Red Cells

and Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 733

Method 2.4. Confirmation of Weak A or B Subgroup by Adsorption and Elution. . 735

Method 2.5. Saliva Testing for A, B, H, Lea,andLe

b. . . . . . . . . . . . . . . . . . . 736

Method 2.6. Slide Test for Determination of Rh Type . . . . . . . . . . . . . . . . . 739

Method 2.7. Tube Test for Determination of Rh Type . . . . . . . . . . . . . . . . . 740

Method 2.8. Microplate Test for Determination of Rh Type . . . . . . . . . . . . . . 741

Method 2.9. Test for Weak D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 741

Method 2.10. Preparation and Use of Lectins . . . . . . . . . . . . . . . . . . . . . . 743

Method 2.11. Use of Sulfhydryl Reagents to Disperse Autoagglutination . . . . . . 744

Method 2.12. Gentle Heat Elution for Testing Red Cells with a Positive DAT . . . . 745

Method 2.13. Dissociation of IgG by Chloroquine for Red Cell Antigen

Testing of Red Cells with a Positive DAT . . . . . . . . . . . . . . . . . . . . . . . . 746

Method 2.14. Acid Glycine/EDTA Method to Remove Antibodies from

Red Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747

Method 2.15. Separation of Transfused from Autologous Red Cells by

Simple Centrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748

Method 2.16. Separation of Transfused Red Cells from Autologous

Red Cells in Patients with Hemoglobin S Disease . . . . . . . . . . . . . . . . . . 749

xviii AABB Technical Manual

3. Antibody Detection, Antibody Identification, and Serologic Compatibility

Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751

Method 3.1. Immediate-Spin Compatibility Testing to Demonstrate

ABO Incompatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751

Method 3.2. Indirect Antiglobulin Test (IAT) for the Detection of Antibodies

to Red Cell Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 752

Method 3.3. Prewarming Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . 754

Method 3.4. Saline Replacement to Demonstrate Alloantibody in the

Presence of Rouleaux . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 755

Method 3.5. Enzyme Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 756

Method 3.6. Direct Antiglobulin Test (DAT) . . . . . . . . . . . . . . . . . . . . . . . 760

Method 3.7. Antibody Titration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 761

Method 3.8. Use of Sulfhydryl Reagents to Distinguish IgM from IgG

Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 764

Method 3.9. Plasma Inhibition to Distinguish Anti-Ch and -Rg from

Other Antibodies with HTLA Characteristics . . . . . . . . . . . . . . . . . . . . . 765

Method 3.10. Dithiothreitol (DTT) Treatment of Red Cells . . . . . . . . . . . . . . 766

Method 3.11. Urine Neutralization of Anti-Sda. . . . . . . . . . . . . . . . . . . . . . 767

Method 3.12. Adsorption Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . 768

Method 3.13. Using the American Rare Donor Program . . . . . . . . . . . . . . . . 769

4. Investigation of a Positive Direct Antiglobulin Test . . . . . . . . . . . . . . . . . 771

Elution Techniques

Method 4.1. Cold-Acid Elution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 772

Method 4.2. Glycine-HCl/EDTA Elution . . . . . . . . . . . . . . . . . . . . . . . . . 772

Method 4.3. Heat Elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 773

Method 4.4. Lui Freeze-Thaw Elution. . . . . . . . . . . . . . . . . . . . . . . . . . . 774

Method 4.5. Methylene Chloride Elution. . . . . . . . . . . . . . . . . . . . . . . . . 775

Immune Hemolytic Anemia Serum/Plasma Methods

Method 4.6. Cold Autoadsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 775

Method 4.7. Determining the Specificity of Cold-Reactive Autoagglutinins. . . . . 776

Method 4.8. Cold Agglutinin Titer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 778

Method 4.9. Autologous Adsorption of Warm-Reactive Autoantibodies . . . . . . . 779

Method 4.10. Differential Warm Adsorption Using Enzyme- or ZZAP-Treated

Allogeneic Red Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781

Method 4.11. One-Cell Sample Enzyme or ZZAP Allogeneic Adsorption . . . . . . 782

Method 4.12. Polyethylene Glycol Adsorption. . . . . . . . . . . . . . . . . . . . . . 783

Method 4.13. The Donath-Landsteiner Test . . . . . . . . . . . . . . . . . . . . . . . 784

Method 4.14. Detection of Antibodies to Penicillin or Cephalosporins by

Testing Drug-Treated Red Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786

Method 4.15. Demonstration of Immune-Complex Formation Involving Drugs. . 788

Method 4.16. Ex-Vivo Demonstration of Drug/Anti-Drug Complexes . . . . . . . . 789

Contents xix

5. Hemolytic Disease of the Fetus and Newborn . . . . . . . . . . . . . . . . . . . . 793

Method 5.1. Indicator Cell Rosette Test for Fetomaternal Hemorrhage . . . . . . . 793

Method 5.2. Acid-Elution Stain (Modified Kleihauer-Betke). . . . . . . . . . . . . . 794

Method 5.3. Antibody Titration Studies to Assist in Early Detection of

Hemolytic Disease of the Fetus and Newborn . . . . . . . . . . . . . . . . . . . . 796

6. Blood Collection, Storage, and Component Preparation. . . . . . . . . . . . . . 799

Method 6.1. Copper Sulfate Method for Screening Donors for Anemia . . . . . . . 799

Method 6.2. Arm Preparation for Blood Collection . . . . . . . . . . . . . . . . . . . 800

Method 6.3. Phlebotomy and Collection of Samples for Processing and

Compatibility Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 801

Method 6.4. Preparation of Red Blood Cells . . . . . . . . . . . . . . . . . . . . . . . 804

Method 6.5. Preparation of Prestorage Red Blood Cells Leukocytes

Reduced . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 805

Method 6.6. Rejuvenation of Red Blood Cells . . . . . . . . . . . . . . . . . . . . . . 806

Method 6.7. Red Cell Cryopreservation Using High-Concentration

Glycerol—Meryman Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 807

Method 6.8. Red Cell Cryopreservation Using High-Concentration

Glycerol—Valeri Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 810

Method 6.9. Checking the Adequacy of Deglycerolization of Red Blood Cells . . . 812

Method 6.10. Preparation of Fresh Frozen Plasma from Whole Blood . . . . . . . . 813

Method 6.11. Preparation of Cryoprecipitated AHF from Whole Blood . . . . . . . 814

Method 6.12. Thawing and Pooling Cryoprecipitated AHF . . . . . . . . . . . . . . 815

Method 6.13. Preparation of Platelets from Whole Blood . . . . . . . . . . . . . . . 815

Method 6.14. Preparation of Prestorage Platelets Leukocytes Reduced

from Whole Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 817

Method 6.15. Removing Plasma from Platelet Concentrates (Volume

Reduction) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 817

7. Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 819

Method 7.1. Quality Control for Copper Sulfate Solution . . . . . . . . . . . . . . . 819

Method 7.2. Standardization and Calibration of Thermometers . . . . . . . . . . . 821

Method 7.3. Testing Blood Storage Equipment Alarms . . . . . . . . . . . . . . . . 823

Method 7.4. Functional Calibration of Centrifuges for Platelet Separation . . . . . 826

Method 7.5. Functional Calibration of a Serologic Centrifuge . . . . . . . . . . . . 828

Method 7.6. Performance Testing of Automatic Cell Washers . . . . . . . . . . . . 830

Method 7.7. Monitoring Cell Counts of Apheresis Components . . . . . . . . . . . 832

Method 7.8. Manual Method for Counting Residual White Cells in

Leukocyte-Reduced Blood and Components . . . . . . . . . . . . . . . . . . . . 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

xx AABB Technical Manual

Appendix 4. Coagulation Factor Values in Platelet Concentrates . . . . . . . . . . . 838

Appendix 5. Approximate Normal Values for Red Cell, Plasma, and

Blood Volumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839

Appendix 6. Blood Group Antigens Assigned to Systems. . . . . . . . . . . . . . . . 840

Appendix 7. Examples of Gene, Antigen, and Phenotype Terms . . . . . . . . . . . 844

Appendix 8. Examples of Correct and Incorrect Terminology . . . . . . . . . . . . . 844

Appendix 9. Distribution of ABO/Rh Phenotypes by Race or Ethnicity . . . . . . . 845

Appendix 10. Suggested Quality Control Performance Intervals . . . . . . . . . . . 846

Appendix 11. Directory of Organizations . . . . . . . . . . . . . . . . . . . . . . . . . 848

Appendix 12. Resources for Safety Information . . . . . . . . . . . . . . . . . . . . . 850

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

Contents xxi

Chapter 1: Quality Systems

Chapter 1

Quality Systems

APRIMARYGOALOFbloodcenters

and transfusion services is to pro-

mote high standards of quality in

all aspects of production, patient care, and

service. This commitment to quality is re-

flected in standards of practice set forth by

the AABB.1(p1) A quality system includes the

organizational structure, responsibilities,

policies, processes, procedures, and re-

sources established by the executive man-

agement to achieve quality.1(p1) A glossary of

quality terms used in this chapter is in-

cluded in Appendix 1-1.

The establishment of a formal quality as-

surance program is required by regulation

under the Centers for Medicare and Medi-

caid Services (CMS)2Clinical Laboratory

Improvement Amendments (CLIA) and the

Food and Drug Administration (FDA)3-5 cur-

rent good manufacturing practice (cGMP).

The FDA regulations in 21 CFR 211.22 re-

quire 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 pro-

cesses 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,1Joint Commission on Accreditation

of Healthcare Organizations (JCAHO),6Col-

lege of American Pathologists (CAP),7and

the Clinical and Laboratory Standards Insti-

tute (formerly NCCLS),8have also estab-

lished requirements and guidelines to ad-

dress quality issues. The International

Organization for Standardization (ISO)

quality management standards (ISO 9001)

are generic to any industry and describe

the key elements of a quality system.9In

addition, the Health Care Criteria for Per-

formance 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

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 compati-

ble with ISO 9001 standards and the FDA

Guideline for Quality Assurance in Blood

Establishments.5Table 1-1 shows a compar-

ison 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 pro-

gress. 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 re-

agent QC, clerical checks, visual inspec-

tions, and measurements such as temper-

ature 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 anal-

ysis of operational performance data to de-

termine if the overall process is in a state of

control and to detect shifts or trends that

require attention. Quality assurance pro-

vides information to process managers re-

garding levels of performance that can be

used in setting priorities for process im-

provement. Examples in blood banking in-

clude record reviews, monitoring of quality

indicators, and internal assessments.

Quality management considers interre-

lated processes in the context of the organi-

zation and its relations with customers and

suppliers. It addresses the leadership role of

executive management in creating a com-

mitment to quality throughout the organi-

zation, the understanding of suppliers and

customers as partners in quality, the man-

agement of human and other resources,

and quality planning. The quality systems

approach described in this chapter encom-

passes all of these activities. It ensures the

application of quality principles through-

out the organization and reflects the chang-

ing 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 pro-

cesses 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 re-

quirements, to develop product and pro-

cess specifications to meet those require-

ments, and to design the process. During

the planning phase, the facility must per-

form the following steps:

1. Establish quality goals for the pro-

ject.

2. Identify the customers.

3. Determine customer needs and ex-

pectations.

4. Develop product and service specifi-

cations to meet customer, opera-

tional, regulatory, and accreditation

requirements.

2 AABB Technical Manual

Chapter 1: Quality Systems 3

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 General requirements

5.1 Management commitment

5.2 Customer focus

5.3 Quality policy

5.4 Planning

5.5 Responsibility, authority, and communication

5.6 Management review

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

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

5. Develop operational processes for

production and delivery, including

written procedures and resources re-

quirements.

6. Develop process controls and vali-

date the process in the operational

setting.

The results of the planning process are

referred to as design output.9

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, produc-

tion, 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 pro-

cess. An important goal in quality manage-

ment is to establish a set of controls that

ensure process and product quality but that

are not excessive. Controls that do not add

valueshouldbeeliminatedinordertocon

serve 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 eval-

uate process performance during the plan-

ning 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 at-

tain 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. Oppor-

tunities to improve may be related to defi-

ciencies in the initial planning process; un-

foreseen factors that are discovered upon

implementation; shifts in customer needs;

or changes in starting materials, environ-

mental factors, and other variables that af-

fect 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 in-

cludes 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 PROCESS OUTPUT

For example, a key process for donor cen-

ters is donor selection. The “input” in-

cludes 1) the individual who presents for

donation and 2) all of the resources re-

quired 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 individ-

ual 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 re-

sults in a deferred donor, the resources

(inputs) associated with that process are

wasted and contribute to the cost of qual-

ity. 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.

4 AABB Technical Manual

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 inven-

tory control are strategies for ensuring that

the inputs to a process meet specifications.

Personnel training and competency assess-

ment, equipment maintenance and con-

trol, management of documents and re-

cords, and implementation of appropriate

in-process controls provide assurance that

the process will operate as intended. End-

product testing and inspection, customer

feedback, and outcome measurement pro-

vide information to help evaluate the qual-

ity 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 pro-

cesses effectively, the facility must under-

stand 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 be-

havior that is not identified during the se-

lection process. The donated product may

test positive for one of the viral marker as-

says, triggering follow-up testing, look-back

investigations, and donor deferral and noti-

fication procedures. Components must be

quarantined and their discard documented.

Staff involved in collecting and processing

theproductareatriskofexposuretoinfec

tious 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 in-

clude not only product manufacture or ser-

vice creation, but also the delivery of a

product or service. Delivery generally in-

volves 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 ap-

propriate when there are differing expec-

tations 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 cus-

tomer 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 cus-

tomer expectations but does not compro-

mise quality. To do this, personnel must

have sufficient knowledge and under-

standing 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 over-

sight and support needed to keep it run-

ning smoothly and effectively. In service,

people are the focus; the underlying pro-

cess provides a foundation that enables

staff to deliver safe and effective services

that meet the needs of the customers in

almost any situation.

Chapter 1: Quality Systems 5

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 knowl-

edge of organizational behavior, as tech-

nology 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 effec-

tive 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 prac-

tical application of quality principles to the

blood bank and transfusion service envi-

ronment. These basic elements include:

■Organizational management

■Human resources

■Customer and supplier relations

■Equipment management

■Process management

■Documents and records

■Deviations and nonconforming prod-

ucts and services

■Monitoring and assessment

■Process improvement

■Work environment

Organizational Management

The facility should be organized in a man-

ner that promotes effective implementa-

tion and management of its quality system.

The structure of the organization must be

documented and the responsibilities for

the provision of blood, components, pro-

ducts, and services must be clearly de-

fined. These should include a description

of the relationships and avenues of com-

munication between organizational units

and those responsible for key quality

functions. Each facility may define its

structure in any format that suits its oper-

ations. Organizational trees or charts that

show the structure and relationships are

helpful.

The facility must define in writing the

authority and responsibilities of manage-

ment to establish and maintain the quality

system. These include oversight of opera-

tions 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. Man-

agement 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 di-

rectly to management. This person has the

responsibility to coordinate, monitor, and

facilitate quality system activities and has

the authority to recommend and initiate

corrective action when appropriate.5The

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 or-

ganization, the designated oversight person

mayworkinadepartment(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). Individ-

uals with dual quality and operational re-

sponsibilities should not provide quality

oversight for operational work they have

performed (21 CFR 211.194).

6 AABB Technical Manual

Quality oversight functions may include

the following5:

■Review and approval of standard op-

erating procedures (SOPs) and train-

ing 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 man-

ufacturing, distribution, or transfusion

of blood and components.

■Review of reports of adverse reactions,

deviations in the manufacturing pro-

cess, nonconforming products and ser-

vices, and customer complaints.

■Participation in decisions to deter-

mine blood and component suitabil-

ity for use, distribution, or recall.

■Review and approval of corrective

action plans.

■Surveillance of problems (eg, error

reports, inspection deficiencies, cus-

tomer complaints) and the effective-

ness of corrective actions implemented

to solve these problems.

■Use of information resources to

identify trends and potential prob-

lems before a situation worsens and

products or patients are affected.

■Preparation of periodic (as specified

by the organization) reports of qual-

ity issues, trends, findings, and cor-

rective 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 evalua-

tion of the facility’s quality activities as pos-

sible. Policies, processes, and procedures

must exist to define the roles and responsi-

bilities of all individuals in the development

and maintenance of these quality goals.

Quality system policies and processes

should be applicable across the entire facil-

ity. 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 sys-

tem must address all matters related to

compliance with federal, state, and local

regulations and accreditation standards ap-

plicable to the organization.

Human Resources

This element of the quality system is aim-

ed at management of personnel, includ-

ing selection, orientation, training, com-

petency assessment, and staffing.

Selection

Each blood bank, transfusion service, or

donor center must have a process to pro-

vide adequate numbers of qualified per-

sonnel to perform, verify, and manage all

activities within the facility.1(p3),3 Qualifica-

tion requirements are determined based

on job responsibilities. The selection pro-

cess should consider the applicant’s qual-

ifications for a particular position as

determined by education, training, exper-

ience, certifications, and/or licensure. For

laboratory testing staff, the standards for

personnel qualifications must be compat-

ible with the regulatory requirements es-

tablished under CLIA.2Job descriptions are

required for all personnel involved in pro-

cesses and procedures that affect the

quality of blood, components, tissues,

and services. Effective job descriptions

clearly define the qualifications, responsi-

bilities, and reporting relationships of the

position.

Chapter 1: Quality Systems 7

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 poli-

cies that address issues such as safety,

quality, computers, security, and confi-

dentiality. The job-related portion of the

orientation program covers the opera-

tional issues specific to the work area.

Training must be provided for each proce-

dure for which employees have responsi-

bility. The ultimate result of the orienta-

tion and training program is to deem new

employees competent to work independ-

ently in performing the duties and re-

sponsibilities defined in their job descrip-

tions. Time frames should be established

to accomplish this goal. Before the intro-

duction 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 docu-

mented and the facility trainer or desig-

nated 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.4It should provide staff

with an understanding of the regulatory ba-

sis for the facility’s policies and procedures

as well as train them in facility-specific ap-

plication of the cGMP requirements as de-

scribedintheirownwrittenoperatingpro

cedures. This training must be provided at

periodic intervals to ensure that staff re-

main familiar with regulatory require-

ments.

To ensure that skills are maintained, the

facility must have regularly scheduled com-

petence evaluations of all staff whose activ-

ities affect the quality of blood, compo-

nents, tissues, or services.2,6 Depending

upon the nature of the job duties, such as-

sessments may include: written evalua-

tions; direct observation of activities; review

of work records or reports, computer re-

cords, 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 meth-

ods. Written tests can be used effectively to

evaluate problem-solving skills and to rap-

idly cover many topics by asking one or

Suggested Reading

CDC Office of Biosafety. Radiation safety manual.

Atlanta, GA: Centers for Disease Control, 1992.

Disaster operations handbook: Coordinating the

nation’s blood supply during disasters and biolog-

ical events. Bethesda, MD: AABB, 2003.

Disaster plan development procedure manual. In:

Developing a disaster plan. Bethesda, MD: AABB,

1998.

Handbook of compressed gases. 3rd ed. Compressed

Gas Association. New York: Chapman and Hall, 1990.

Heinsohn PA, Jacobs RR, Concoby BA, eds. Biosafety

reference manual. 2nd ed. Fairfax, VA: American

Industrial Hygiene Association Biosafety Commit-

tee, 1995.

Liberman DF, ed. Biohazards management hand-

book. 2nd ed. New York: Marcel Dekker, Inc, 1995.

NIH guide to waste disposal. Bethesda, MD: Na-

tional Institutes of Health, 2003. [Available at http://

www.nih.gov/od/ors/ds/wasteguide.]

Prudent practices for handling hazardous chemi-

cals in laboratories. Washington, DC: National

Academy Press, 1981.

Risk management and safety procedure manual.

In: Developing a disaster plan. Bethesda, MD:

AABB, 1998.

Vesley D, Lauer JL. Decontamination, sterilization,

disinfection and antisepsis. In: Fleming DO, Rich-

ardson JH, Tulis JJ, Vesley D, eds. Laboratory

safety, principles and practices. 2nd ed. Washing-

ton, DC: American Society for Microbiology Press,

1995:219-37.

70 AABB Technical Manual

Chapter 2: Facilities and Safety 71

Appendix 2-1. Safety Regulations and Recommendations Applicable to Health-Care

Settings

Agency/Organization Reference Title

Federal Regulations and Recommendations

Nuclear Regulatory Com-

mission (NRC)

10 CFR 20 Standards for Protection Against

Radiation

Guide 8.29 Instruction Concerning Risks

from Occupational Radiation

Exposure

Department of Labor, Occu-

pational Safety and

Health Administration

(OSHA)

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 Haz-

ardous Chemicals in Labora-

tories

Department of Transporta-

tion (DOT)

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 Precau-

tions in Hospitals

Food and Drug Administra-

tion (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)

72 AABB Technical Manual

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 Identifica-

tion System (HMIS) Imple-

mentation Manual

International Air Traffic

Association (IATA)

Dangerous Goods Regulations

Appendix 2-1. Safety Regulations and Recommendations Applicable to Health-Care

Settings (cont'd)

Chapter 2: Facilities and Safety 73

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 re-

quired coverings should be appropriate for the type and amount of hazard exposure. Plastic dis-

posable 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 be-

cause 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)

74 AABB Technical Manual

■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

mouth and nose should be protected.5Permanent 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 avail-

able; eliciting staff input on comfort and selection can improve compliance on use.

Appendix 2-2. General Guidelines for Safe Work Practices, Personal Protective

Equipment, and Engineering Controls (cont'd)

Chapter 2: Facilities and Safety 75

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. Res-

pirators 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 hazard-

ous 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 main-

tains for surface disinfectants, consumers should request data from the manufacturer to support

advertising claims.

Eye Washes

Laboratory areas that contain hazardous chemicals must be equipped with eye wash stations.3,6

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 rou-

tinely 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 proce-

dures, 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.

Appendix 2-2. General Guidelines for Safe Work Practices, Personal Protective

Equipment, and Engineering Controls (cont'd)

(cont’d)

76 AABB Technical Manual

1. Code of federal regulations. Occupational exposure to bloodborne pathogens, final rule. Title 29 CFR

Part 1910.1030. Fed Regist 1991;56(235):64175-82.

2. 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.

3. Clinical and Laboratory Standards Institute. Clinical laboratory safety; approved guideline. NCCLS

document GP17-A. Wayne, PA: CLSI, 1996.

4. 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.]

5. Inspection checklist: General laboratory. Chicago, IL: College of American Pathologists, 2001.

6. American National Standards Institute. American national standards for emergency eyewash and

shower equipment. ANSI Z358.1-1998. New York: ANSI, 1998.

Appendix 2-2. General Guidelines for Safe Work Practices, Personal Protective

Equipment, and Engineering Controls (cont'd)

Chapter 2: Facilities and Safety 77

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. Clinical and Laboratory Standards Institute. Clinical laboratory safety; approved guideline. NCCLS

document GP17-A. Wayne, PA: CLSI, 1996.

2. 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.

78 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 79

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: ___________________________________________________________

____________________________________________________________________________

Appendix 2-4. Sample Hazardous Chemical Data Sheet (cont'd)

80 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 81

Chemical Hazard

Sodium phosphate Irritant, hygroscopic

Sulfosalicylic acid Toxic, corrosive

Trichloroacetic acid (TCA) Corrosive, toxic

Trypsin Irritant, sensitizer

Xylene Highly flammable, toxic, irritant

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

82 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 chemical-

resistant-rated gloves

and gowns as recom-

mended

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 concen-

trated acids to 1 liter

Post cautions for materials

in the area

Report changes in appear-

ance (perchloric acid

may be explosive if it be-

comes 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

Leave valve safety covers on

until use

Open valves slowly for use

Label empty tanks

Transport using hand trucks

or dollies

Place cylinders in a stand or

secure them to prevent

falling

Store in well-ventilated sep-

arate rooms

Oxygen should not be

stored close to combusti-

ble gas or solvents

Check connections for leaks

with soapy water

Liquid nitrogen Freeze injury

Severe burns to

skin or eyes

Use heavy insulated gloves

and goggles when work-

ingwithliquidnitrogen

The tanks should be se-

curely supported to avoid

being tipped over

The final container of liquid

nitrogen (freezing unit)

must be securely sup-

ported to avoid tipping

over

Chapter 2: Facilities and Safety 83

Chemical

Category Hazard Precautions Special Treatment

Flammable

solvents

Classified accord-

ingtoflash

point—see

MSDS

Classified accord-

ing to volatility

Use extreme caution when

handling

Post NO SMOKING signs in

working area

Have a fire extinguisher and

solvent cleanup kit in the

room

Pour volatile solvents under

suitable hood

Use eye protection when

pouring and chemical-re-

sistant neoprene gloves

No flame or other source of

possible ignition should

be in or near areas where

flammable solvents are

being poured

Label as FLAMMABLE

Make every attempt to re-

place hazardous materi-

als with less hazardous

materials

Store containers larger than

1galloninaflammable

solvent storage room or

in a fire safety cabinet

Ground metal containers by

connecting the can to a

water pipe or ground

connection; if recipient

container is also metal, it

should be electrically

connected to the delivery

container while pouring

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

Chemicals (cont'd)

84 AABB Technical Manual

Appendix 2-7. Incidental Spill Response*

Chemicals 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 poison-

ous gas

Nitric, perchloric, and sulfuric

acids are water-reactive oxi-

dizers

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; impervi-

ous 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 irri-

tation of the eyes or skin

Toxicity due to alkalinity, possi-

ble 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 (neo-

prene boots for emergency

response releases)

Self-contained breathing appara-

tus (emergency response

releases)

Chlorine control powder

Absorbent pillow

Absorbent

Absorbent boom

Drain mat

Vapor barrier

Leakproof container

Shovel or paddle

Chapter 2: Facilities and Safety 85

Cryogenic gases

Carbon dioxide

Nitrous oxide

Liquid nitrogen

Contact with liquid

nitrogen can produce

frostbite

Asphyxiation (displaces oxygen)

Anesthetic effects

(nitrous oxide)

Full face shield or goggles; neo-

prene boots; gloves (insu-

lated 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 oxy-

gen-deficient

atmosphere

Face shield and goggles; neo-

prene 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 flam-

mable 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% formalde-

hyde)

Gloves (double set 4H

undergloves and butyl or

nitrile overgloves); impervi-

ous apron or coveralls; gog-

gles; impervious foot covers

Aldehyde neutralizer/absorbent

Absorbent boom

Absorbent pillow

Shovel or pallet (nonsparking)

Drain mat

Leakproof container

(cont’d)

86 AABB Technical Manual

Chemicals Hazards PPE Control Materials

Mercury

Cantor tubes

Thermometers

Barometers

Sphygmomanometers

Mercuric chloride

Mercury and mercury vapors are

rapidly absorbed in respira-

tory tract, GI tract, skin

Short-term exposure may cause

erosion of respiratory/GI

tracts, nausea, vomiting,

bloody diarrhea, shock, head-

ache, metallic taste

Inhalation of high concentrations

can cause pneumonitis, chest

pain, dyspnea, coughing

stomatitis, gingivitis, and sali-

vation

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; imper-

vious 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 chem-

ical-specific MSDS for more complete information. GI = gastrointestinal; MSDS = material safety data sheet; PPE = personal protective equipment.

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

Chapter 2: Facilities and Safety 87

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 ig-

nition within 10 feet of spilled hazardous material. For flammable liq-

uids, remove all sources of ignition.

Gases: Remove all sources of heat and ignition within 50 feet for flamma-

ble 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

PPE

See Appendix 2-2 for recommended PPE.

Contain the spill Liquids or mercury: Stop the source of spill if possible.

Gases: Assess the scene; consider the circumstances of the release (quan-

tity, location, ventilation). If circumstances indicate it is an emergency

response release, make appropriate notifications; if release is deter-

mined to be incidental, contact supplier for assistance.

Confine the spill Liquids: Confine spill to initial spill area using appropriate control equip-

ment 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 mate-

rials. 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 wa-

ter. 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 re-

move absorbent. Collect all contaminated disposal equipment and put

into hazardous waste container. Collect supplies and remove gross con-

tamination; place them into a separate container for equipment that will

be thoroughly washed and decontaminated.

(cont’d)

88 AABB Technical Manual

Actions Instructions for Hazardous Liquids, Gases, and Mercury

Disposal Liquids: For material that was neutralized, dispose of it as solid waste. Fol-

low 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 ap-

plicable.

Mercury: Label with appropriate hazardous waste label and DOT diamond

label.

Report Follow appropriate spill documentation and reporting procedures. Investi-

gate the spill; perform root cause analysis if needed. Act on opportuni-

ties for improving safety.

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

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

Chapter 3: Blood Utilization Management

Chapter 3

Blood Utilization

Management

THE 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 us-

age review. Although regional blood cen-

ters 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 utiliza-

tion management, emphasizing the trans-

fusion service.

Minimum and Ideal

Inventory Levels

Transfusion services should establish both

minimum and ideal inventory levels. In-

ventory levels should be evaluated period-

ically 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 when-

ever a significant change is planned or

observed. Examples of significant change

may include adding more beds; perform-

ing new surgical procedures; or changing

practices in oncology, transplantation, neo-

natology, or cardiac surgery.

Determining Inventory

Levels

The ideal inventory level provides ade-

quate supplies of blood for routine and

emergency situations and minimizes out-

dating. Forecasting is an attempt to pre-

dict future blood product use from data

collected about past usage. The optimal

number of units to keep in inventory can

be estimated using mathematical formu-

las, computer simulations, or empirical

calculations. Three less complicated meth-

ods of estimating minimum inventory are

described below. When the minimum in-

ventory level has been calculated, a buffer

margin for emergencies should be added

to obtain an ideal inventory level.

Average Weekly Use Estimate

This method gives an estimate of the av-

erage 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 num-

ber 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

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 aver-

age daily use calculation more helpful

when blood shipments are made once or

more per day.

Moving Average Method

Themovingaveragemethodcanbeuse

ful 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).1The data

from this report showed that approxi-

mately 2,190,000 total components out-

dated in 2001, a 6.6% decrease from 1999.

Whole-blood-derived platelet concen-

trates 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 af-

fected by many factors other than inven-

tory 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).

90 AABB Technical Manual

In addition to establishing both mini-

mum and ideal inventory levels, maximum

inventory levels may assist staff in deter-

mining when to arrange for return or trans-

fer of in-date products to avoid outdating.

Both transfusion services and donor cen-

ters should establish record-keeping sys-

tems that allow personnel to determine the

number of units ordered and the number of

units received or shipped. The responsibil-

ity for ordering may be centralized, and or-

ders should be based on established poli-

cies 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 regu-

lar 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 flexi-

ble, to allow use of fresher blood when indi-

cated (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

Chapter 3: Blood Utilization Management 91

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

States in 20011

Component

Units

Processed

Units

Transfused

Units

Outdated

Percent

Outdated

RBCs (allogeneic,

nondirected)

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-blood-

derived)

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

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

maximum surgical blood order schedules

(MSBOS),2as well as monitoring cross-

match-to-transfusion (C:T) ratios may be

helpful. A C:T ratio greater than 2.0 usu-

ally indicates excessive crossmatch re-

quests. In some situations, it may be use-

ful to determine C:T ratios by service to

identify areas with the highest ratio.

Some institutions define those proce-

dures that normally do not use blood in a

“type and screen” guideline. Both the T/S

guidelineandtheMSBOSusedataabout

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 Ta-

ble 3-2 are derived by reviewing a facility’s

blood use over a suitable period. Conclu-

sions can then be drawn about the likeli-

hood 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, where-

as the MSBOS often defines the number of

units needed to meet the needs of 80% to

90% of patients undergoing a specific surgi-

cal procedure.3

An institution’s guidelines must reflect

local patterns of surgical practice and pa-

tient 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 Chap-

ter1.)OncetheT/S,MSBOS,SBO,orother

92 AABB Technical Manual

Table 3-2. Example of a Maximum

Surgical Blood Order Schedule

Procedure Units*

General Surgery

Breast biopsy T/S

Colon resection 2

Exploratory laparotomy T/S

Gastrectomy 2

Laryngectomy 2

Mastectomy, radical T/S

Pancreatectomy 4

Splenectomy 2

Thyroidectomy T/S

Cardiac-Thoracic

Aneurism resection 6

Redo coronary artery bypass graft 4

Primary coronary artery bypass graft 2

Lobectomy T/S

Lung biopsy T/S

Vascular

Aortic bypass with graft 4

Endarterectomy T/S

Femoral-popliteal bypass with graft 2

Orthopedics

Arthroscopy T/S

Laminectomy T/S

Spinal fusion 3

Total hip replacement 3

Total knee replacement T/S

OB-GYN

Abdomino-perineal repair T/S

Cesarean section T/S

Dilation and curettage T/S

Hysterectomy, abdominal/

laparoscopic

T/S

Hysterectomy, radical 2

Urology

Bladder, transurethral resection T/S

Nephrectomy, radical 3

Radical prostatectomy, perineal 2

Prostatectomy, transurethral T/S

Renal transplant 2

*Numbers may vary with institutional practice.

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 in-

tended for typical circumstances. Surgeons

or anesthesiologists may individualize spe-

cific requests and override the system to ac-

commodate 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 ap-

propriate 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 addi-

tional crossmatched units more rapidly if

required. This capability can allow such fa-

cilities 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 nec-

essary.

Routine vs Emergency Orders

Transfusion services should establish pro-

cedures that define ideal stocking levels for

each blood type and critical levels at which

emergency orders are indicated. Transfu-

sion 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 immedi-

ately 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 im-

mediately be met.

Transfusion services should develop pol-

icies 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 be-

fore 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. Individ-

ual units must be visually inspected for

signs of contamination or atypical ap-

pearance 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. Unpro-

cessed or incompletely processed units,

autologous units, and unsuitable units

must be clearly segregated (quarantined)

from routine stock.4(p15) Most institutions or-

ganize 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.

Chapter 3: Blood Utilization Management 93

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, in-

correctly placed among group O Rh-nega-

tive units, is issued without careful check-

ing in an emergency situation.

Special Product Concerns

Platelets

Few articles address the management of

platelet inventory. Optimal levels are diffi-

cult to determine because demand is epi-

sodicandtheshelflifeisshort.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, HLA-

matched, or cytomegalovirus (CMV)-sero-

negative 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 cen-

ter 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 antici-

pate needs and place orders in advance.

Transfusion services with high use may find

it helpful to maintain platelets in inventory.

Transfusion services should define selec-

tion and transfusion guidelines for ABO

group, Rh type, irradiation, CMV serologic

testing, and leukocyte reduction of platelet

products. The formulas described in the

beginning of the chapter may be used to

estimate ideal inventory ranges.

Platelet usage often increases the day af-

ter a holiday because elective procedures

and oncology transfusions will have been

postponed. Planning ahead to stock plate-

lets helps meet postholiday demand.

Frozen Plasma Products

Because plasma components can be stored

frozen up to 1 year, these inventories are

easier to manage. Optimal inventory lev-

els are determined by assessing statistics

on patient populations and usage pat-

terns. 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 col-

lected by apheresis to increase general

stock and provide for special needs.

Cryoprecipitated AHF is a labor-inten-

sive product to prepare and supplies can-

not 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 inven-

tory, their management becomes a signif-

icant 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 ar-

ranged alphabetically by the intended re-

94 AABB Technical Manual

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, allo-

geneic 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 physi-

cians.

Special Inventories

Donor centers and transfusion services

are faced with requests for specialty prod-

ucts such as CMV-reduced-risk units,

HLA-matched platelets, antigen-matched

red cells, or irradiated components. The

appropriate use of these products is dis-

cussed 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 gen-

eral stock as they near their outdate be-

cause 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.

2. Friedman BA, Oberman HA, Chadwick AR, et

al. The maximum surgical blood order sched-

ule and surgical blood use in the United States.

Transfusion 1976;16:380-7.

3. Devine P, Linden JV, Hoffstadter L, et al. Blood

donor-, apheresis-, and transfusion-related

activities: Results of the 1991 American Asso-

ciation of Blood Banks Institutional Member-

ship Questionnaire. Transfusion 1993;33:779-

82.

4. Silva MA, ed. Standards for blood banks and

transfusion services. 23rd ed. Bethesda, MD:

AABB, 2005:15.

Chapter 3: Blood Utilization Management 95

Chapter 4: Allogeneic Donor Selection and Blood Collection

Chapter 4

Allogeneic Donor Selection

and Blood Collection

BLOOD CENTERS AND transfusion

services depend on volunteer do-

nors to provide the blood neces-

sary to meet the needs of the patients

they serve. To attract volunteer donors

and encourage their continued participa-

tion, it is essential that conditions sur-

rounding blood donation be as pleasant,

safe, and convenient as possible. To pro-

tect 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, ac-

cessible, and open at hours convenient

for donors. It must be well lighted, com-

fortably ventilated, orderly, and clean.

Personnel should be friendly, understand-

ing, professional, and well trained. The area

mustprovideadequatespaceforprivate

and accurate examinations of individuals

to determine their eligibility as blood do-

nors, and for the withdrawal of blood

from donors with minimum risk of con-

tamination 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 re-

cords.1(p13) Some facilities require photo-

graphic identification. Current informa-

tion must be obtained and recorded for

each donation. Selected portions of dona-

tion records must be kept indefinitely and

must make it possible to notify the donor

of any information that needs to be con-

veyed.1(p69) The following information should

be included:

1. Date and time of donation.

2. Name: Last, first (and middle initial

if available).

3. Address: Residence and/or business.

4. Telephone: Residence and/or busi-

ness.

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 de-

ferrals, if any. Persons who have been

placed on a deferral or surveillance

list must be identified before any

unitdrawnfromthemismadeavail

able for release. Ideally, a donor de-

ferral registry should be available to

identify ineligible donors before

blood is drawn. If such a registry is

notavailable,theremustbeaproce-

dure to review prior donation re-

cords and/or deferral registries be-

fore releasing the components from

quarantine.2

The following information may also be

useful:

1. Additional identification such as so-

cial security or driver’s license num-

beroranyothernameusedbythe

donor on a previous donation. The

Social Security Act specifically al-

lows the use of the social security

number for this purpose. These data

are required for information retrieval

in some computerized systems. Iden-

tification of other names used by a

donor is particularly important to

ensure that the appropriate donor

file is accessed or that a deferral sta-

tusisaccurate.

2. Name of patient or group to be ac-

knowledged.

3. Race. Although not required, this in-

formation 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 popu-

lations understand the medical im-

portance and scientific applications

of this information.3,4

4. Unique characteristics of the donor.

Certain information about the donor

may enable the blood bank to make

optimal use of the donation. For ex-

ample, blood from donors who are

seronegative for cytomegalovirus

(CMV), or who are group O, Rh neg-

ative, is often designated for neona-

tal patients. The blood center may

specify that blood from these indi-

viduals be drawn routinely into col-

lection 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.

5. A record of special communications

to a donor, special drawing of blood

samples for studies, etc.

6. If the donation is directed to a spe-

cific patient, information about when

and where the intended recipient will

be hospitalized should be obtained.

An order from the intended recipi-

ent’s physician should be provided

to the blood center staff. The in-

tended recipient’s date of birth, so-

cial security number, or other identi-

fiers may be required by the transfusion

service. If the donor is a blood relative

of the intended recipient, this infor-

mation must be noted so that cellular

components can be irradiated.1(p43)

Information Provided to the Prospective

Donor

All donors must be given educational ma-

terials informing them of significant risk

98 AABB Technical Manual

of the procedure, the clinical signs and

symptoms associated with human immu-

nodeficiency virus (HIV) infection and

AIDS, high-risk activities for transmission,

and the importance of refraining from do-

nating blood if they have engaged in these

activities or experienced associated signs

or symptoms. Before donating, the pro-

spectivedonorsmustdocumentthatthey

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 lo-

cal 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 hepati-

tis 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 possi-

bility that testing may fail to identify infec-

tive individuals in an early seronegative

stage of infection should be included as

well.6The 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.5Pro-

visionsshouldbemadeforthehearing-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 state-

ment should be obtained. In some loca-

tions, 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 alter-

native sites or other mechanisms to obtain

HIV tests should be available to all prospec-

tive 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 med-

ical history and behavior or events that

put a person at risk for transmissible dis-

ease or at personal medical risk. It is,

therefore, imperative that proper guide-

lines 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

Chapter 4: Allogeneic Donor Selection and Blood Collection 99

under the physician’s direction after appro-

priate training.7Donor selection criteria are

established through regulations, recom-

mendations, and standards of practice.

When a donor’s condition is not covered or

addressed by any of these, a qualified phy-

sician 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 recipi-

ent.1(p17) The medical history questions (in-

cluding questions pertaining to risk behav-

ior 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-ap-

proved computer-generated questionnaire.

The interview and physical examination

must be performed in a manner that en-

sures 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 doc-

umented by the staff on the donor form.

Resultsofobservationsmadewhenaphys

ical examination is given and when tests

are performed must be recorded concur-

rently.

Donors must understand the informa-

tion 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 elimi-

nating ineligible donors from the donor

pool. Of equal importance is the training of

donor center staff. Screening can be effec-

tive 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 re-

lations skills are essential for job

competency. Because donor center staff are

in constant contact with donors, knowl-

edgeable personnel and effective commu-

nication 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 neces-

sary to ensure that, to the greatest extent

possible, it is safe for the donor to donate

and for the blood to be transfused. The in-

terviewer should review and evaluate all

responses to determine eligibility for do-

nation and document the decision. To be

sure that all the appropriate questions are

asked and that donors are given a consis-

tent message, use of the most recent FDA-

approved AABB donor history question-

naire is recommended. The most recent

FDA-approved version is found on the AABB

Web site. (See Appendices 4-1 through 4-3,

whichwerecurrentatthetimeofthiswrit-

ing.)

One area of the medical history—medi-

cations and drugs taken by the donor—of-

ten requires further investigation. New pre-

scription 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 man-

ual. 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 with-

out approval from a donor center physician.

Prospective donors who have taken iso-

tretinoin (Accutane) or finasteride (Proscar

100 AABB Technical Manual

or Propecia) within the 30 days preceding

donation; dutasteride (Avodart) within the

6 months preceding donation; acitretin

(Soriatane) within the 3 years preceding do-

nation; or etretinate (Tegison) at any time

must be deferred.1(p62) The Armed Services

Blood Program Office makes its drug defer-

ral 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

Donorsmaybegiventheopportunityto

indicate confidentially whether their blood

is or is not suitable for transfusion to oth-

ers. This should be done by a mechanism

that allows the donor to avoid face-to-

face 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 col-

lected 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

results. Counseling or referral must be pro-

vided 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 physi-

cian must approve exceptions to routinely

acceptable findings. For special donor

categories, the medical director may pro-

vide policies and procedures to guide de-

cisions. Other donors may require indi-

vidual 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 dona-

tion.1(p61) This amount shall include

samples for testing. If it is necessary

to draw a smaller amount than ap-

propriate for a standard collection

container, then the amount of anti-

coagulant in the container must be

adjusted appropriately. The formula

inTable4-1maybeusedtodeter

mine the amount of anticoagulant to

remove. The volume of blood drawn

must be measured carefully and ac-

curately.

3. Temperature: The donor’s tempera-

ture must not exceed 37.5 C (99.5 F)

if measured orally, or its equivalent if

measured by another method. Lower

Chapter 4: Allogeneic Donor Selection and Blood Collection 101

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.

than normal temperatures are usu-

ally of no significance in healthy in-

dividuals; however, they should be

repeated for confirmation.

4. Pulse: The pulse rate should be counted

for at least 15 seconds. It should ex-

hibit no pathologic irregularity, and

the frequency should be between 50

and 100 beats per minute. If a pro-

spective donor is a known athlete

with high exercise tolerance, a pulse

rate below 50 may be noted and

should be acceptable. A donor cen-

ter physician should evaluate marked

abnormalities of pulse and recom-

mend acceptance, deferral, or refer-

ral for additional evaluation.

5. Blood pressure: The blood pressure

should be no higher than 180 mm Hg

systolic and 100 mm Hg diastolic. Pro-

spective donors whose blood pres-

sure is above these values should not

be drawn without individual evalua-

tion by a qualified physician.

6. 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 in-

tended to prevent collection of blood

from a donor with anemia, it does

not ensure that the donor has an ad-

equatestoreofiron.Table4-2gives

the lower limits of hemoglobin for

accepting allogeneic donors. Indi-

viduals with unusually high hemo-

globin or hematocrit levels may need

to be evaluated by a physician be-

cause the elevated levels may reflect

pulmonary, hematologic, or other

abnormalities. Methods to evaluate

hemoglobin concentration include

1) specific gravity determined by cop-

per sulfate (see Method 6.1), 2) spec-

trophotometric measurement of he-

moglobin or determination of the

hematocrit, or 3) alternate accepted

methods to rule out erroneous re-

sults that may lead to rejection of a

donor. Earlobe puncture is not an

acceptable source for a blood sam-

ple.1(p61),10

7. 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 rea-

son for indefinite exclusion of a pro-

spective donor. Mild skin disorders

or the rash of poison ivy should not

be cause for deferral unless unusu-

ally extensive and/or present in the

antecubital area. Individuals with

boils, purulent wounds, or severe

skin infections anywhere on the

102 AABB Technical Manual

Table 4-2. Minimum Levels of Hemoglobin, Hematocrit, and Red Cell Density for

Accepting an Allogeneic Blood Donor

Donor Test Method Minimal Acceptable Value1

Hemoglobin

Hematocrit

Copper sulfate

12.5 g/dL

38%

1.053 sp gr

body should be deferred, as should

anyone with purplish-red or hemor-

rhagic 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 physi-

cal examination, medical history, or post-

donation laboratory testing.11 Abnormalities

found before donation may be explained

verbally by qualified personnel. Test results

obtained after donation that preclude fur-

ther donation may be reported in person,

by telephone, or by letter. Donors should be

asked to report any illness developing with-

in a few days after donation and, especially,

to report a positive HIV test or the occur-

rence of hepatitis or AIDS that develops

within 12 months after donation.

Donor Consent

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.

Theproceduremustbeexplainedin

terms that donors can understand, and

theremustbeanopportunityforthepro

spective 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 mean-

ing to the following is suggested:

“I have read and understand the infor-

mation provided to me regarding the spread

of the AIDS virus (HIV) by blood and

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 test-

ing may not be performed. If this testing in-

dicates that I should no longer donate blood

or plasma because of the risk of transmitting

an infectious disease, my name will be en-

tered on a list of permanently deferred do-

nors. I understand that I will be notified of a

positive laboratory test result(s). If, instead,

the results of the testing are not clearly nega-

tive 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.

Special Donor Categories

Exceptions to the usual eligibility require-

ments may be made for special donor cat-

egories:

1. Autologous donors: The indications

for collection and variations from

usual donor procedures are discus-

sed in Chapter 5.

2. Hemapheresis: Special requirements

and recommendations for cytaphere-

sisdonorsorfordonorsinaplasma

pheresis program are detailed in

Chapter 6.

3. Recipient-specific “designated” dona-

tions: Under certain circumstances,

it may be important to use blood or

components from a specific donor

for a specific patient. Examples in-

clude the patient with an antibody

to a high-incidence antigen or a

combination of antibodies that makes

Chapter 4: Allogeneic Donor Selection and Blood Collection 103

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 physi-

cian and approved by the donor cen-

ter physician. The donor must meet

all the usual requirements for dona-

tion, except that the frequency of

donation can be as often as every 3

days, as long as the predonation he-

moglobin level meets or exceeds the

minimum value for routine allo-

geneic blood donation.

The blood must be processed ac-

cording to AABB Standards for Blood

Banks and Transfusion Services.1(pp24-32)

Special tags identifying the donor

unit number and the intended recip-

ient 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.

4. Directed donors: The public’s con-

cern about the safety of transfusion

has generated demands from poten-

tial 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 re-

quested by a potential blood recipi-

ent or ordered by a physician. Despite

logistic and philosophic problems

associated with these “directed” do-

nations, most blood centers and

hospitals provide this service. The

selection and testing of directed do-

nors 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 misunderstand-

ings, it is important to establish SOPs

that define the interval required be-

tween collection of the blood and its

availability to the recipient; the policy

about determining ABO group before

collection; and the policy for releas-

ing 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 col-

lection 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 addi-

tional venipuncture unless the SOP allows

the use of an FDA-approved device to at-

tach a new needle while preserving steril-

ity. 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-ap-

proved container that is pyrogen-free and

sterile and contains sufficient anticoagu-

lant for the quantity of blood to be col-

104 AABB Technical Manual

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 manu-

facturer’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 disposi-

tion of each component. A numeric or al-

phanumeric system must be used that

identifies, and relates to, the source do-

nor, 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 re-

cords and labels should be checked be-

fore use for printing errors. If duplicate

numbers are found, they must be re-

moved and may be investigated to ascer-

tain 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, at-

tached satellite bags, and tubes for

donor blood samples. Attaching the

numbers at the donor chair, rather

than during the examination proce-

dures, 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

collection of blood. Tubes may be at-

tached in any convenient manner to

theprimarybagorintegraltubing.

4. 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 tour-

niquet 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 de-

vice should be released before the skin

site is prepared.

There is no way to make the veni-

puncture site completely aseptic, but surgi-

cal 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 prepara-

tion 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 pres-

sure device has again been inflated. Dur-

ing collection, the blood should be mixed

with the anticoagulant. The amount of

blood collected should be monitored care-

fully so that the total, including samples,

does not exceed 10.5 mL per kilogram of

donor weight per donation.1(p61) When the

appropriate amount has been collected,

segments and specimen tubes must be

filled. The needle and any blood-contam-

inatedwastemustbedisposedofsafelyin

Chapter 4: Allogeneic Donor Selection and Blood Collection 105

accordance with universal precaution

guidelines. The needle must not be re-

capped unless a safety recapping device is

used. Disposal of the needle must be in a

puncture-proof container. After collec-

tion, 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 in-

dividuals 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 in-

structed 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

bedorinthedonorchairforafew

minutes under close observation by

staff.

3. Allow the donor to sit up under ob-

servation until his or her condition

appears satisfactory. The donor should

be observed in the upright position

before release to the observation/re-

freshment area. Staff should monitor

donors in this area. The period of ob-

servation and provision of refreshment

should be specified in the SOP manual.

4. Give the donor instructions about

postphlebotomy care. The medical

directormaywishtoincludesome

or all of the following recommenda-

tions or instructions:

a. Eat and drink something before

leaving the donor site.

b. Do not leave until released by a

staff member.

c. 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 do-

norcenterorseeadoctor.

i. Resume all normal activities if

asymptomatic. Donors who work

in certain occupations (eg, con-

struction workers, operators of

machinery) or persons working

at heights should be cautioned

that dizziness or faintness may

occur if they return to work im-

mediately after giving blood.

j. Remove bandage after a few

hours.

k. Maintain high fluid intake for

several days to restore blood

volume.

5. 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.

6. Note on the donor record any ad-

verse reactions that occurred. If the

donor leaves the area before being

released, note this on the record.

106 AABB Technical Manual

Adverse Donor Reactions

Most donors tolerate giving blood very

well, but adverse reactions occur occa-

sionally. Personnel must be trained to rec-

ognize adverse reactions and to provide

initial treatment.

Donor room personnel should be train-

ed in cardiopulmonary resuscitation (CPR).

Special equipment to handle emergency

situations must be available.

Syncope (fainting or vasovagal syn-

drome)maybecausedbythesightof

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 neuro-

physiologic response to blood donation, the

symptoms may include weakness, sweat-

ing, dizziness, pallor, loss of consciousness,

convulsions, and involuntary passage of fe-

ces 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 sig-

nificantly. This can be useful in distinguish-

ing between vasovagal attack and cardio-

genic or hypovolemic shock, in which cases

the pulse rate rises. This distinction, al-

though characteristic, is far from abso-

lute.

Rapid breathing or hyperventilation may

cause the anxious or excited donor to lose

excessive amounts of carbon dioxide. This

may cause generalized sensations of suffo-

cation or anxiety, or localized problems

such as tingling or twitching.

The donor center physician must pro-

vide written instructions for handling do-

nor reactions, including a procedure for ob-

taining emergency medical help. Sample

instructions might be as follows:

1. General.

a. Remove the tourniquet and with-

draw the needle from the arm if

signs of a reaction occur during

the phlebotomy.

b. If possible, remove any donor

who experiences an adverse re-

action 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.

2. 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 ade-

quate airway.

e. Monitor blood pressure, pulse,

and respiration periodically un-

til the donor recovers.

Note: Some donors who experi-

ence prolonged hypotension may re-

spondtoaninfusionofnormalsa-

line. The decision to initiate such

therapy should be made by the do-

nor center physician either on a case-

by-case basis or in a policy stated in

the facility’s SOP manual.

3. Nausea and vomiting.

a. Make the donor as comfortable

as possible.

b. Instruct the donor who is nau-

seated 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 clean-

sing tissues or a damp towel

ready. Be sure the donor’s head

Chapter 4: Allogeneic Donor Selection and Blood Collection 107

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.

4. Twitching or muscular spasms. Ex-

tremely nervous donors may hyper-

ventilate, causing faint muscular

twitching or tetanic spasm of their

hands or face. Donor room person-

nel 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.

5. 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 pres-

sure 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 min-

utes, if desired.

d. Should an arterial puncture be

suspected, immediately with-

draw needle and apply firm

pressure for 10 minutes. Apply

pressure dressing afterwards.

Check for the presence of a ra-

dial pulse. If pulse is not palpa-

ble or is weak, call a donor cen-

ter physician.

6. Convulsions.

a. Call for help immediately. Pre-

vent 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 pre-

vent injury to the donor and to

yourself.

b. Be sure the donor has an ade-

quate airway. A padded device

should separate the jaws after

convulsion has ceased.

c. Notify the donor center physi-

cian.

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 neces-

sary supplies and drugs. Most donor cen-

ters 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

andamounttobekeptonhandmust

be specified in writing. In addition,

the medical director must provide

written policies stating when and by

108 AABB Technical Manual

whom any of the above medical sup-

plies or drugs may be used.

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2. Code of federal regulations. Title 21 CFR

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Chicago, IL: American Society of Clinical Patholo-

gists, 1997.

Schmuñis GA. Trypanosoma cruzi,theetiologic

agent of Chagas’ disease: Status in the blood sup-

ply in endemic and nonendemic countries. Trans-

fusion 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 medi-

cine. 2nd ed. Baltimore, MD: Williams and Wilkins,

1995:871-9.

Tan L, Williams MA, Khan MK, et al. Risk of trans-

mission of bovine spongiform encephalopathy to

humans in the United States. JAMA 1999;281:2330-8.

Chapter 4: Allogeneic Donor Selection and Blood Collection 109

110 AABB Technical Manual

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

Chapter 4: Allogeneic Donor Selection and Blood Collection 111

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

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 113

Appendix 4-2. Medication Deferral List*

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

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 follow-

ing 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

AUTOLOGOUS BLOOD TRANSFU-

sion is an alternative therapy for

many patients anticipating trans-

fusion. Different categories of autologous

transfusion are:

1. Preoperative collection (blood is drawn

and stored before anticipated need).

2. Perioperative collection and admin-

istration.

a. Acute normovolemic hemodi-

lution (blood is collected at the

start of surgery and then in-

fused during or at the end of

the procedure).

b. Intraoperative collection (shed

blood is recovered from the

surgical field or circulatory de-

vice and then infused).

c. Postoperative collection (blood

is collected from drainage de-

vices and reinfused to the pa-

tient).

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 transfu-

sion practices, transfusion practices of

other similarly situated institutions, and its

own capabilities to determine the appropri-

ate 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 asymptom-

atic infection with HIV is a disability pro-

tected under the Americans with Disabili-

ties Act.1Therefore, if institutions offer

autologous services to any patient, they

should consider offering such services to

HIV-positive patients.2Patients who are

likely to require transfusion therapy and

whoalsomeetthedonationcriteriashould

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 auto-

117

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 disad-

vantages of preoperative autologous blood

donation (PAD) are summarized in Table

5-1. Candidates for preoperative collec-

tion are stable patients scheduled for sur-

gical procedures in which blood transfu-

sion is likely. For procedures that are

unlikely to require transfusion (ie, a maxi-

mal 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, preopera-

tive collection of autologous blood can sig-

nificantly reduce exposure to allogeneic

blood. PAD collections should be considered

for patients likely to receive transfusion,

such as patients undergoing major ortho-

pedic 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.4Autologous 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 per-

formed for patients who would not ordi-

narily be considered for allogeneic dona-

tion. The availability of medical support is

important in assessing patient eligibility.

With suitable volume modification, pa-

rental cooperation, and attention to prep-

aration and reassurance, pediatric patients

118 AABB Technical Manual

Table 5-1. Autologous Blood Donation

Advantages Disadvantages

1. Prevents transfusion-transmitted disease. 1. Does not eliminate risk of bacterial contam-

ination.

2. Prevents red cell alloimmunization. 2. Does not eliminate risk of ABO incompati-

bility error.

3. Supplements the blood supply. 3. Is more costly than allogeneic blood.

4. Provides compatible blood for patients with

alloantibodies.

4. Results in wastage of blood that is not

transfused.

5. Prevents some adverse transfusion reac-

tions.

5. Increased incidence of adverse reactions to

autologous donation.

6. Provides reassurance to patients con-

cerned about blood risks.

6. Subjects patients to perioperative anemia

and increased likelihood of transfusion.

can participate in preoperative collection

programs.6The successful use of autolo-

gous blood in a patient with sickle cell

disease has been reported,7and 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 he-

moglobin A. Red cells containing hemo-

globin 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,9the risks that

are associated with autologous blood dona-

tion10 in these patients are probably greater

than the current estimated risks of allo-

geneic transfusion.11,12 Table 5-2 summa-

rizes the contraindications to a patient’s

participation in an autologous blood dona-

tion program.13

The collection of autologous blood from

women during routine pregnancy is unwar-

ranted,14 because blood is so seldom needed.

Many centers give serious consideration to

autologous collection for women with allo-

antibodies to multiple or high-incidence

antigens, placenta previa, or other conditions

placing them at high risk for ante- or intra-

partum hemorrhage.5Apolicyshouldbe

developed for situations in which maternal

red cells are considered for transfusion to

the infant.

Voluntary Standards

AABB Standards for Blood Banks and Trans-

fusion Services offers uniform standards to

be followed in determining patient eligi-

bility; collecting, testing, and labeling the

unit; and pretransfusion testing.15(pp18,39,51)

These AABB standards apply to preopera-

tive autologous blood collection. Stan-

dards for Perioperative Autologous Blood

Collection and Administration have been

established to enhance the quality and

safety of perioperative autologous trans-

fusion activities (intra- and postoperative

blood recovery, perioperative autologous

component production, and intraopera-

tive acute normovolemic hemodilution).16

Compliance Considerations

Food and Drug Administration (FDA) re-

quirements 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 pre-

vious 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 in-

fection resulting from the following com-

Chapter 5: Autologous Blood Donation and Transfusion 119

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 steno-

sis.

3. Unstable angina.

4. Uncontrolled seizure disorder.

5. Myocardial infarction or cerebrovascular

accident within 6 months of donation.

6. Patients with significant cardiac or pulmo-

nary disease who have not yet been cleared

for surgery by their treating physician.

7. High-grade left main coronary artery dis-

ease.

8. Cyanotic heart disease.

9. Uncontrolled hypertension.

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 in-

clude nucleic acid tests for HCV and HIV.21

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 dona-

tions 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 reac-

tive by a required screening test must be re-

tested 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 Stan-

dards15(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 dis-

ease 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,itmustbelabeled“DONORTESTED

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 la-

beled appropriately [21 CFR 610.40(d)]. If

distributed on a common carrier not un-

der the direct control of the collection fa-

cility, the transportation of the product

must meet provisions for shipping an in-

fectious 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 ex-

ist for establishing a new program, moni-

toring utilization, or improving an exist-

ing 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

120 AABB Technical Manual

with the patient’s physician and the medi-

cal director of the transfusion service. The

patient’s physician initiates the request

for autologous services, which must be

approved by the transfusion service phy-

sician. There should be a transfusion

medicine physician available to help as-

sess patients whose medical history sug-

gests a risk for complications if a donor

reaction occurs during blood collection.

Supplemental Iron

The patient should be advised about tak-

ing supplemental iron. Ideally, supple-

mental 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 collec-

tion from regular volunteer donors, but

numerous special considerations exist.

Requests for autologous blood collection

aremadeinwritingbythepatient’sphysi

cian; a request form (which may be a sim-

ple prescription or a form designed for

the purpose) is kept by the collecting fa-

cility. The request should include the

patient’s name, a unique identification

number, the number of units and kind of

component requested, the date of sched-

uled 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 col-

lected. A sufficient number of units should

be drawn, whenever possible, so that the

patient can minimize exposure to allo-

geneic blood. However, excessive collection

and/or collection close to the date of sur-

gery increases the patient’s likelihood of re-

quiring 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 comp-

ensatory 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 dona-

tion early in the known preoperative inter-

val, 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 be-

fore the scheduled surgery and preferably

longer, to allow time for adequate volume

repletion. Programs should notify the re-

questing physician of the total number of

units donated when the requested number

of units cannot be collected. Each program

should establish a policy regarding re-

scheduling of surgery beyond the expira-

tion date of autologous units and whether

discarding or freezing the unit are options.

Donor Screening

Because of the special circumstances re-

garding autologous blood transfusion,

rigid criteria for donor selection are not

required. In situations where require-

ments for allogeneic donor selection or

collection are not applied, alternative re-

quirements must be established by the

medical director and recorded in the pro-

cedures manual. The hemoglobin con-

centration of the donor should be no less

Chapter 5: Autologous Blood Donation and Transfusion 121

than 11.0 g/dL and the hematocrit, if sub-

stituted, should be no less than 33%. Indi-

vidual deviations from the alternate re-

quirements must be approved by the blood

bank medical director, usually in consul-

tation with the donor-patient’s physician.

Medical Interview

The medical interview should be struc-

tured to meet the special needs of autolo-

gous donors. For example, more attention

should be given to questions about medi-

cations, associated medical illnesses, and

cardiovascular risk factors.10 Questions

should elicit any possibility of intermittent

bacteremia. Because crossover is not rou-

tinely permitted, a substantially shortened

set of interview questions can be used for

autologous donations; for example, ques-

tions related to donor risks for transfusion-

transmitted diseases are not necessary.

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 do-

nor 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/

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.

Labeling

Units should be clearly labeled with the

patient’s name and an identifying num-

ber, 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,

122 AABB Technical Manual

Table 5-3. Timing and Red Cell Regeneration During Preoperative Autologous

Donation26

Time from Donation to

Surgery (days)

No. of

Patients

Mean RBC Units

Regenerated

5% CI

of Mean

6-13 39 0.52 0.25-0.79

14-20 127 0.54 0.40-0.68

21-27 128 0.75 0.61-0.90

28-34 48 1.16 0.96-1.36

35-41 30 1.93 1.64-2.2

the container of each component must be

similarly labeled. A biohazard label must

be applied when indicated by FDA require-

ments (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 col-

lected 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 stor-

age 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 autol-

ogous 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, freez-

ing and thawing add to the cost of the

program, reduce the volume of red cells

through processing losses, and compli-

cate blood availability during the peri-

operative 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 edu-

cated about the importance of selecting

autologous components before allogeneic

units are given, and a policy should be in

placeregardingtheissueofautologous,

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) Re-

cords 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. Autolo-

gous transfusions have a lower risk of in-

fectious and immune complications but

carry a similar risk of bacterial contami-

nation, volume overload, and misadminis-

tration 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

been identified for PAD practices.5The

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 pros-

tatectomy, a well-designed program may

result in 50% of collected units being un-

used (Fig 5-1).5,27 Nevertheless, as much as

25% of autologous blood is collected for

procedures that seldom require transfu-

sion, such as vaginal hysterectomies and

normal vaginal deliveries. Up to 90% of

units collected for these procedures are

wasted.14 The additional costs associated

Chapter 5: Autologous Blood Donation and Transfusion 123

with the collection of autologous units,

along with advances in the safety of allo-

geneic blood, have altered the cost-effec-

tiveness of PAD in many situations.28 Such

cost-effectiveness analyses do not consider

an immunomodulatory effect of avoiding

allogeneic leukocytes, which remains

controversial.29,30

Criteria can be established to monitor

the appropriateness of autologous transfu-

sions.Thesecriteriamaybethesameas,or

different from, those established for allo-

geneic units.24 As with allogeneic blood,

transfusion of preoperatively donated auto-

logous blood carries the same risks associ-

ated with administrativeerrororbacterial

contamination. Autologous programs should

be monitored for unavailability of autolo-

gous 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 transfu-

sion based on the baseline level of hemo-

globin and on the type of procedure show

some promise. In a Canadian study using

a point score system, 80% of patients un-

dergoing orthopedic procedures were

identified to be at low risk (<10%) for

transfusion, so autologous blood procure-

ment for these patients would not be rec-

ommended.31 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 proce-

dures performed even by the same sur-

124 AABB Technical Manual

Figure 5-1. Autologous RBC collection and transfusion data from 1980 to 2001 in the United States il-

lustrate 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)

Percent of Total RBCs that Were PAD RBCs

1980 1986 1989 1992 1994 1997 1999 2001

Collected 0.3 1.5 4.8 8.5 7.8 4.9 4.7 4.0

Transfused <1.0 3.1 5.0 4.3 3.7 3.0 2.6

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 erythro-

poiesis increases the production of red

cells.25,34-36 The endogenous erythropoietin

response and compensatory erythropoie-

sis are suboptimal under “standard” con-

ditions of one blood unit donated weekly.

Weekly PAD is accompanied by an 11%

(with no oral iron supplementation) to 19%

(with oral iron supplementation) expan-

sion in red cell volume over a 3-week pe-

riod, which is not sufficient to prevent in-

creasing 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 dona-

tion of autologous blood may be harm-

ful.37 This outcome was confirmed in a

study of patients undergoing hysterecto-

mies,38 in which it was shown that PAD

resulted in perioperative anemia and an

increased likelihood of any blood transfu-

sion. A published mathematical model37

illustrates the relationship between antic-

ipated surgical blood losses, the level of

hematocrit that the physician may want

to maintain perioperatively, and the need

for autologous blood donation for indivi-

dual patients (Fig 5-2). Models such as

this may be helpful in designing autolo-

gous procurement programs or monitor-

ing their value through quality assurance.

In contrast to autologous blood donation

under “standard” conditions, studies of “ag-

gressive” 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-

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 phlebot-

omy has been approved in Canada and Ja-

panbutnotintheUnitedStates.

Transfusion Trigger

Disagreement exists about the proper he-

moglobin/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 simi-

lar because the additional mortality risks

of allogeneic blood now approach the risks

of mortality from administrative errors as-

sociated with both autologous and allo-

geneic 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 ques-

tioned 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 pro-

grams without sacrificing safety.45

Chapter 5: Autologous Blood Donation and Transfusion 125

Preoperative Collection of Components

Some workers believe that preoperative or

intraoperative collection of platelet-rich

plasma during cardiopulmonary bypass

surgery may improve hemostasis and de-

crease allogeneic exposures, but others

have found no benefit.46 Preoperative col-

lection of autologous platelets, especially

in cardiac surgery, is often impractical be-

cause patients may be taking antiplatelet

drugs; surgery is often scheduled on an

emergency basis; and relatively low num-

bers 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 es-

tablish 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 pa-

tient, with concurrent restoration of the

circulating blood volume with an acellular

fluid shortly before an anticipated signifi-

cant surgical blood loss. To minimize the

manual labor associated with hemodilu-

tion, the blood should be collected in

standard blood bags containing anticoag-

126 AABB Technical Manual

Figure 5-2. Relationship of estimated blood loss (EBL) and minimum (nadir) hematocrit during hospi-

talization 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 Co-

hen and Brecher.37)

ulant on a tilt-rocker with automatic cut-

off 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, albu-

min, 1 mL for each 1 mL of blood with-

drawn) have been recommended.50

Subsequent intraoperative fluid manage-

ment is based on the usual surgical require-

ments. Blood units are reinfused in the

reverse order of collection. The first unit

collected, and therefore the last unit trans-

fused, has the highest hematocrit and con-

centration of coagulation factors and plate-

lets. 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 alterna-

tive method of autologous blood procure-

ment.51 Augmented hemodilution (replace-

ment 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 Mathe-

matical modeling has suggested that se-

vere ANH to preoperative hematocrit lev-

els of less than 20%, accompanied by

substantial blood losses, would be re-

quired before the red cell volume “saved”

by ANH would become clinically impor-

tant.53 However, the equivalent of one blood

unit54 can be “saved” by ANH,55 which ap-

proaches the red cell volume expansion

generated by PAD under standard condi-

tions (Table 5-3).

Improved Oxygenation

Withdrawal of whole blood and replace-

ment with crystalloid or colloid solution

decrease arterial oxygen content, but com-

pensatory hemodynamic mechanisms and

the existence of surplus oxygen-delivery

capacity make ANH safe. A sudden de-

crease in red cell concentration lowers

blood viscosity, thereby decreasing pe-

ripheral resistance and increasing cardiac

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.

output. If cardiac output can effectively

compensate, oxygen delivery to the tis-

sues at a hematocrit of 25% to 30% is as

good as, but no better than, oxygen deliv-

ery at a hematocrit of 30% to 35%.56

Preservation of Hemostasis

Because blood collected by ANH is stored

at room temperature and is usually re-

turned 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 ortho-

pedicorurologicsurgerybecauseplasma

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

hip replacement60 suggest that ANH can

be considered equivalent to PAD as a

method of autologous blood procure-

ment. Additional, selected clinical trials of

ANH are summarized in Table 5-5.61-67 Re-

views68,69 and commentaries70 on the mer-

its 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 associ-

ated 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. Be-

cause 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 impor-

tant 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 hemo-

dilution hematocrit, and other phys-

iologic variables.

2. The institution’s policy and proce-

dures and the mechanisms for edu-

cating staff should be established

and periodically reviewed.

3. Thereshouldbecarefulmonitoring

of the patient’s circulating volume

and perfusion status during the pro-

cedure.

4. Blood must be collected in an asep-

tic 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 ex-

ceed 8 hours. Units maintained at

room temperature should be re-

infused in the reverse order of col-

lection to provide the maximum

number of functional platelets and

coagulation factors in the last units

infused. If more time elapses be-

tween collection and transfusion,

128 AABB Technical Manual

Chapter 5: Autologous Blood Donation and Transfusion 129

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

Estimated Blood Loss (mL)

Postoperative

Hematocrit (%)

Allogeneic RBC-Containing

Units or Liters Transfused

Surgery Control ANH p Value Control ANH p Value Control ANH p Value Reference

Vascular 2250 2458 NS NR 33.0 NR 6.0 2.6 <0.01 61

Liver resection 1479 1284 NS 37.9 33.8 <0.01 3.8 0.4 <0.001 62

Hip arthroplasty 1800 2000 NS 38.4 32.4 NS (2.1) (0.9) NR 63

Spinal fusion 5490 1700 <0.005 NR 28.7 NR 8.6 <1.0 <0.001 64

Colectomy NR NR NR 37.0 35.0 NR 2.4 0 NR 65

Prostate 1246 1106 NS 35.5 31.8 <0.001 0.16 0 NS 66

Prostate 1717 1710 NS 29.5 27.9 <0.5 0.30 0.13 NS 67

Modified from Brecher and Rosenfeld.53 NR = not reported; NS = not significant.

the blood should be stored in a

monitored refrigerator. ANH blood

collected from an open system (eg,

from a central venous line or an ar-

terial 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 anesthesiolo-

gists in conjunction with the operat-

ing room’s nursing staff and the hos-

pital’s blood bank and transfusion

services.68 These will include indica-

tions for ANH, monitoring require-

ments, endpoints for blood with-

drawal 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 pa-

tient 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-trans-

port properties of recovered red cells are

equivalent to stored allogeneic red cells.

The survival of recovered blood cells ap-

pears to be at least comparable to that of

130 AABB Technical Manual

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.

transfused allogeneic red cells.72 Intra-

operative collection is contraindicated

when certain procoagulant materials (eg,

topical collagen) are applied to the surgi-

cal field because systemic activation of

coagulation may result. Microaggregate

filters (40 microns) are used most often

because recovered blood may contain tis-

sue 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

embolusisapotentiallyseriousproblem.

Three fatalities from air embolus were re-

ported 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 recom-

mend a maximum vacuum setting of no

more than 150 torr. One study found that

vacuumsettingsashighas300torrcould

be used when necessary, without causing

excessive hemolysis.74 The clinical impor-

tance of free hemoglobin in the concentra-

tions usually seen has not been established,

although excessive free hemoglobin may

indicate inadequate washing. Positive bac-

terial cultures from recovered blood are

sometimes observed; however, clinical in-

fection is rare.75

Most programs use machines that col-

lect 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%. Pa-

tients exhibit a level of plasma-free hemo-

globin that is usually higher than after

allogeneic transfusion. Sodium and chlo-

ride concentrations are the same as in the

saline wash solution, and potassium con-

centration is low. The infusate contains

minimal coagulation factors and platelets.

Clinical Studies

As with PAD and ANH, collection and re-

covery of intraoperative autologous blood

should undergo scrutiny with regard to

both safety and efficacy. Controlled stud-

ies in cardiothoracic surgery have re-

ported conflicting results when transfu-

sion requirements and clinical outcome

were followed.75,76 Although the collection

of a minimum of one blood unit equiva-

lent is possible for less expensive (with

unwashed blood) methods, it is generally

agreed that at least two blood unit equiva-

lents need to be recovered using a cell-re-

covery instrument (with washed blood) in

order to achieve cost-effectiveness.77 The

value of intraoperative blood collection

has been best defined for vascular surger-

ies with large blood losses, such as aortic

aneurysm repair and liver transplanta-

tion.78 However, a prospective randomized

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.

trial79 of intraoperative recovery and re-

infusion in patients undergoing aortic an-

eurysm repair showed no benefit in the

reduction of allogeneic blood exposure. A

mathematical model of cell recovery sug-

gests that when it is combined with normo-

volemic 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 consider-

ations in patients with substantial blood

losses.

Medical Controversies

Collection devices that neither concentrate

nor wash shed blood before reinfusion in-

crease 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

coagulation.81 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 anticoagu-

lant used.

3. The extent of contact between blood

and serosal surfaces.

4. The extent of contact between blood

and artificial surfaces.

5. Thedegreeofturbulenceduringcol

lection.

In general, blood collected at low flow rates

or during slow bleeding from patients who

are not systemically anticoagulated will

have undergone coagulation and fibrinoly-

sis and will not contribute to hemostasis

upon reinfusion.

The high suction pressure and surface

skimming during aspiration and the turbu-

lence 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 im-

paired 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, anes-

thesiologists, transfusion medicine spe-

cialists, 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 wash-

ed in a separate cell washer.

3. High-speed machines that automat-

ically concentrate and wash recov-

ered red cells.

Some hospitals develop their own pro-

grams, whereas others contract with outside

services. Each hospital’s needs should dic-

tate whether blood collection and recovery

are used and how they are achieved.

Processing Before Reinfusion

Several devices automatically process re-

covered blood before reinfusion. Vacuum

suction and simultaneous anticoagula-

tion are used for collection. To minimize

hemolysis, the vacuum level should ordi-

narily 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

withavolumeofsalinethatvariesbe

tween 500 and 1500 mL. If not infused im-

mediately, the unit must be labeled with

the patient’s name and identification

132 AABB Technical Manual

number, the date and time collection was

initiated, and the statement “For Autolo-

gous Use Only.”

An alternative approach is to collect

blood in a canister system designed for di-

rect 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 sys-

tems generally consist of a suction cathe-

ter attached to a disposable collection bag

or rigid plastic canister, to which antico-

agulant (citrate or heparin) may have

been added. Blood is suctioned into the

holding canister before being reinfused

through a microaggregate filter. Low-vac-

uum 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 expira-

tion, 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)

Hospitals with collection and recovery

programs should establish written policies

and procedures that are regularly reviewed

by a physician who has been assigned re-

sponsibility for the program. Transfusion

medicine specialists should play an active

role in design, implementation, and opera-

tion of the program. Written policies must

be in place for the proper collection, label-

ing, and storage of intraoperative autolo-

gous blood. Equipment and techniques for

collection and infusion must ensure that

the blood is aseptic. Quality management

should include evaluation of the appropri-

ate use of blood collection and recovery

services and adequate training of person-

nel. Written protocols, procedure logs, ma-

chine maintenance, procedures for han-

dling 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, postopera-

tive shed blood is collected into sterile

canisters and reinfused, without process-

ing, through a microaggregate filter. Re-

covered 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 re-

quirements, quality assurance, and review

consistent with AABB standards.16(p2)

Chapter 5: Autologous Blood Donation and Transfusion 133

Clinical Studies

The evolution of cardiac surgery has been

accompaniedbyabroadexperiencein

postoperative conservation of blood. Post-

operative autologous blood transfusion is

practiced widely, but not uniformly. Pro-

spective and controlled trials have dis-

agreed over the efficacy of postoperative

blood recovery in cardiac surgery pat-

ients; at least three such studies have

demonstrated lack of efficacy,82-84 but at

least two studies have shown benefit.85,86

The disparity of the results in these stud-

ies may be explained, in part, by differences

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 des-

cribed the successful recovery and reinfu-

sion of washed87 and unwashed88,89 wound

drainage from patients undergoing arthro-

plasty. Red cells recovered in this setting

appear to have normal survival in the circu-

lation.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-

134 AABB Technical Manual

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

Collection

Type

Storage

Temperature Expiration

Special

Conditions

Acute normovolemic

hemodilution

Room tempera-

ture

8 hours from start

of collection

None

1-6 C 24 hours from start

of collection

Storage at 1-6 C shall

begin within 8 hours

of start of collection

Intraoperative blood

recovered with

Room tempera-

ture

4 hours from end of

collection

None

processing 1-6 C 24 hours from start

of collection

Storage at 1-6 C shall

begin within 4 hours

of start of collection

Intraoperative blood

recovered without

processing

Room tempera-

ture or 1-6 C

4 hours from end of

collection

None

Shed blood under post-

operative or post-

traumatic conditions

with or without pro-

cessing

N/A 6 hours from start

of collection

None

Non-red-cell component

preparation

Room tempera-

ture

Shall be used before

leaving the oper-

ating room

None

placement.89 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 reinfu-

sion in patients undergoing total knee or

hip replacement found no differences in

perioperative hemoglobin levels or allo-

geneic blood transfusions between patients

who did or did not have joint drainage de-

vices.92 The safety of reinfused unwashed

orthopedic wound drainage has been con-

troversial. Theoretical concerns have been

expressed regarding infusion of potentially

harmful materials in recovered blood, in-

cluding free hemoglobin, red cell stroma,

marrow fat, toxic irritants, tissue or metha-

crylate debris, fibrin degradation products,

activated coagulation factors, and comple-

ment. Although two small studies have

reported complications,93,94 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

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 replace-

ment, 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 addi-

tional cost of processing technology.96,97 As

in the selection of patients who can bene-

fit from PAD and ANH, prospective identi-

fication of patients who can benefit from

intra- and postoperative autologous blood

recovery is possible if anticipated surgical

blood losses and the perioperative “trans-

fusion trigger” are taken into account (Fig

5-2).

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mathematical analysis. Transfusion 1995;35:640-4.

38. Kanter MH, Van Maanen D, Anders KH, et al.

Preoperative autologous blood donation be-

fore elective hysterectomy. JAMA 1996;276:

798-801.

39. GoodnoughLT,RudnickS,PriceTH,etal.In

creased preoperative collection of autologous

blood with recombinant human erythropoie-

tin therapy. N Engl J Med 1989;321:1163-8.

40. Goodnough LT, Price TH, Rudnick S, Soegiarso

RW. Preoperative red blood cell production in

patients undergoing aggressive autologous

blood phlebotomy with and without erythro-

poietin therapy. Transfusion 1992;32:441-5.

41. Goodnough LT, Price TH, Friedman KD, et al.

A phase III trial of recombinant human eryth-

ropoietin therapy in non-anemic orthopedic

patients subjected to aggressive autologous

blood phlebotomy: Dose, response, toxicity,

efficacy. Transfusion 1994;34:66-71.

42. Goodnough LT, Monk TG, Andriole GL. Eryth-

ropoietin therapy. N Engl J Med 1997;336:

933-8.

43. LindenJV,WagnerK,VoytovichAE,Sheehan

J.TransfusionerrorsinNewYorkState:An

analysis of 10 years’ experience. Transfusion

2000;40:1207-13.

44. Hébert PC, Wells G, Blajchman MA, et al. A

multicenter, randomized, controlled clinical

trial of transfusion requirements in critical

care. N Engl J Med 1999;340:409-17.

136 AABB Technical Manual

45. Kruskall MS, Yomtovian R, Dzik WH, et al. On

improving the cost effectiveness of autolo-

gous blood transfusion practices. Transfusion

1994;34:259-64.

46. Triulzi DJ, Gilmor GD, Ness PM, et al. Efficacy

of autologous fresh whole blood or platelet-

rich plasma in adult cardiac surgery. Transfu-

sion 1995;35:627-34.

47. Kevy SV, Jacobson MS. Comparison of meth-

ods for point of care preparation of autolo-

gous platelet gel. J Extra Corpor Technol

2004;36:28-35.

48. Crovetti G, Martinelli G, Issi M, et al. Platelet

gel for healing cutaneous chronic wounds.

Transfus Apheresis Sci 2004;30:145-51.

49. Mazzucco L, Medici D, Serra M, et al. The use

of autologous platelet gel to treat difficult to

heal wounds: A pilot study. Transfusion 2004;

44:1013-8.

50. Goodnough LT, Brecher ME, Monk TG. Acute

normovolemic hemodilution in surgery. He-

matology 1992;2:413-20.

51. GoodnoughLT,BrecherME,KanterMH,

AuBuchon JP. Transfusion medicine. Second

of two parts. Blood conservation. N Engl J

Med 1999;340:525-33.

52. Messmer K, Kreimeier M, Intagliett A. Present

state of intentional hemodilution. Eur Surg

Res 1986;18:254-63.

53. Brecher ME, Rosenfeld M. Mathematical and

computer modeling of acute normovolemic

hemodilution. Transfusion 1994;34:176-9.

54. Goodnough LT, Bravo J, Hsueh Y, et al. Red

blood cell volume in autologous and homolo-

gous units: Implications for risk/benefit

assessment for autologous blood “crossover”

and directed blood transfusion. Transfusion

1989;29:821-2.

55. GoodnoughLT,GrishaberJE,MonkTG,

Catalona WJ. Acute normovolemic hemodilu-

tion in patients undergoing radical supra-

pubic prostatectomy: A case study analysis.

Anesth Analg 1994;78:932-7.

56. Weiskopf RB. Mathematical analysis of isovo-

lemic hemodilution indicates that it can de-

crease the need for allogeneic blood trans-

fusion. Transfusion 1995;35:37-41.

57. Petry AF, Jost T, Sievers H. Reduction of ho-

mologous blood requirements by blood pool-

ing at the onset of cardiopulmonary bypass. J

Thorac Cardiovasc Surg 1994;1097:1210-14.

58. Monk TG, Goodnough LT, Brecher ME, et al. A

prospective, randomized trial of three blood

conservation strategies for radical prostatec-

tomy. Anesthesiology 1999;91:24-33.

59. Goodnough LT, Merkel K, Monk TG, Despotis

GJ. A randomized trial of acute normovo-

lemic hemodilution compared to preopera-

tive autologous blood donation in total knee

arthroplasty. Vox Sang 1999;77:11-16.

60. Goodnough LT, Despotis GJ, Merkel K, Monk

TG. A randomized trial of acute normo-

volemic hemodilution compared to preoper-

ative autologous blood donation in total hip

arthroplasty. Transfusion 2000;40:1054-7.

61. Davies MJ, Cronin KD, Domanique C. Hae-

modilution for major vascular surgery using

3.5% polygeline (Haemaccel). Anaesth Inten-

sive Care 1982;10:265-70.

62. Sejourne P, Poirier A, Meakins JL, et al. Effects

of haemodilution on transfusion requirements

in liver resection. Lancet 1989;ii:1380-2.

63. Rosenberg B, Wulff K. Regional lung function

following hip arthroplasty and preoperative

normovolemic hemodilution. Acta Anaesthe-

siol Scand 1979;23:242-7.

64. Kafer ER, Isley MR, Hansen T, et al. Auto-

mated acute normovolemic hemodilution re-

duces blood transfusion requirements for

spinal fusion (abstract). Anesth Analg 1986;

65(Suppl):S76.

65. Rose D, Coustoftides T. Intraoperative normo-

volemic hemodilution. J Surg Res 1981;31:375-81.

66. Ness PM, Bourke DL, Walsh PC. A random-

ized trial of perioperative hemodilution ver-

sus transfusion of preoperatively deposited

autologous blood in elective surgery. Transfu-

sion 1991;31:226-30.

67. Monk TG, Goodnough LT, Birkmeyer JD, et al.

Acute normovolemic hemodilution is a cost-

effective alternative to preoperative autolo-

gous blood donation by patients undergoing

radical retropubic prostatectomy. Transfu-

sion 1995;35:559-65.

68. Shander A. Acute normovolemic hemodilu-

tion. In: Spence RK, ed. Problems in general

surgery. Philadelphia: Lippincott Williams &

Wilkins, 1999;17:32-40.

69. Monk TG, Goodnough LT, Brecher ME, et al.

Acute normovolemic hemodilution can re-

place preoperative autologous blood dona-

tion as a standard of care for autologous

blood procurement in radical prostatectomy.

Anesth Analg 1997;85:953-8.

70. Goodnough LT, Monk TG, Brecher ME. Acute

normovolemic hemodilution should replace

preoperative autologous blood donation be-

fore elective surgery. Transfusion 1998;38:

473-7.

71. Segal JB, Blasco-Colmenares E, Norris EJ,

Guallar E. Preoperative acute normovolemic

hemodilution: A meta-analysis. Transfusion

2004;44:632-44.

72. Williamson KR, Taswell HF. Intraoperative

blood salvage: A review. Transfusion 1991;31:

662-75.

Chapter 5: Autologous Blood Donation and Transfusion 137

73. Domen RE. Adverse reactions associated with

autologous blood transfusion: Evaluation

and incidence at a large academic hospital.

Transfusion 1998;38:296-300.

74. Gregoretti S. Suction-induced hemolysis at

various vacuum pressures: Implications for

intraoperative blood salvage. Transfusion

1996;36:57-60.

75. Bell K, Stott K, Sinclair CJ, et al. A controlled

trial of intra-operative autologous transfu-

sion in cardiothoracic surgery measuring ef-

fect on transfusion requirements and clinical

outcome. Transfus Med 1992;2:295-300.

76. TempeDK,BanjerjeeA,VirmaniS,etal.

Comparison of the effects of a cell saver and

low dose aprotinin on blood loss and homol-

ogous blood use in patients undergoing valve

surgery. J Cardiothorac Vasc Anesth 2001;15:

326-30.

77. Bovill DF, Moulton CW, Jackson WS, et al. The

efficacy of intraoperative autologous transfu-

sion in major orthopaedic surgery: A regres-

sion analysis. Orthopedics 1986;9:1403-7.

78. Goodnough LT, Monk TG, Sicard G, et al.

Intraoperative salvage in patients undergoing

elective abdominal aortic aneurysm repair.

An analysis of costs and benefits. J Vasc Surg

1996;24:213-8.

79. Claggett GP, Valentine RJ, Jackson MR, et al. A

randomized trial of intraoperative transfu-

sion during aortic surgery. J Vasc Surg 1999;

29:22-31.

80. Waters JH, Karafa MT. A mathematical model

of cell salvage efficiency. Anesth Analg 2002;

95:1312-7.

81. de Haan J, Boonstra P, Monnink S, et al. Re-

transfusion of suctioned blood during cardio-

pulmonary bypass impairs hemostasis. Ann

Thorac Surg 1995;59:901-7.

82. WardHB,SmithRA,CandisKP,etal.Apro

spective, randomized trial of autotransfusion

after routine cardiac surgery. Ann Thorac

Surg 1993;56:137-41.

83. Thurer RL, Lytle BW, Cosgrove DM, Loop FD.

Autotransfusion following cardiac opera-

tions: A randomized, prospective study. Ann

Thorac Surg 1979;27:500-6.

84. Roberts SP, Early GL, Brown B, et al. Auto-

transfusion of unwashed mediastinal shed

blood fails to decrease banked blood require-

ments in patients undergoing aorta coronary

bypass surgery. Am J Surg 1991;162:477-80.

85. SchaffHV,HauerJM,BellWR,etal.Auto

transfusion of shed mediastinal blood after

cardiac surgery. A prospective study. J Thorac

Cardiovasc Surg 1978;75:632-41.

86. EngJ,KayPH,MurdayAJ,etal.Post-opera

tive autologous transfusion in cardiac sur-

gery. A prospective, randomized study. Eur J

Cardiothorac Surg 1990;4:595-600.

87. Semkiw LB, Schurman OJ, Goodman SB,

Woolson ST. Postoperative blood salvage us-

ing the cell saver after total joint arthroplasty.

J Bone Joint Surg (Am) 1989;71A:823-7.

88. Faris PM, Ritter MA, Keating EM, Valeri CR.

Unwashed filtered shed blood collected after

knee and hip arthroplasties. J Bone Joint Surg

(Am) 1991;73A:1169-77.

89. MartinJW,WhitesideLA,MillianoMT,Reedy

ME. Postoperative blood retrieval and trans-

fusion in cementless total knee arthroplasty. J

Arthroplasty 1992;7:205-10.

90. 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.

91. Umlas J, Foster RR, Dalal SA, et al. Red cell

loss following orthopedic surgery: The case

against postoperative blood salvage. Transfu-

sion 1994;34:402-6.

92. Ritter MA, Keating EM, Faris PM. Closed

wound drainage in total hip or knee replace-

ment: A prospective, randomized study. J

Bone J Surg 1994;76:35-8.

93. 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.

94. Woda R, Tetzlaff JE. Upper airway oedema

following autologous blood transfusion from

a wound drainage system. Can J Anesth 1992;

39:290-2.

95. Blevins FT, Shaw B, Valeri RC, et al. Re-

infusion of shed blood after orthopedic pro-

cedures in children and adolescents. J Bone

Joint Surg (Am) 1993;75A:363-71.

96. Goodnough LT, Verbrugge D, Marcus RE. The

relationship between hematocrit, blood lost,

and blood transfused in total knee replace-

ment: Implications for postoperative blood

salvage and reinfusion. Am J Knee Surg 1995;

8:83-7.

97. Jackson BR, Umlas J, AuBuchon JP. The cost-

effectiveness of postoperative recovery of

RBCs in preventing transfusion-associated

virus transmission after joint arthroplasty.

Transfusion 2000;40:1063-6.

138 AABB Technical Manual

Chapter 6: Apheresis

Chapter 6

Apheresis

APHERESIS, FROM THE Greek

pheresis meaning “to take away,”

involves the selective removal of

blood constituents from blood donors or

patients.

The AABB provides standards1for volun-

tary compliance for apheresis activities.

The Food and Drug Administration (FDA)

has established specific requirements that

aresetforthintheCode of Federal Regula-

tions2for apheresis activities. The American

Society for Apheresis (ASFA)3has 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 per-

form 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 centri-

fuged 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, centrifu-

gal force separates blood into components

on the basis of differences in density. A

measured amount of anticoagulant solu-

tion 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 com-

139

ponents occurs on the basis of their den-

sities. The desired fraction is diverted and

the remaining elements are returned to the

donor (or patient) by intermittent or con-

tinuous flow.

All systems require prepackaged dispos-

able sets of sterile bags, tubing, and centrif-

ugal devices unique to the instrument. Each

system has a mechanism to allow the sepa-

ration 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 separa-

tion chamber. Depending on the procedure

and device used, the apheresis procedure

time varies from 30 minutes to several hours.

Each manufacturer supplies detailed in-

formation and operational protocols. Each

facility must have, in a manual readily

available to nursing and technical person-

nel, 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 re-

moval of all plasma constituents. Centrif-

ugal devices can be adapted to protocols

that selectively remove specific soluble

plasma constituents by exploiting the

principles of affinity chromatography.5Se-

lective removal of low-density lipoproteins

(LDLs) in patients with familial hypercho-

lesterolemia has been accomplished using

both immunoaffinity (anti-LDL) and che-

mical affinity (eg, dextran sulfate) columns.6

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. Immuno-

adsorption can be performed online, or

the plasma can be separated from the cel-

lular components, passed through an off-

line column, and then reinfused.

Component Collection

Whenever components intended for trans-

fusion are collected by apheresis, the do-

nor must give informed consent. Although

apheresis collection and preparation pro-

cesses are different from those used for

whole-blood-derived components, stor-

age conditions, transportation require-

ments, and some quality control steps are

thesame.SeeChapter8formoredetailed

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 pat-

ients’ family members, or from donors with

matched HLA or platelet antigen pheno-

types. Because large numbers of platelets

can be obtained from a single individual,

collection by apheresis reduces the num-

ber of donor exposures for patients. AABB

Standards requires the component to

contain at least 3 ×1011 platelets in 90% of

sampled units.1(p31) When a high yield is

obtained, the original apheresis unit may

be divided into multiple units, each of which

must meet minimum standards inde-

pendently. Some instruments are pro-

grammed to calculate the yield from the

donor’s hematocrit, platelet count, height,

and weight. For alloimmunized patients

who are refractory to random allogeneic

140 AABB Technical Manual

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 post-

transfusion platelet increment. Within the

United States, the use of apheresis plate-

lets has been steadily increasing over the

last 25 years. Currently, it is estimated that

77% of therapeutic platelet doses are

transfused as apheresis platelets.7

Donor Selection and Monitoring

Plateletpheresis donors may donate more

frequently than whole blood donors but

must meet all other donor criteria. The in-

terval between donations should be at

least 2 days, and donors should not un-

dergo 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 pla-

teletpheresis, at least 8 weeks should elapse

before a subsequent plateletpheresis pro-

cedure, 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 compo-

nent is expected to be of particular value

to a specific intended recipient, and if a

physician certifies in writing that the do-

nor’s health will not be compromised (eg,

an HLA-matched donor). Donors who

have taken aspirin-containing medica-

tions within 36 hours of donation are usu-

ally deferred because the platelets ob-

tained 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 antico-

agulant are common (see Complications,

later in this chapter). Serious reactions

occur less often among apheresis donors

than among whole blood donors.

Plateletpheresis donors should meet

usual donor requirements, including he-

moglobin 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 platelet-

pheresis 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 phy-

sician. The FDA specifies that the total vol-

ume of plasma collected should be no more

than 500 mL (or 600 mL for donors weighing

more than 175 pounds).8The 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 col-

lection. Apheresis can also be used to col-

lect 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.9A total serum or plasma

protein determination and a quantitative

determination of IgG and IgM (or a serum

protein electrophoresis) must be deter-

mined at 4-month intervals for donors un-

dergoing large-volume plasma collection, if

the total annual volume of plasma collected

exceeds 12 liters (14.4 L for donors weigh-

ing more than 175 pounds) or if the donor

is a frequent (more often than every 4

weeks) plasma donor.10 AABB Standards re-

quires that the donor’s intravascular volume

deficit must be less than 10.5 mL per kilo-

gram of body weight at all times.1(p24)

Chapter 6: Apheresis 141

Laboratory Testing

Tests for ABO group and Rh type, unex-

pected alloantibodies, and markers for

transfusion-transmitted diseases must be

performed by the collecting facility in the

same manner as for other blood compo-

nents. 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 re-

peated only at 30-day intervals.1(p34)

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.8In

some instances, it may be desirable for the

donorplasmatobeABO-compatiblewith

the recipient’s red cells—for example, if the

recipient is a child or an ABO-mismatched

allogeneic progenitor cell transplant recipi-

ent. In order to be considered leukocyte-

reduced, apheresis platelets must contain

less than 5 ×106leukocytes and must meet

the specifications of the apheresis device

manufacturer. Chapter 8 describes addi-

tional quality control measures that apply

to all platelet components.

Records

Complete records (see Chapter l) must be

kept for each procedure. All adverse reac-

tions should be documented along with

the results of their investigation and fol-

low-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.8Facilities must have

policies and procedures in place to ensure

that donor red cell loss during each pro-

cedure does not exceed acceptable limits.

Plasma

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; per-

sonnel who perform serial plasmapheresis

must be familiar with both AABB stan-

dards and FDA requirements. If plasma is

intended for transfusion, testing require-

ments are the same as those for red cell

components. Plasma collected for manu-

facture of plasma derivatives is subject to

different requirements for infectious dis-

ease testing.

A distinction is made between “occa-

sional plasmapheresis,” in which the donor

undergoes plasmapheresis no more often

than once in 4 weeks, and “serial plasma-

pheresis,” in which donation is more fre-

quent 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 plasma-

pheresis using either automated instru-

ments or manual techniques, the following

principles apply:

1. Donors must provide informed con-

sent. They must be observed closely

during the procedure and emergency

medicalcaremustbeavailable.

2. Red cell losses related to the proce-

dure, including samples collected

for testing, must not exceed 25 mL

perweek,sothatnomorethan200

mL of red cells are removed in 8

weeks. If the donor’s red cells cannot

be returned during an apheresis pro-

cedure, hemapheresis or whole blood

donation should be deferred for 8

weeks.

3. In manual plasma collection systems,

there must be a mechanism to en-

sure safe reinfusion of the autologous

red cells. Before the blood container

142 AABB Technical Manual

is separated from the donor for pro-

cessing, there should be two sepa-

rate, independent means of identifi-

cation, 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 iden-

tification number.

4. 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 do-

nors who weigh more than 80 kg are

600 mL and 1200 mL, respectively.

For automated procedures, the allow-

able volume has been determined

for each instrument by the FDA.9

5. At least 48 hours should elapse be-

tween successive procedures; ordi-

narily, donors should not undergo

more than two procedures within a

7-day period. Exceptions are permis-

sible when plasma is expected to

have special therapeutic value for a

single recipient.

6. Atthetimeofinitialplasmapheresis

and at 4-month intervals thereafter

for donors undergoing plasmaphere-

sis more often than once every 4

weeks, serum or plasma must be

tested for total protein and serum

protein electrophoresis or quantita-

tive immunoglobulins. Results must

be within normal limits.2

7. A qualified, licensed physician, know-

ledgeable in all aspects of hema-

pheresis, must be responsible for the

program.

Red Cells

Both AABB standards and FDA-approved

protocols address the removal of two allo-

geneic or autologous Red Blood Cell units

every 16 weeks by an automated aphere-

sis method. Saline infusion is used to min-

imizevolumedepletion,andtheproce

dure is limited to persons who are larger

and have higher hematocrits than current

minimum standards for whole blood do-

nors (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 transfu-

sion are controversial (see Chapter 21). A

meta-analysis of randomized controlled

trials of granulocyte transfusion indicates

that effectiveness depends on an adequate

dose (>1 ×1010 granulocytes/day) and

crossmatch compatibility (no recipient

antibodies to granulocyte antigens).12

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 ab-

sent.

Drugs Administered for Leukapheresis

A daily dose of at least 1 ×1010 granulo-

cytes 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 con-

sent should include specific permission

for any drugs or sedimenting agents to be

used.

Hydroxyethyl Starch. A common sedi-

menting agent, hydroxyethyl starch (HES),

causes red cells to aggregate and thereby

sediment more completely. Sedimenting

Chapter 6: Apheresis 143

agents enhance granulocyte harvest and re-

sult in minimal red cell content. Because

HES can be detected in donors for as long

as a year after infusion, AABB Standards re-

quires facilities performing granulocyte col-

lections to have a process to control the

maximal cumulative dose of any sedi-

menting agent administered to the donor

within a given interval.1(p24) Because HES is a

colloid, it acts as a volume expander, and

donors who have received HES may experi-

ence headaches or peripheral edema because

of expanded circulatory volume.

Corticosteroids. Corticosteroids can

double the number of circulating granulo-

cytes by mobilizing them from the marginal

pool. A protocol using 60 mg of oral predni-

sone as a single or divided dose before do-

nation gives superior granulocyte harvests

with minimal systemic steroid activity.16 Al-

ternatively, 8 mg of oral dexamethasone may

be used. Before administration of cortico-

steroids, donors should be questioned about

any history or symptoms of hypertension,

diabetes, cataracts,17 and peptic ulcer.

Growth Factors. Recombinant hemato-

poietic growth factors—specifically, granulo-

cyte colony-stimulating factor (G-CSF)—

can effectively increase granulocyte yields.

Hematopoietic growth factors alone can re-

sult in collection of up to 4 to 8 ×1010 granu-

locytes per apheresis procedure.13 Typical

doses of G-CSF employed are 5 to 10 µg/kg

given 8 to 12 hours before collection.18 Pre-

liminary evidence suggests that in-vivo re-

covery and survival of these granulocytes

are excellent and that growth factors are

well tolerated by donors.13

Laboratory Testing

Testing for ABO and Rh, alloantibodies,

and infectious disease markers on a sam-

pledrawnatthetimeofphlebotomyare

required. Red cell content in granulocyte

concentrates is inevitable; the red cells

should be ABO-compatible with the re-

cipient’s plasma and, if more than 2 mL

are present, the component should be

crossmatched. Ideally, D-negative recipi-

ents should receive granulocyte concentrates

from D-negative donors. Leukocyte (HLA)

matching is recommended in alloim-

munized patients.

Storage and Infusion

Because granulocyte function deteriorates

during storage, concentrates should be

transfused as soon as possible after prep-

aration. 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 im-

munodeficient recipients and will proba-

bly be indicated for nearly all recipients

because of their primary disease. Infusion

through a microaggregate or leukocyte re-

duction 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 re-

mission, and various lymphomas (see

Chapter 25). Cytapheresis procedures can

also be used to collect donor lymphocytes

forinfusionasanimmunetherapyinthese

patients (see Chapter 25). The AABB has

published Standards for Cellular Therapy

Product Services.19 Additional requirements

are reviewed in Chapter 25.

Therapeutic Apheresis

Therapeutic apheresis has been used to

treat many different diseases. Cells, plasma,

or plasma constituents may be removed

from the circulation and replaced by nor-

mal plasma, crystalloid, or colloid solu-

144 AABB Technical Manual

tions of starch or albumin. The term

“therapeutic apheresis” is used for the

general procedure and the term “thera-

peutic plasma exchange” (TPE) is used for

procedures in which the goal is the re-

moval of plasma, regardless of the solu-

tion used as replacement.

The theoretical basis for therapeutic

apheresisistoreducethepatient’sloadofa

pathologic substance to levels that will al-

low clinical improvement. In some condi-

tions, 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 solu-

tions and/or saline should be used as re-

placement fluids. Other possible outcomes

of therapeutic apheresis include alteration

of the antigen-to-antibody ratio, modifica-

tion of mediators of inflammation or im-

munity, and clearance of immune com-

plexes. Some perceived benefit may result

from a placebo effect. Despite difficulties in

documentation, there is general agreement

that therapeutic apheresis is effective treat-

ment 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 per-

sonal physician and by the apheresis phy-

sician. Close consultation between these

physicians is important, especially if the

patient is small or elderly, has poor vascu-

lar access or cardiovascular instability, or

has a condition for which apheresis is of

uncertain benefit. The apheresis physi-

cian should make the final determination

about appropriateness of the procedure

and eligibility of the patient (see Table

6-1).3,20-22 When therapeutic apheresis is

anticipated, those involved with the pa-

tient’s care should establish a treatment

plan and the goal of therapy. The end-

point 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 ac-

ceptable goals in the patient’s medical re-

cord. The nature of the procedure, its ex-

pected benefits, its possible risks, and the

available alternatives should be explained

to the patient by a knowledgeable individ-

ual, 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 reac-

tions, including equipment, medications,

and personnel trained in managing seri-

ous 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 ef-

fective are rigid-wall, large-bore, double-

lumen catheters placed in the subclavian,

femoral, or internal jugular vein. Cathe-

ters of the type used for temporary hemo-

dialysis 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

canbeusedwhenlong-termapheresisis

anticipated.

Removal of Pathologic Substances

During TPE, plasma that contains the

pathologic substance is removed and a re-

placement 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 es-

timate depends on the following assump-

Chapter 6: Apheresis 145

146 AABB Technical Manual

Table 6-1. Indication Categories for Therapeutic Apheresis20

Disease Procedure

Indication

Suggested Reading

AABB, American Red Cross, and America’s Blood

Centers. Circular of information for the use of hu-

man blood and blood components. Bethesda, MD:

AABB, 2002.

174 AABB Technical Manual

Chapter 8: Components from Whole Blood Donations

Chapter 8

Collection, Preparation,

Storage, and Distribution of

Components from Whole

Blood Donations

DONOR 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 man-

ufacturing practice regulations, all pro-

cesses involved in the collection, testing,

preparation, storage, and transport of blood

and components are monitored for qual-

ity, including procedures, personnel, re-

agents, equipment, and the contents of the

components themselves. Processes should

ensure the potency and purity of the final

product, minimize microbial contamina-

tion and proliferation, and prevent or de-

lay 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

Components1for more detailed indica-

tions and contraindications for transfusion.

Whole Blood

Fresh Whole Blood contains all blood ele-

ments plus the anticoagulant-preservative

in the collecting bag. It is used commonly

as a source for component production.

After 24-hour storage, it essentially be-

comes red cells suspended in a protein

solution equivalent to liquid plasma, with

a minimum hematocrit of approximately

33%.

175

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-phosphate-

dextrose (CPD), citrate-phosphate-dextrose-

dextrose (CP2D), or citrate-phosphate-

dextrose-adenine (CPDA-1), the final

hematocrit must be ≤80%. Additive red

cell preservative systems consist of a pri-

mary collection bag containing an antico-

agulant-preservative with at least two sat-

ellite bags integrally attached; one is

empty and one contains an additive solu-

tion (AS). AS contains sodium chloride,

dextrose, adenine, and other substances that

supportredcellsurvivalandfunctionup

to 42 days2(seeTable8-1).Thevolumeof

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

intheprimarybagaftermostofthe

plasma has been removed. This allows

blood centers to use or recover a maxi-

mum amount of plasma, yet still prepare

a red cell component with a final hemato-

crit between 55% and 65%, a level that fa-

cilitates 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 manufac-

turer, generally within the first 72 hours of

storage. Shelf life at 1 to 6 C storage de-

pends 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. Plate-

let-rich plasma (PRP) is separated within

4 hours after completion of the phlebot-

omy or within the time frame specified in

the directions for the use of the blood col-

lecting, processing, and storage system—

typically8hours.

3The platelets are concen-

trated by an additional centrifugation step

and the removal of most of the superna-

tant 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 ade-

quate to maintain an acceptable pH; gen-

erally, 40 to 70 mL is used. Although not

approved in the United States, platelet

concentrates are commonly manufac-

176 AABB Technical Manual

Table 8-1. Content of Additive Solutions (mg/100 mL)

AS-1

(Adsol)

AS-3

(Nutricel)

AS-5

(Optisol)

Dextrose 2200 1100 900

Adenine 27 30 30

Monobasic sodium phosphate 0 276 0

Mannitol 750 0 525

Sodium chloride 900 410 877

Sodium citrate 0 588 0

Citric acid 0 42 0

tured in Europe using buffy coat as an in-

termediate 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 “soft-

spin” to remove the white cells or, more

commonly, pooled before storage (not

currently allowed by the FDA), then soft-

spun as a pooled concentrate, with ex-

pression of the PRP.4-6

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 af-

ter 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

frozen within 8 hours of collection.3See

Methods 6.10 and 6.13.

Blood centers often convert plasma and

liquid plasma to an unlicensed component,

“Recovered Plasma (plasma for manufac-

ture),” which is usually shipped to a frac-

tionator and processed into derivatives

such as albumin and/or immune globulins.

To ship recovered plasma, the collecting fa-

cility must have a “short supply agreement”

with the manufacturer.7Because recovered

plasma has no expiration date, records for

this component must be retained indefi-

nitely.

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

inthefreshplasma.CRYOcontainsboth

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 phle-

botomy. 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 com-

ponent has a 12-month expiration date

fromthedateofcollection.

8(p59) This com-

ponent is used primarily in the treatment

of thrombotic thrombocytopenic purpura.9

Granulocytes

Granulocytes are usually collected by aphere-

sis techniques; however, buffy coats har-

vested from fresh Whole Blood units can

provide a source (<1 ×109)ofgranulocytes

in urgent neonatal situations. Their effec-

tiveness is controversial, however.10 Granu-

locytes should be transfused as soon as

possible after collection but may be

stored at 20 to 24 C without agitation for

Chapter 8: Components from Whole Blood Donations 177

up to 24 hours. Arrangements for pre-

collection 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 col-

lection time is usually 4 to 10 minutes,

one study has shown platelets and fresh

frozen plasma to be satisfactory after col-

lection times of up to 15 minutes.11 The fa-

cility’s written procedures should be fol-

lowed regarding these collection times.

There should be frequent, gentle mixing

of the blood with the anticoagulant. If pre-

storage filtration is not intended after col-

lection, the tubing to the donor arm may be

stripped into the primary collection bag, al-

lowed to fill, and segmented, so that it will

represent the contents of the donor bag for

compatibility testing. Blood is then cooled

toward1to6Cunlessitistobeusedfor

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 anticoagulant-

preservative solution designed to prevent

clotting and to maintain cell viability and

function during storage. Table 8-2 com-

pares some common solutions. Citrate

prevents coagulation by chelating cal-

cium, thereby inhibiting the several cal-

cium-dependent steps of the coagulation

cascade.2

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 specifi-

cally 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-

178 AABB Technical Manual

Table 8-2. Anticoagulant-Preservative Solutions (mg in 63 mL) for 450 mL

Collections

CPD CP2D CPDA-1

Ratio (mL solution to blood) 1.4:10 1.4:10 1.4:10

FDA-approved shelf life (days) 21 21 35

Content

Sodium citrate 1660 1660 1660

Citric acid 206 206 206

Dextrose 1610 3220 2010

Monobasic sodium phosphate 140 140 140

Adenine 0 0 17.3

With 500 mL collections, the volume is 70 mL and the content 10% to 11% higher.

servative.Thevolumeofanticoagulant-pre

servative in 500 mL ±50 mL bags is 70 mL.

The allowable range of whole blood col-

lected 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 col-

lected into a blood bag designed for a

450-mL8(p28) collection, the red cells may be

used for transfusion provided the unit is la-

beled “Red Blood Cells Low Volume.” How-

ever, other components should not be pre-

pared from these low-volume units. If a

whole blood collection of less than 300 mL

is planned, the volume of anticoagulant-

preservative 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 pre-

paration 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 compo-

nents must not exceed the time frame

specified in the directions for use of the

blood collection,3processing, 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.5Set design is

based on intended use: RBCs, platelets,

FFP, Cryoprecipitated AHF, or neonatal

aliquots. Refer to Methods Section 6 for

specific component preparation proce-

dures. Whole blood must be separated

andpreparedcomponentsplacedintotheir

required storage temperatures within the

anticoagulant-preservative manufacturer’s

recommended times of collection.3Re-

cords of component preparation should

identify each individual performing a sig-

nificant 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 compo-

nents prepared in like fashion and for each

different type of collection bag. Times in-

clude the time of acceleration and “at

speed,” not deceleration time. Once the op-

erating variables are identified for compo-

nent production, timer accuracy, rpm, and

temperature (if appropriate), centrifuges

should be monitored periodically to verify

equipment performance.

Another practical way to assess centrifu-

gation 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

areinconsistent,theentireprocessshould

be evaluated. Factors affecting the quality

assessment are the calibration of the centri-

fuge, the initial platelet count on the whole

blood donations, storage time, conditions

between blood collection and platelet prep-

aration, sampling technique, and counting

methods.

Large centrifuges rotate at high speeds,

exerting gravitational forces of thousands of

pounds on blood bags. Weaknesses in these

Chapter 8: Components from Whole Blood Donations 179

blood bags or the seals between tubing seg-

ments can cause rupture and leakage. The

addition of filters for blood sets presents

different challenges for cup insertion.

Blood bags may be overwrapped with plas-

tic bags to contain any leaks. Bags should

be positioned so that a broad surface faces

the outside wall of the centrifuge to reduce

thecentrifugalforceonbloodbagseams.

Contents in opposing cups of the centri-

fuge must be equal in weight to improve

centrifuge efficiency and prevent damage to

the rotor. Dry balancing materials are pref-

erable 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.

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 reduc-

tion filter contained in the collection sys-

tem. The filtered whole blood may be

manufactured into leukocyte-reduced

RBCs. Whole blood leukocyte reduction

filters retain the platelets to a variable de-

gree, so platelet concentrates cannot be

routinely prepared. However, newly de-

signed platelet sparing filters are under

investigation. This filtration should occur

within the time specified by the filter

manufacturer.

A leukocyte reduction filter can be at-

tached to a unit of Whole Blood or RBCs

using a sterile connection device. Ideally,

such filtration should occur as early as pos-

sible after collection but must conform to

the manufacturer’s recommendations for

the specific filter in use. This method may

be preferable if special units are to be se-

lected for leukocyte reduction.

During inline filtration of red cells, plate-

lets 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 al-

lowed time frame for the specific filter in

use.

Leukocyte-reduced platelets may be pre-

pared from PRP using inline leukocyte re-

duction filtration.14 FFP manufactured using

this intermediate step typically will have a

leukocyte content of <5 ×106.

Freezing

Acellular Components

When stored at –18 C or colder, FFP con-

tains maximum levels of labile and non-

labile clotting factors (about 1 IU per mL)

and has a shelf life of 12 months from the

date of collection. FFP frozen and main-

tainedat–65Cmaybestoredupto7years.

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-anti-

freeze 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

180 AABB Technical Manual

disappears if the unit thaws. Remove

tube(s).

2. Freezing the plasma bag in a flat,

horizontal position but storing it up-

right. Air bubbles trapped along the

bag’s uppermost broad surface dur-

ing freezing will move to the top if

the unit is thawed in a vertical posi-

tion.

3. Placing a rubber band around the

liquid plasma bag and removing it

after freezing to create an indenta-

tion 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 require-

ments 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 guide-

lines apply.

Cellular Components

Frozen storage can significantly extend the

shelf life of red cell components. Unfortu-

nately, the process can also cause cell dam-

age 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 be-

fore 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 cryo-

protective agents must be used.

Cryoprotective agents are classified as

penetrating and nonpenetrating. Penetrat-

ing agents such as glycerol are small mole-

cules that freely cross the cell membrane

into the cytoplasm. The intracellular cryo-

protectant provides an osmotic force that

prevents water from migrating outward as

extracellular ice is formed. A high concen-

tration of cryoprotectant prevents forma-

tion of ice crystals and consequent mem-

brane damage.15 Glycerol, a trihydric alcohol,

is a colorless, sweet-tasting, syrup-like fluid

that is miscible with water. Pharmacologi-

cally, glycerol is relatively inert.

Nonpenetrating cryoprotective agents

(eg, hydroxyethyl starch) are large mole-

cules that do not enter the cell. The mole-

cules protect the cells by a process called

“vitrification” because they form a non-

crystalline “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 effec-

tively 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 post-

thaw 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 dis-

cussed elsewhere.16 Several instruments are

available that partially automate glyceroli-

zation and deglycerolization of red cells.

The manufacturer of each instrument pro-

videsdetailedinstructionsforitsuse.

Chapter 8: Components from Whole Blood Donations 181

Blood intended for freezing can be col-

lected into CPD, CP2D, or CPDA-1 and stored

as Whole Blood or RBCs (including AS-

RBCs). Ordinarily, red cells are glycerolized

andfrozenwithin6daysofcollectionorre

juvenated 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

removalofmostoftheplasmaoradditive

from the RBCs; others do not. The concen-

tration of glycerol used for freezing is hyper-

tonic to blood. Its rapid introduction can

cause osmotic damage to red cells, which

manifests as hemolysis only after thawing.

Therefore, when initiating the cryopreser-

vation process, glycerol should be intro-

duced 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 pri-

mary collection container suitable for

freezing (see Method 6.8). Because the cyto-

protective agent for freezing is transferred

directly into the primary collection con-

tainers, there is less chance of contamina-

tion 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

mayoccurinpolyolefinthaninsomepoly

vinyl chloride (PVC) bags. Contact between

red cells and the PVC bag surface may cause

an injury that slightly increases hemolysis

upon deglycerolization. In addition, polyo-

lefin bags are less brittle at –80 C and less

likely to break during shipment and han-

dling than PVC bags.

Freezing Process. Red cells frozen within

6 days of collection with a final glycerol

182 AABB Technical Manual

Table 8-3. Comparison of Two Methods of Red Blood Cell Cryopreservation

Consideration

High-Concentration

Glycerol

Low-Concentration

Glycerol

Final glycerol concentration (wt/vol) Approx. 40% Approx. 20%

Initial freezing temperature –80 C –196 C

Freezing rate Slow Rapid

Freezing rate controlled No 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 No

Deglycerolizing time 20-40 minutes 30 minutes

Hematocrit 55-70% 50-70%

concentration of 40% (wt/vol) must be

stored at –65 C or colder. Red cells are usu-

ally placed in canisters to protect the plastic

bagduringfreezing,storage,andthawing.

Although up to 18 hours at room tem-

perature may elapse between glycerolizing

and freezing without increased postthaw

hemolysis, an interval not exceeding 4

hours is recommended.16 With current 40%

(wt/vol) glycerol methods, controlled rate

freezing is unnecessary; freezing is accom-

plished 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 ex-

pected 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), 2,3-

diphosphoglycerate (2,3-DPG), or in-vivo

survival,19 and RBCs subjected three times

to glycerolizing, freezing, and thawing ex-

hibited a 27% loss of total hemoglobin.20

AABB Standards for Blood Banks and Trans-

fusion Services does not address refreezing

thawed units because this should not be

considered a routine practice. If thawed

units are refrozen, the records should docu-

mentthevaluablenatureofsuchunitsand

the reasons for refreezing them.

Freezing of Platelets. Perhaps because of

their greater complexity, platelets appear to

sustain greater injury during cryopreserva-

tion than red cells, although several proto-

cols have successfully used dimethyl sul-

foxide 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 wide-

spread. The primary use of this procedure

is to freeze autologous platelets for future

use. Platelet cryopreservation is essentially

a research technique.

Irradiation

Cellular blood components may be irradi-

ated before storage to prevent transfusion-

associated graft-vs-host disease (GVHD).

This does not shorten the shelf life of pla-

telets 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 at-

tach additional bags and compatible tub-

ing 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 com-

pleteness, integrity, leakage, and air bub-

bles; procedures must address the action

to take if the weld is not satisfactory. Re-

cord-keeping should include documenta-

tion of the products welded, weld quality

control, and lot numbers of disposables.23

Cryoprecipitated AHF

Units of CRYO may be pooled before la-

beling, freezing, and storage. If pooled

promptly after preparation using aseptic

technique and refrozen immediately, the

resulting component is labeled “Cryopre-

cipitated 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 Components1instead 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)

Chapter 8: Components from Whole Blood Donations 183

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 re-

quire transfusion within 4 hours for pooled

thawed components stored at 20 to 24

C.8(p58)

Platelets

Prestorage pooling of PRP whole-blood-

derived platelets is possible using a sterile

connection device and a storage con-

tainer suitable for storage of a high-yield

platelet concentrate. Platelet concentrates

can be leukocyte-reduced using inline fil-

tration14 or they can be pooled into a

pooling container, then subsequently leu-

kocyte-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 leu-

kocyte-reduced pooled platelet concen-

trates 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 re-

quire transfusion of pooled platelets within

4 hours of pooling.

As indicated earlier, buffy-coat-derived

platelets are commonly pooled before stor-

age with filtration of the PRP after centri-

fugation.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 tem-

peratures continuously and record them

at least every 4 hours, and an alarm sys-

tem with an audible signal that activates

before blood reaches unacceptable storage

temperatures.

Interiors should be clean, adequately

lighted, and well organized. Clearly desig-

nated and segregated areas are needed for:

1) unprocessed blood; 2) labeled blood

suitable for allogeneic transfusion; 3) re-

jected, 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, tis-

sues, 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 pres-

ent. It is usually most practical to make

blood bank personnel responsible for mon-

itoring 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 medi-

cal director may wish to extend the stor-

age 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 docu-

mented. As units are put into long-term

storage, many consider it prudent to freeze

samples of serum or plasma for subse-

quent testing should new donor screening

testsbeintroducedinthefuture.Thetype

of any specimen saved, date of collection,

date of freezing, and specimen location, if

necessary, should be included in records

of frozen blood.

184 AABB Technical Manual

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 in-

dicate that the unit has not been com-

pletely tested and should identify the miss-

ing test(s) results.

Bloodfreezershavethesametempera

ture monitoring and alarm requirements as

blood refrigerators and must be kept clean

and well organized. Freezers designated for

plasma storage must maintain tempera-

tures colder than –18 C (many function at

–30 C or colder); RBC freezers must main-

tain temperatures colder than –65 C (many

maintain temperatures colder than –80 C).

Self-defrosting freezers must maintain ac-

ceptable temperatures throughout their

defrost cycle.

Freezer alarm sensors should be accessi-

ble and located near the door, although

older units may have sensors located be-

tween the inner and outer freezer walls

where they are neither apparent nor acces-

sible. In such cases, the location of the sen-

sor 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

foreasieruse.

Liquid nitrogen tanks used for blood

storage also have alarm system require-

ments. The level of liquid nitrogen should

be measured and the sensor placed some-

where above the minimum height needed.

Room Temperature Storage

Platelets require gentle, continuous agita-

tion during storage because stationary

platelets display increased lactate pro-

duction and a decrease in pH. Elliptical,

circular, and flat-bed agitators are avail-

able for tabletop or chamber use. Ellipti-

cal 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, pro-

vided the temperature is recorded every 4

hours during storage. Because room tem-

peratures fluctuate, “environmental” or

“platelet chambers” have been developed to

provide consistent, controlled room temper-

atures. 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 re-

duction 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 mas-

sively transfused recipients. Hemoglobin

becomes fully saturated with oxygen in

the lungs but characteristically releases

only some of its oxygen at the lower oxy-

gen pressure (pO2) of normal tissues. The

relationship between pO2and oxygen satu-

ration of hemoglobin is shown by the oxy-

gendissociationcurve(seeFig8-1).Re

lease of oxygen from hemoglobin at a

given pO2is 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-

Chapter 8: Components from Whole Blood Donations 185

186 AABB Technical Manual

Table 8-4. Biochemical Changes in Stored Non-Leukocyte-Reduced Red Blood Cells

CPD CPDA-1 AS-1 AS-3 AS-5

Variable Whole Blood

Whole

Blood

Red Blood

Cells

Whole

Blood

Red Blood

Cells

Red Blood

Cells

Red Blood

Cells

Red Blood

Cells

Days of Storage 0 21 0 0 35 35 42 42 42

% Viable cells

(24 hours

posttransfusion)

100 80 100 100 79 71 76 (64-85) 84 80

pH (measure at 37 C) 7.20 6.84 7.60 7.55 6.98 6.71 6.6 6.5 6.5

ATP (% of initial

value)

100 86 100 100 56 (± 16) 45 (± 12) 60 59 68.5

2,3-DPG (% of initial

value)

100 44 100 100 <10 <10 <5 <10 <5

Plasma K+ (mmol/L) 3.9 21 4.20 5.10 27.30 78.50* 50 46 45.6

Plasma hemoglobin 17 191 82 78 461 658.0* N/A 386 N/A

% Hemolysis N/A N/A N/A N/A N/A N/A 0.5 0.9 0.6

*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.

tive change in anemia; lower red cell lev-

els of 2,3-DPG increase the affinity of he-

moglobin 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 intra-

cellular pH caused by lactic acid, which

increases the activity of a diphosphatase.26

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 re-

generate half their 2,3-DPG levels, and

about 24 hours for complete restoration of

2,3-DPG and normal hemoglobin func-

tion.26,27

Red cells lose potassium and gain so-

dium 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 co-

efficient and functions poorly in the cold.

Supernatant levels of potassium in a unit of

CPDA-1 RBCs have been reported to in-

crease from 5.1 mmol/L on the day of col-

lection to 23 mmol/L on day 7 and 75

mmol/L on day 35. Intracellular levels of

potassium will be replenished after transfu-

sion.

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 consid-

ered 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,

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 pO2(40 mm Hg), 25% to 30% of the oxygen is normally re-

leased. In stored RBCs, this will decrease to 10% to 15%.

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 pa-

tients, 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 Forbetterpreserva

tion of Factors V and VIII and platelets,

whole blood is separated into its compo-

nent 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

countriesis5days(Table8-5).Thistime

limitation is partly related to concerns

about storage-related deterioration in

product potency28 and partly to the poten-

tial for bacteria to grow rapidly in this

temperature range.29-30 With regard to po-

tency, 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 pla-

telet shape from discoid to spherical; the

generation of lactic acid from glycolysis,

with an associated decrease in pH; the re-

lease of cytoplasmic and granule con-

tents; a decrease in various in-vitro mea-

sures of platelet function, particularly

osmotic challenge; shape changes in-

duced by adenosine diphosphate; and re-

duction 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 via-

bility and function.33 Attempts to date to

define the biochemical nature of the

platelet storage lesion have not been con-

clusive. These observed changes may rep-

resent 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. Rest-

ing platelets derive substantial energy

from β-oxidation of fatty acids.34 Alter-

ation in mitochondrial integrity would re-

sult 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 genera-

tion of efficient ATP and result in a de-

crease in the metabolic pool of ATP and,

therefore, the energy charge of the pla-

telet.35 This decrease in the energy charge

wouldbeexpectedtoaffectmembrane

integrity, resulting in a leakage of cyto-

plasmic contents, a diminished response

to physiologic stimuli, and an inability to

repair oxidized membrane lipids, with

subsequent distortions in platelet mor-

phology.36

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 an-

ticoagulant-preservative require that at

least 75% of the original red cells (from a

normal allogeneic donor) be in the recipi-

ent’s circulation 24 hours after transfusion.

For other components, their shelf life is

based on functional considerations. Stor-

age times are listed in Table 8-5.

188 AABB Technical Manual

Chapter 8: Components from Whole Blood Donations 189

Table 8-5. Expiration Dates for Selected Blood Components8(pp53-60)

Category Expiration

Whole Blood ACD/CPD/CP2D – 21 days

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)

Poststorage Processing

Additional discussion of some of the fol-

lowing 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) re-

moval needed to meet the 5 ×106specifi-

cation.37 Red cell leukocyte reduction fil-

ters contain multiple layers of synthetic

nonwoven fibers that retain white cells and

platelets, allowing red cells to flow through.

Leukocyte reduction filters are commer-

cially available in a number of set config-

urations 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, pre-

storage leukocyte reduction is preferred.39

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

(within24hours)inthecaseofRBCsmay

reduce the likelihood of significant bacte-

rial contamination.40 Bedside filtration, par-

ticularly of platelets, may not be as effective

in preventing reactions in multitransfused

patients and is less desirable for this reason

than prestorage leukocyte reduction.41 Bed-

side filtration has also caused hypotensive

reactions.42 Cytokines and other substances

that accumulate during storage (particu-

larly 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

190 AABB Technical Manual

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

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.

Table 8-5. Expiration Dates for Selected Blood Components8(pp53-60) (cont'd)

Thawing

Thawing FFP

FFP is thawed either at temperatures be-

tween 30 and 37 C or in an FDA-cleared

device.8(p59) It is then known as “FFP

Thawed” and should be transfused imme-

diately 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 con-

taminated with water. This can be accom-

plished by wrapping the container in a

plastic overwrap, or by positioning the con-

tainer 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 in-

dicate if the temperature rises unaccept-

ably.Aswithanydevice,thereshouldbea

procedure for the quality control of indi-

cated functions.

When whole-blood-derived FFP pre-

pared in a closed system is thawed but not

transfused within 24 hours, the label must

be modified. This product should be la-

beled “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 re-

duction 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 re-

main at 30 to 37 C once thawed, so that

degradation of Factor VIII is minimized.

As with FFP, entry ports should be pro-

tected from water contamination if the

unit is thawed in a waterbath. Single-unit

thawed CRYO must be transfused imme-

diately or can be stored at room tempera-

ture (20 to 24 C) for no more than 6 hours.

All pooled CRYO, whether prepared in an

open or a closed system, must be trans-

fused 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 im-

mersed in a 37 C waterbath. Units frozen

in the primary collection bag system

should be thawed at 42 C.16 The thawing

process takes at least 20 to 25 minutes and

should not exceed 40 minutes. Gentle agi-

tation may be used to speed thawing.

Thawed cells contain a high concentra-

tion of glycerol that must be reduced grad-

ually 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 de-

crease in osmolarity of the washing solu-

tions causes osmotic swelling of the cells,

so each solution must be added slowly, with

adequate time allowed for mixing and os-

motic equilibration. Any of the commer-

cially available instruments for batch or

continuous-flow washing can be used to

deglycerolize red cells frozen in a high con-

centration of glycerol. Because there are

many potentially important variations in

deglycerolization protocols for each instru-

Chapter 8: Components from Whole Blood Donations 191

ment, personnel in each facility should not

only follow the manufacturer’s instructions,

but also validate the local process. The pro-

cess selected must ensure adequate re-

moval of cryoprotectant agents, minimal

free hemoglobin, and recovery of greater

than 80% of the original red cell volume af-

ter 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 degly-

cerolized unit if it is different from the col-

lection facility.

When glycerolized frozen red cells from

persons with sickle cell trait are suspended

in hypertonic wash solutions during de-

glycerolization 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, dona-

tions are screened for the presence of he-

moglobin S before being frozen.

When glycerolization or deglyceroliza-

tion involves entering the blood bag, the

system is considered “open” and the result-

ing suspension of deglycerolized cells can

be stored for only 24 hours at 1 to 6 C. A

method for glycerolization and degly-

cerolization 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 concentra-

tions of potassium and hemoglobin in the

supernatant fluid. Red cells that have un-

dergone 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 trans-

fused T lymphocytes in recipients at risk

of acquiring, or from donors at risk of caus-

ing, GVHD. The AABB and FDA recom-

mend a minimum 25 Gy dose of gamma

radiation to the central portion of the

container, with no less than 15 Gy deliv-

ered to any part of the bag.8(p26)

Irradiation is accomplished using ce-

sium-137 or cobalt-60, in self-contained

blood irradiators or hospital radiation ther-

apy machines. More recently, an x-ray de-

vice 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 expo-

sure time as the radioactive source decays

should be addressed in the facility’s proce-

dures.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 be-

fore irradiation. When exposed to an ade-

quate amount of radiation, the film portion

of the label darkens, indicating that the

component has been exposed to an ade-

quate radiation dosage. Because irradiation

damages red cells and reduces the overall

viability (24-hour recovery),49 red cell com-

ponents that have been irradiated expire on

their originally assigned outdate or 28 days

from the date of irradiation, whichever co-

mes first. Platelets sustain minimal damage

from irradiation, so their expiration date

192 AABB Technical Manual

does not change.50 Irradiated blood is es-

sential for patients at risk from transfu-

sion-associated GVHD, including fetuses

receiving intrauterine transfusion, select

immunocompetent or immunocompro-

mised recipients, recipients who are under-

going hematopoietic transplantation, recip-

ients of platelets selected for HLA or platelet

compatibility, and recipients of donor units

from blood relatives.

Washing

Washing a unit of RBCs with 1 to 2 L of

sterile normal saline removes about 99%

of plasma proteins, electrolytes, and anti-

bodies. 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 sys-

tem, and removal of the anticoagulant-

preservative solution compromises long-

term preservation of cell viability and

function.

Platelets can be washed with normal sa-

line 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 prod-

uct should be labeled “Platelets Pooled.”

If Platelets contain a significant number

of red cells and ABO groups are mixed,

plasma antibodies should be compatible

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

storageat20to24C.

Volume Reduction

Platelets may be volume-reduced in order

to decrease the total volume of the com-

ponent transfused or partially remove

supernatant substances, such as ABO allo-

antibodies. 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 expi-

ration of the product becomes 4 hours. If

sterility is not broken (eg, a sterile con-

nection device is used23), removal of the

supernatant still reduces glucose avail-

ability 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-vol-

ume 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 al-

tered by aliquoting, unlike volume reduc-

tion. 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

Chapter 8: Components from Whole Blood Donations 193

dependent on the storage bag, plasma

volume, and storage environment. Re-

moving aliquots of platelets from the

“mother” bag changes the storage envi-

ronment of the platelets remaining in the

“mother” bag. If the altered storage envi-

ronment does not meet the storage bag

manufacturer’s requirements, the expira-

tion 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-li-

censed solution containing pyruvate,

inosine, phosphate, and adenine (see

Method 6.6). RBCs may be rejuvenated af-

ter 1 to 6 C storage up to 3 days after expi-

ration; then, they may be glycerolized and

frozeninthesamemannerasfreshred

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 ac-

tion taken, and the identity of personnel

involved. Visual inspections cannot al-

ways detect contamination or other dele-

terious conditions; nonetheless, blood

components that look abnormal must not

be shipped or transfused.

Deleterious conditions should be sus-

pected if52: 1) segments appear much

lighter in color than what is in the bag (for

AS-RBCs),2)theredcellmasslookspurple,

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, pur-

ple, brown, or red. A green hue from

light-induced changes in bilirubin pig-

ments need not cause the unit to be re-

jected. Mild lipemia, characterized by a

milky appearance, does not render a dona-

tion 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 re-

sponsible person determines its disposi-

tion. 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 alter-

ations may be present in the undisturbed

red cells. If, after resuspension, resettling,

and careful examination, the blood no lon-

ger 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 evi-

dence of thawing and refreezing and for ev-

idence of cracks in the tubing or plastic

bag. Unusual turbidity in thawed compo-

nents may be cause for discard.

All platelet components should be in-

spected 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

194 AABB Technical Manual

swirl phenomenon correlates well with pH

values associated with adequate platelet

in-vivo viability.53 Some platelet compo-

nents have been noted to contain small

amounts of particulate matter. These com-

ponents are suitable for use.

Bacterial contamination of transfusion

components is rare because of the use of

aseptic technique and screening for bacte-

ria in platelets, the availability of closed sys-

tems for collection and preparation, and

careful control of storage conditions. Steril-

ity 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 reac-

tion 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 cen-

ter staff on sample requirements and appro-

priate test methods for detecting potential

blood contaminants, including cryophilic mi-

croorganisms. Making cultures directly from

the contents of the bag and from the recipi-

ent can provide useful diagnostic information.

Any facility that collected a reportedly

contaminated component should be noti-

fied so that donor bacteremia, potentially

inadequate donor arm preparation, or im-

proper 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 re-

quires additional packaging. Transport

containers or coolers and packaging pro-

cedures must be validated before use to

verify that they are able to maintain blood

components at required temperatures for

the intended time and conditions. Con-

tainersmustalsobeabletowithstand

leakage, pressure, and other conditions

incidental to routine handling. Refer to

Chapter 2 for more information on ship-

ping regulations and guidelines.

Simple exposure to temperatures outside

the acceptable range does not necessarily

render blood unsuitable for transfusion. Ex-

ceptions may be made under unusual cir-

cumstances 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 in-

clude the length of time in shipment, mode

of transportation, magnitude of variance

above or below the acceptable range, pres-

ence of residual ice in the shipping box, ap-

pearance 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 fa-

cility 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 tempera-

ture of 25 C. Smaller units, as are com-

monly used for pediatric patients, can

warm even more quickly.

Wet ice, securely bagged to prevent leak-

age, is the coolant of choice to maintain re-

quired 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-

Chapter 8: Components from Whole Blood Donations 195

port temperatures. Super-cooled ice (eg,

large blocks of ice stored at –18 C) in con-

tact 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 ship-

ment. 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 enve-

lopes or newspaper are excellent insula-

tors. For very long distances or travel

times in excess of 24 hours, double-insu-

lated 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 us-

ing a suitable quantity of dry ice in well-

insulated containers or in standard ship-

ping cartons lined with insulation mate-

rial, 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 op-

timal conditions for shipping frozen com-

ponents, which depend on the temperature

requirements of the component, the dis-

tance to be shipped, the shipping container

used, and ambient temperatures to be en-

countered. Procedures and shipping con-

tainers should be validated and periodically

monitored. The receiving facility should al-

ways observe the shipment temperature

and report unacceptable findings to the

shipping facility.

Red cells cryopreserved with high-con-

centration 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 posttrans-

fusion survival, so transport with dry ice is

acceptable. Blood shipments containing

dry ice as a coolant are considered “danger-

ous goods,” and special packaging and la-

beling requirements apply (see Chapter 2).

Shipping boxes containing dry ice must not

be completely sealed so that the carbon di-

oxide gas released as the dry ice sublimes

can escape without risk of explosion.

Disposition

Both blood collection sites and transfu-

sion 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 re-

turned 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 techni-

ques, donor screening methods, or the

handling of units during processing, stor-

age, or transport.

Disposal procedures must conform to the

local public health code for biohazardous

waste. Autoclaving or incineration is rec-

ommended. If disposal is carried out off-

site, 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 biohazar-

dous 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

196 AABB Technical Manual

concerns and institutional preferences.

However, blood should never unnecessar-

ily be allowed to reach temperatures out-

side 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 reis-

sued 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 main-

tained continuously between 1 and

10 C, preferably 1 to 6 C. Blood cen-

ters and transfusion services usually

donotreissueRBCunitsthathave

remained out of a monitored refrig-

erator or a validated cooler for lon-

ger 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 inte-

gral donor tubing remains attached

to the red cell component, if the

blood has left the premises of the is-

suing 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 com-

ponents requires applying the principles

of quality assurance to all aspects of com-

ponent collection, preparation, testing,

storage, and transport. All procedures and

equipment in use must be validated be-

fore their implementation and periodi-

cally monitored thereafter. Staff must be

appropriately trained and their compe-

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 con-

sideration 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 mon-

itoring sensors connected to recording

charts or digital readout systems for easy

surveillance. Digital recording devices

measure the differences in potential gen-

erated by a thermocouple; this difference

is converted to temperature. Because warm

air rises, temperature-recording sensors

should be placed on a high shelf and im-

mersed in a volume of liquid not greater

than the volume of the smallest compo-

nent stored. Either a glass container or a

plastic blood bag may be used. Recording

charts and monitoring systems must be in-

spected 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 temper-

ature should be explained in writing on the

chart beside the tracing or on another doc-

ument 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

Chapter 8: Components from Whole Blood Donations 197

alarm monitoring system that monitors all

equipment continuously and simultane-

ously 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 equip-

ment reaches its predetermined tempera-

ture alarm point and indicates the equip-

ment in question. Blood storage equipment

so monitored does not require a separate

independent recording chart.

Thermometers

Visual thermometers in blood storage equip-

ment provide ongoing verification of tem-

perature accuracy. One should be immersed

in the container with the continuous mon-

itoring sensor. The temperature of the

thermometer should be compared peri-

odically with the temperature on the re-

cording 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 Tech-

nology (NIST) and suitable corrective ac-

tion should be taken (see Method 7.2). (A

2 C variation between calibrated ther-

mometers allows for the variation that

may occur between thermometers cali-

brated against the NIST thermometers.)

Thermometers also help verify that the

temperature is appropriately maintained

throughout the storage space. Large refrig-

erators or freezers may require several ther-

mometers to assess temperature fluctua-

tions. 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 low-

est shelf on which blood is stored. The tem-

perature in both areas must be within the

required range at all times.

Either liquid-in-glass (analog) thermom-

eters or electronic and thermocouple (digi-

tal) devices can be used for assessing stor-

age temperatures, as long as their accuracy

is calibrated against a NIST-certified ther-

mometer or a thermometer with a NIST-

traceable calibration certificate (see Method

7.2). Of equal importance is that they be

used as intended, according to the manu-

facturer’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 tempera-

ture 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; ei-

ther a continuously rechargeable battery

or an independent electrical circuit served

by an emergency generator is acceptable.

Method 7.3.1 provides a detailed proce-

dure to test the temperatures of activation

for refrigerator alarms. Suggestions for

freezer alarms are in Method 7.3.2 Ther-

mocouple devices that function at freezer

temperatures are especially useful for de-

termining the temperature of activation

with accuracy when sensors are accessible.

When they are not, approximate activation

temperatures can be determined by check-

ing a freezer’s thermometer and recording

chart when the alarm sounds after it is shut

down for periodic cleaning or mainte-

nance. 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 in-

structions for personnel to follow when the

alarm sounds. These instructions should

include steps to determine the immediate

cause of the temperature change and ways

198 AABB Technical Manual

to handle temporary malfunctions, as well

as steps to take in the event of prolonged

failure. It is important to list the names of

keypeopletobenotifiedandwhatsteps

should be taken to ensure that proper stor-

age 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 vali-

dation and periodically confirmed by

quality control procedures. RBCs Leuko-

cytes Reduced should contain <5 ×106re-

sidual 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.)

Quality Control of Platelets

The quality of every method of platelet

preparation must be assessed periodi-

cally.Datamustshowthatatleast90%of

components tested contain an acceptable

number of platelets (5.5 ×1010 for whole-

blood-derived platelets) and have a plasma

pH of 6.2 or higher at the end of the allow-

able storage period.8(p36)

Prestorage leukocyte-reduced Platelets

derived from filtration of platelet-rich

plasma must contain less than 8.3 ×105re-

sidual leukocytes per unit to be labeled as

leukocyte reduced. Validation and quality

control should demonstrate that at least

95% of units sampled meet this require-

ment.8(p31)

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

mg of fibrinogen.8(p30) Each pool must have

aFactorVIIIcontentofatleast80mg

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.)

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52. Kim DM, Brecher ME, Bland LA, et al. Visual

identification of bacterially contaminated red

cells. Transfusion 1992;32:221-5.

53. Bertoloni F, Murphy S. A multicenter inspec-

tion of the swirling phenomenon in platelet

concentrates prepared in routine practice.

Transfusion 1996;36:128-32.

54. Dumont LJ, Dzik WH, Rebulla P, Brandwein

H, and the Members of the BEST Working

Party of the ISBT. Practical guidelines for pro-

cess validation and process control of white

cell-reduced blood components: Report of

theBiomedicalExcellenceforSaferTransfu-

sion (BEST) Working Party of the Interna-

tional Society of Blood Transfusion (ISBT).

Transfusion 1996;36:11-20.

Chapter 8: Components from Whole Blood Donations 201

202 AABB Technical Manual

Appendix 8-1. Component Quality Control

Component

Specifications

and Standards*

AABB

Standards

†

Red Blood Cells Hematocrit ≤80% (in all) 5.7.5.1

Red Blood Cells

Leukocytes

Reduced

Retain 85% of original red cells, 95% of tested

units <5 ×106leukocytes in the final con-

tainer

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 ×105leukocytes in 95% of units tested

5.7.5.17

Platelets Pheresis ≥3.0 ×1011 platelets in final container of compo-

nents tested; and pH ≥6.2 in 90% of units

tested

5.7.5.19

Platelets Pheresis

Leukocytes

Reduced

<5.0 ×106leukocytes 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 com-

ponents tested

5.7.5.21

Irradiated compo-

nents

25 Gy delivered to the central portion of the con-

tainer; 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 thresh-

old. The manufacturing procedures used should be validated as capable of meeting these standards before implementa-

tion 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

PROTEINS ARE MACROMOLECULES

composed of amino acids, the se-

quences of which are determined

by genes. Lipids and carbohydrates are

not encoded directly by genes; genetic de-

termination of their assembly and func-

tional structures results from the action of

different protein enzymes. Blood group

antigens can be considered gene prod-

ucts, either directly, as polymorphisms of

membrane-associated proteins, or indi-

rectly, as carbohydrate configurations cat-

alyzed 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 chro-

mosome consists of long molecules or

strands of deoxyribonucleic acid (DNA).

DNA is composed of the sugar deoxyribose,

a phosphate group, the purine bases ade-

nine (A) and guanine (G), and the pyrimi-

dine 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 com-

plementary (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 configu-

ration with the sugar-phosphate back-

bone on the outside and the paired bases

ontheinside(seeFig9-1).DNAsynthesis

is catalyzed by DNA polymerase, which

adds a deoxyribonucleotide to the 3′end

oftheexistingchain.The3′and 5′nota-

tion refers to the carbon position of the

deoxyribose linkage to the phosphate

group. Phosphate groups bridge the sugar

groups between the fifth carbon atom of

one deoxyribose molecule and the third

carbon atom of the adjacent deoxyribose

203

molecule and thus create the backbone of

the DNA strand. DNA polymerase-depend-

ent synthesis always occurs in the direc-

tion 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 infor-

mation encoded in the DNA sequence

must be copied into RNA (transcription)

and transported to the cytoplasmic organ-

elles called ribosomes where protein as-

sembly takes place (translation). Tran-

scription 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 re-

placement of thymine by uracil (U), which

also pairs with adenine. DNA transcrip-

tion is catalyzed by the enzyme RNA poly-

merase. RNA polymerase binds tightly to a

specific DNA sequence called the pro-

moter, which contains the site at which

RNA synthesis begins (see Fig 9-3). Pro-

teins called transcription factors are re-

quired for RNA polymerase to bind to

DNA and for transcription to occur. Regu-

lation of transcription can lead to in-

creased, decreased, or absent expression

of a gene. For instance, a single base-pair

mutation in the transcription factor bind-

ing site of the Duffy gene promoter im-

pairs the promoter activity and is respon-

sible for the Fy(a–b–) phenotype.1

After binding to the promoter, RNA poly-

merase opens up the double helix of a local

region of DNA, exposing the nucleotides on

each strand. The nucleotides of one ex-

posed DNA strand act as a template for com-

plementary base pairing; RNA is synthe-

sized by the addition of ribonucleotides to

theelongatingchain.AsRNApolymerase

movesalongthetemplatestrandofDNA,

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 en-

zyme halts synthesis and releases both the

DNAtemplateandthenewRNAchain.

mRNA Processing

Shortly after the initiation of transcrip-

tion, the newly formed chain is capped at

204 AABB Technical Manual

Figure 9-1. Schematic representation of the base

pairing of double-stranded DNA.

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 cy-

toplasm. 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 se-

quence 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 re-

quired for efficient translation.

In eukaryotic cells, the nucleotide se-

quence of a gene often contains certain re-

gions that are represented in the mRNA and

other regions that are not represented. The

Chapter 9: Molecular Biology in Transfusion Medicine 205

Figure 9-2. Model of a nucleotide sequence. The sequence is fictitious.

↓

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 nucleo-

tides in introns; uppercase letters represent coding bases in exons. The sequence is fictitious.

regions of the gene that are represented in

mRNA are called exons, which specify the

protein-coding sequences and the se-

quences of the untranslated 5′and 3′re-

gions. The regions that are not represented

in the mRNA are called intervening se-

quences or introns. In the initial transcrip-

tion of DNA to RNA, the introns and exons

are copied in their entirety, and the result-

ing 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).

RNAsplicingdependsonthepresenceof

certain highly conserved sequences con-

sisting 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 cleav-

age 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 inaccu-

rate RNA splicing (see Fig 9-4). Splicing of

pre-RNA is highly regulated during differ-

entiation and is tissue-specific. For example,

acetylcholinesterase, which bears the Cart-

wright blood group antigen, is spliced in a

manner to produce a glycosylphosphati-

dylinositol-linked protein in red cells, but it

is a transmembrane protein in nerve cells.2

Alternative splicing may also occur in a sin-

gletissuetypeandmayresultinthepro

ductionofmorethanoneproteinfromthe

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 combina-

tion 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 “degen-

eracy” or “redundancy” in the genetic

code. For example, lysine can be specified

206 AABB Technical Manual

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).

by either AAA or AAG. Methionine has

only the single codon AUG. Three codons

(UAA, UGA, and UAG) function as stop sig-

nals; when the translation process en-

counters one of them, peptide synthesis

stops.

For the translation of codons into amino

acids, cytoplasmic mRNA requires the as-

sistance 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)groupandendswithatermi-

nal carboxylic acid (COOH) group. Protein

synthesis occurs on ribosomes, which are

large complexes of RNA and protein mole-

cules; ribosomes bound to the rough endo-

plasmic reticulum are the site of synthesis

of membrane and secretory proteins, where-

asfreeribosomesarethesiteofsynthesisof

cytosolicproteins.Theribosomebindsto

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 tran-

scription process, addition of the poly-A tail,

transportation of mRNA to the cytosol, ini-

tiation of translation, and elongation of the

polypeptide chain. Moreover, tissue-specific

and differentiation or stage-specific tran-

scriptionfactors,aswellashormonere

sponse elements and regulatory elements

at the 5´ and 3´ ends of mRNA, can affect

gene expression.

Genetic Mechanisms that

Create Polymorphism

Despite the redundancy inherent in de-

generacy of the genetic code, molecular

events such as substitution, insertion, or

deletion of a nucleotide may have far-

reaching 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 re-

sultant protein. Many blood group anti-

gens 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

sequencefromaTtoaCatagivenposi

tion 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

wouldbenoeffectontheprotein

because the codon would still be

translated as serine.

Chapter 9: Molecular Biology in Transfusion Medicine 207

2. Missense response. The substitution

of a G with an A in the second posi-

tion 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 mole-

cule. 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 substitu-

tion in exon 6 of the Kell (KEL)gene.

3. Nonsense response. The substitu-

tion of a G with a T in the second po-

sition 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 syn-

thesis 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 func-

tion 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 nucleo-

tide substitution of the DAF gene

creates a stop codon at position 53,

andtheredcellhasnoDAFexpres

sion.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 glyco-

phorin B (GPB)6or in the failure to produce

a normal amount of protein, as in the

Dr(a–) phenotype of Cromer.7In 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 re-

sults in a frameshift, described as +1, be-

cause a nucleotide is being added. Nucle-

otide deletion causes a –1 frameshift. A

peptide may be drastically altered by the

insertion or deletion of a single nucleo-

tide. For example, the insertion of an A af-

ter the second nucleotide of the noncoding

DNA strand of Fig 9-2 would change the

readingframeofthemRNAtoUAUCAG

AAG CUG CCC UGG and represent the

polypeptide isoleucine-valine-phenylala-

nine-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. Exam-

ples 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 anti-

gens 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 en-

coded by the nucleotides at the junction

of the hybrid gene may result in epitopes

recognized by antibodies in human se-

rum and are known to occur in at least

three blood group systems: Rh, MNS, and

Gerbich.

208 AABB Technical Manual

Gene Conversion

The process of gene conversion is thought

to consist of crossover, a general DNA re-

combination process, and DNA repair

duringmeiosis.Theresultisthatnucleo

tides from one homologous gene are in-

serted into another gene without recipro-

cal exchange. At the site of chromosome

crossover during meiosis, a heteroduplex

joint can form; this is a staggered joint be-

tween nucleotide sequences on two par-

ticipating DNA strands (see Fig 9-6). A

second type of gene conversion occurs

in meiosis when the DNA polymerase

switches templates and copies informa-

tion from a homologous sequence. This

event is usually the result of mismatch

repair; nucleotides removed from one

strand are replaced by repair synthesis us-

ing the homologous strand as a template.

Molecular Techniques

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 foren-

sic science, the generation of recombi-

nant proteins, and the production of func-

tional 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 purifica-

tion of nucleic acid, either DNA or RNA.

For applications of interest to the blood

banking community, the desired nucleic

Chapter 9: Molecular Biology in Transfusion Medicine 209

Figure 9-5. Single crossover: exchange of nucleotides between misaligned homologous genes. The

products are reciprocal.

acid is typically human genomic DNA and

mRNA. Genomic DNA is present in all nu-

cleated cells and can be isolated from pe-

ripheral 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 pu-

rity 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-

210 AABB Technical Manual

Figure 9-6. Gene conversion: a heteroduplex joint forms between homologous sequences on two genes.

DNA polymerase repairs the double strands. Any excess single-stranded DNA is degraded by nu-

cleases, producing a hybrid gene on one chromosome but not on the other.

ing 1.8. Low ratios (<1.6) indicate that the

DNA is contaminated with protein or mate-

rials 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 con-

centration, it is best quantitated by electro-

phoresis in agarose gel along with DNA

standards of known concentration, fol-

lowed 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 cru-

cial, 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-molec-

ular-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 µgof

DNA. Commercial DNA isolation kits typi-

cally yield in excess of 15 µgofDNAper

milliliter of whole blood processed.

Polymerase Chain Reaction

The introduction of the PCR technique has

revolutionized the field of molecular ge-

netics.8This 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 se-

quence under study, provided a part of the

nucleotide sequence is known. The inves-

tigator 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′)

and a reverse primer (3′). These are de-

signed 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 subse-

quent cloning or labeled to facilitate its

detection. Labels may incorporate radio-

activity, or, more frequently, a nonradio-

active 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 na-

tive 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

Theamplificationtechniqueissimpleand

requires very little DNA (typically <100 ng

ofgenomicDNA).TheDNAunderstudy

is mixed together with a reaction buffer,

excess nucleotides, the primers, and poly-

merase (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 amplifi-

cation of the DNA. The target DNA is ini-

tially denatured by heating the mixture,

which separates the double-stranded DNA

into single strands. Subsequent cooling in

the presence of excess quantities of sin-

gle-stranded forward and reverse primers

allows them to bind or anneal with com-

plementary sequences on the single-

stranded template DNA. The specific cool-

ing temperature is calculated to be appro-

priate for the primers being used. The re-

action mixture is then heated to the

optimal temperature for the thermo-sta-

ble DNA polymerase, which generates a

new strand of DNA on the single-strand

template, using the nucleotides as build-

Chapter 9: Molecular Biology in Transfusion Medicine 211

212 AABB Technical Manual

Figure 9-7. The polymerase chain reaction results in the exponential amplification of short DNA se-

quences 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.)

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. Alter-

natively, the DNA sample can be blotted

onto a membrane and hybridized to a la-

beled, 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 devel-

oped 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 tech-

nology, 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 seg-

ment. Other PCR variations include multi-

plex 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.

Applications of PCR

Amplification of minute quantities of DNA

to detectable levels may significantly af-

fect 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

WestNilevirusRNAhasbeenperformed

as an investigational test. Detection of vi-

ral RNA involves three steps: extraction,

amplification, and detection. Extraction

may occur following centrifugation steps

to concentrate the virus and remove con-

taminants. 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 parti-

cles 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 ac-

complished by the enzyme, reverse tran-

scriptase (RT). Amplification of the DNA

then can occur through multiple interme-

diates. In the case of PCR, the amplified

product is DNA (and is synthesized using

thermostable Taq DNA polymerase),

whereas, in the case of transcription-me-

diated amplification, the amplified prod-

uct 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 nitro-

cellulose or by enzyme immunoassay or

chemiluminescence. Currently, NAT for

blood donor screening has been imple-

mented for HIV and hepatitis C; NAT for

hepatitis B is under development. NAT for

Chapter 9: Molecular Biology in Transfusion Medicine 213

other agents (eg, human T-cell lympho-

tropic virus, hepatitis A virus, parvovirus

B19) is not widely available.

PCR is being used for prenatal determi-

nation of many inheritable disorders, such

as sickle cell disease, in evaluating hemolytic

disease of the fetus and newborn, to type

fetal amniocytes,9to 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 appli-

cability in transfusion medicine because of

its powerful ability to detect genetic vari-

ants. In the field of transplantation, PCR us-

ing sequence-specific oligonucleotide

probes or sequence-specific primers is

used to determine HLA types (see Chapter

17).

Restriction Endonucleases

The discovery of bacterial restriction endo-

nucleases provided the key technique for

DNA analysis. These enzymes, found in

different strains of bacteria, protect a bac-

teria 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 nucleo-

tides. These enzymes cleave the DNA

strand wherever the recognized sequence

occurs, generating a number of DNA frag-

ments 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 re-

flects its host bacterium, eg, Eco RI is iso-

lated from Escherichia coli, Hind III is

from Hemophilus influenzae,andHpaIis

from Hemophilus parainfluenzae.

Restriction Fragment Length Polymorphism

Analysis

The unique properties of restriction endo-

nucleases make analysis of restriction

fragment length polymorphism (RFLP)

suitable for the detection of a DNA poly-

morphism. The changes in nucleotide se-

quence described above (substitution, in-

sertion, 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 hy-

bridization (see Fig 9-9).

The isolated DNA is cleaved into frag-

ments by digestion with one or more re-

striction endonucleases. The DNA frag-

ments 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 oligo-

nucleotide or may derive from cloned com-

plementary 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. Un-

bound excess probe is washed off, and hy-

bridized DNA is visualized as one or more

bands of specific size, dictated by the spe-

cific nucleotide sequence. If several indi-

viduals 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 transplan-

tation, paternity testing, and forensic sci-

ence.

214 AABB Technical Manual

Chapter 9: Molecular Biology in Transfusion Medicine 215

Figure 9-8. Transcription-mediated amplification cycle.

Step 1: Promoter-primer binds to rRNA target.

Step 2: Reverse transcriptase (RT) creates DNA copy of rRNA target.

Step 3: RNA:DNA duplex.

Step 4: RNAse H activities of RT degrade the rRNA.

Step 5: Primer 2 binds to the DNA and RT creates a new DNA copy.

Step 6: Double-stranded DNA template with a promoter sequence.

Step 7: RNA polymerase (RNA Pol) initiates transcription of RNA from DNA template.

Step 8: 100-1000 copies of RNA amplicon are produced.

Step 9: Primer 2 binds to each RNA amplicon and RT creates a DNA copy.

Step 10: RNA:DNA duplex.

Step 11: RNAse H activities of RT degrade the rRNA.

Step 12: 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.

DNA Profiling

Regions of DNA that show great allelic vari-

ability (“minisatellites” and “microsatel-

lites”) can be studied by the application of

RFLP mapping and/or PCR analysis (a pro-

cess sometimes called DNA profiling, DNA

typing, or DNA fingerprinting). Minisatel-

lites 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 re-

peats of a short (typically four base pair)

sequence. These regions are almost al-

ways 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 num-

bers of repeats will be shared by two indi-

viduals, even if related. The VNTR and/or

STR patterns observed at four to eight dif-

ferent loci may be unique for an individ-

ual and thus constitute a profile or “fin-

gerprint” 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 mea-

surementofthesizeofthePCRproducts

produced. PCR products can be separated

by size by electrophoresis through poly-

acrylamide gel and detected by silver stain-

ing, or, if the PCR products incorporate a

216 AABB Technical Manual

Figure 9-9. Southern blotting: a technique for the detection of polymorphism by gel-transfer and hy-

bridization with known probes.

fluorescent tag, by fluorescent detection

systems including those designed for auto-

mated DNA sequencing. Comparison of the

VNTR and STR PCR products with a stan-

dard-size ladder distinguishes the alleles

present in the sample.

DNA profiling is a technique that is ex-

tremely powerful for identifying the source

of human DNA; therefore, it has applica-

tions 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 ge-

netic 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.TheenzymeRTisusedtogenerate

a single strand of DNA, which serves as a

template for a second strand generated by

DNA polymerase. The product is comple-

mentary DNA (cDNA) and it is the DNA

molecule of choice for cloning and se-

quencing.

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 cir-

cular molecules of double-stranded DNA

that occur naturally in bacteria and typi-

cally confer antibiotic resistance. After the

gene has been inserted into the DNA of the

vector, the recombinant DNA can be intro-

duced into a bacterial host where it under-

goes replication. Because many vectors

carry genes for antibiotic resistance, this

characteristic can be exploited by growing

the host bacteria in the presence of the ap-

propriate antibiotic; only bacteria that have

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 recombi-

nant vectors is called a DNA library. Librar-

ies can be obtained from many commercial

sources or can be produced by the individ-

ual investigator. The library can be probed

through a technique similar to Southern

blotting, with an oligonucleotide probe

basedonpartofaknownsequence.Posi

tive clones can be selected and a pure cul-

ture grown in large quantities. Once puri-

fied, 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, includ-

ing bacteria, plants, invertebrates (such as

insects), and vertebrates (such as mam-

mals), permits not only gene characteriza-

tion 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

thecompletenucleotidesequenceofthe

human genome as well as the genomes of

several other key organisms. The initiative

also improved DNA sequencing technol-

ogy. 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 pro-

teins that are relevant in transfusion med-

icine. In turn, this information has helped

Chapter 9: Molecular Biology in Transfusion Medicine 217

in the development of recombinant pro-

teins for use as transfusion components and

in-vitro test reagents. It also plays a role in

clarifying the disorders that afflict trans-

fusion recipients.

Advances in DNA sequencing have taken

thefieldalongwayfromthecumbersome

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 op-

timized for specialty applications such as

heterozygote detection and sizing of PCR

fragments. Automated DNA sequencers us-

ing capillary electrophoresis are especially

useful for the rapid sequencing of short

DNA templates. DNA sequence can also be

obtained using mass spectrometry. Auto-

mated sequencers will become increasingly

common in clinical laboratories as this

technology evolves. If it can be made

cost-effective for routine use, then DNA se-

quencing could become a routine genotyp-

ing method.

DNA Microarrays

The complexity of the human genome re-

quires that the differential expression of

multiple genes be analyzed at once to un-

derstand normal biologic processes as well

as changes in diseases. A powerful tech-

nique to accomplish this goal is DNA

microarrays or gene chips.15 In this method,

tens of thousands of separate DNA mole-

cules are spotted or synthesized on a small

area of a solid support, often a glass slide.

The DNA can be generated by PCR or oligo-

nucleotide 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 pic-

ture of the gene expression profile of the

tissue for all of the genes on the micro-

array. 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 bac-

teria, 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 con-

taining 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 can-

not 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 re-

combinant protein to be effective; for

instance, granulocyte colony-stimulating

factor (G-CSF) is produced in a nonglyco-

sylated form in E. coli (filgrastim) and in a

glycosylated form in yeast (lenograstim).

Recombinant proteins are finding multi-

ple uses in transfusion medicine, as thera-

peutic agents and vaccine components, in

component preparation, in virus diagnosis,

and in serologic testing. Recombinant hu-

man erythropoietin,17 G-CSF and GM-CSF,18

interferon-alpha, interleukin-2, interleu-

kin-11, and Factors VIII,19 IX,20 VII, and VIIa21

218 AABB Technical Manual

are all available and finding clinical accep-

tance. For instance, recombinant erythro-

poietin can be used to increase red cell pro-

duction 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 man-

agement 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 thrombo-

cytopenic patients.24 In the coagulation

arena, recombinant proteins such as pro-

tein C, antithrombin, and hirudin are ap-

provedbytheFoodandDrugAdministra-

tion 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 hemato-

poietic 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. Alphagalactosi-

dase, an enzyme that is capable of convert-

ing 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 recombi-

nant proteins and other products under de-

velopment will undoubtedly affect the vari-

ety of transfusion components that will

become available in the future.

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 an-

tibodies. Cells transfected with appropriate

vectors can be induced to express recombi-

nant proteins on the membrane surface

and, as such, may become useful as geneti-

cally 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 ther-

apy 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 over-

all have been disappointing largely due to

problems with delivery (transfection) of

the genetic material (transgene) into cells

and the limited lifespan of the success-

fully transfected cells.

There are different types of vectors (gene-

delivery vehicles) that deliver genes to cells,

including viral vectors (retrovirus, adeno-

virus, adeno-associated virus, vaccinia, her-

pes simplex), naked DNA, and modified DNA.

Genetic material can also be transferred to

cells by physical means such as electro-

poration (use of an electric field). As meth-

ods for gene delivery improve, it is antici-

pated that the benefits of gene therapy will

become more apparent.

Gene therapy can be used to replace a

defective gene, leading to increased pro-

duction of a specific protein as in the re-

placement of Factor VIII or IX for patients

with hemophilia,31 or it can be used to

downregulate (reduce) expression of an un-

desirable gene. The latter can be accom-

Chapter 9: Molecular Biology in Transfusion Medicine 219

plished by the introduction of DNA se-

quences (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 ex-

ample is imatinib mesylate (Gleevec,

Novartis Pharmaceuticals, East Hanover,

NJ), which binds specifically to the Bcr-

Abl protein and blocks its tyrosine kinase

activity in malignant white cells. It specif-

ically blocks the binding site for adeno-

sine 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 un-

affected.

Another promising therapy is RNA inter-

ference, which has its historical roots as a

research tool used to characterize the func-

tion of known genes. RNA interference is

based on an antiviral mechanism in which

dsRNA is delivered to a cell and is subse-

quently processed into small (21-25 bp) in-

terfering 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-re-

lated genes or infectious diseases like viral

hepatitis.33

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Kim C. Disruption of a GATA motif in the

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2. Li Y, Camp S, Taylor P. Tissue-specific expres-

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3. Le Van Kim C, Mitjavila MT, Clerget M, et al.

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4. LeeS,WuX,ReidM,etal.Molecularbasisof

the Kell (K1) phenotype. Blood 1995;85:912-

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5. Lublin DM, Mallinson G, Poole J, et al. Molec-

ular basis of reduced or absent expression of

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6. Kudo S, Fukuda M. Structural organization of

glycophorin A and B genes: Glycophorin B

gene evolved by homologous recombination

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U S A 1989;86:4619-23.

7. Lubin DM, Thompson ES, Green AM, et al.

Dr(a–) polymorphism of decay accelerating

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specific transfection. J Clin Invest 1991;87:

1945-52.

8. Erlich HA. Principles and applications of the

polymerase chain reaction. Rev Immuno-

genet 1999;1:127-34.

9. BennettPR,LeVanKimC,ColinY,etal.Pre-

natal determination of fetal RhD type by DNA

amplification. N Engl J Med 1993;329:607-10.

10. Friedmann T. Overcoming the obstacles to

gene therapy. Sci Am 1997;276:80-5.

11. Hillyer CD, Klein HG. Immunotherapy and

gene transfer in the treatment of the oncol-

ogy patient: Role of transfusion medicine.

Transfus Med Rev 1996;10:1-14.

12. International Human Genome Sequencing

Consortium. Initial sequencing and analysis

of the human genome. Nature 2001;409:860-

921.

13. VenterJC,AdamsMD,MyersEW,etal.The

sequence of the human genome. Science

2001;291:1304-51.

14. Griffin HG, Griffin AM. DNA sequencing. Re-

cent innovations and future trends. Appl Bio-

chem Biotechnol 1993;38:147-59.

15. Duggan DJ, Bittner M, Chen Y, et al. Expres-

sion profiling using cDNA microarrays. Nat

Genet 1999;21:S10-14.

16. Lubon H, Paleyanda RK, Velander WH,

Drohan WN. Blood proteins from transgenic

animal bioreactors. Transfus Med Rev 1996;

10:131-43.

17. CazzolaM,MercurialiF,BrugnaraC.Useof

recombinant human erythropoietin outside

the setting of uremia. Blood 1997;89:4248-67.

220 AABB Technical Manual

18. Ganser A, Karthaus M. Clinical use of hema-

topoietic growth factors. Curr Opin Oncol 1996;

8:265-9.

19. VanAken WG. The potential impact of recom-

binant factor VIII on hemophilia care and the

demand for blood and blood products. Trans-

fus Med Rev 1997;11:6-14.

20. White GC II, Beebe A, Nielsen B. Recombi-

nant factor IX. Thromb Haemost 1997;78:

261-5.

21. Lusher JM. Recombinant factor VIIa (Novo-

Seven) in the treatment of internal bleeding

in patients with factor VIII and IX inhibitors.

Haemostasis 1996;26(Suppl 1):124-30.

22. Braga M, Gianotti L, Gentilini O, et al. Ery-

thropoietic response induced by recombi-

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patients candidate to major abdominal sur-

gery. Hepatogastroenterology 1997;44:685-90.

23. Cazenave JP, Irrmann C, Waller C, et al. Epo-

etin alfa facilitates presurgical autologous

blood donation in non-anaemic patients

scheduled for orthopaedic or cardiovascular

surgery. Eur J Anaesthesiol 1997;14:432-42.

24. Kuter DJ. What ever happened to thrombo-

poietin? Transfusion 2002;42:279-83.

25. Ketley NJ, Newland AC. Haemopoietic growth

factors. Postgrad Med J 1997;73:215-21.

26. Growe GH. Recombinant blood components:

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27. Kumar R. Recombinant hemoglobins as

blood substitutes: A biotechnology perspec-

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28. Lenny LL, Hurst R, Zhu A, et al. Multiple-unit

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1995;35:899-902.

29. Ellis RW. The new generation of recombinant

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31. Kaufman RJ. Advances toward gene therapy

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32. Druker BJ, Tamura S, Buchdunger E, et al. Ef-

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sine kinase on the growth of Bcr-Abl positive

cells. Nat Med 1996;2:561-6.

33. Radhakrishnan SK, Layden TJ, Gartel AL. RNA

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34. Farese AM, Schiffer CA, MacVittie TJ. The im-

pact of thrombopoietin and related mpl-lig-

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35. Barbara JA, Garson JA. Polymerase chain re-

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36. Power EG. RAPD typing in microbiology—a

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65.

37. LarsenSA,SteinerBM,RudolphAH,WeissJB.

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21.

38. WeissJB.DNAprobesandPCRfordiagnosis

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41. PenaSD,PradoVF,EpplenJT.DNAdiagnosis

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42. van Belkum A. DNA fingerprinting of medi-

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43. Siegel DL. Research and clinical applications

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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,

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lar biology—an overview. Transfus Med Rev 1997;

11:209-23.

Chapter 9: Molecular Biology in Transfusion Medicine 221

222 AABB Technical Manual

Appendix 9-1. Molecular Techniques in Transfusion Medicine

Technique Applications Examples References

PCR Infectious disease

testing

Viruses, bacteria,

parasites

37, 38

Polymorphism detec-

tion

HLA, blood group

antigens

1, 39

Prenatal detection Rh 9

Recombinant proteins/

DNA cloning

Therapy Erythropoietin,

thrombopoietin,

G-CSF

17, 18, 34

Apheresis G-CSF 39

Infectious disease

testing

Viral testing 40

Recombinant

components

Coagulation factors 19

Hemoglobin 27

Component processing Alpha-galactosidase 28

Vaccine production Hepatitis, malaria, HIV 27-29

DNA profiling Human identification

Bacterial identification

Chimerism, forensic

science

Bacterial typing

DNA sequencing Polymorphism detec-

tion, heterozygote

detection

HLA 14

Phage display/

repertoire cloning

Monoclonal antibody

production

Anti-D 43

RFLP Polymorphism

detection

HLA, blood group

antigens

PCR = polymerase chain reaction; G-CSF = granulocyte colony-stimulating factor; HIV = human immunodefi-

ciency virus; RFLP = restriction fragment length polymorphism.

Chapter 10: Blood Group Genetics

Chapter 10

Blood Group Genetics

LANDSTEINER’S DISCOVERY OF

the ABO blood group system dem-

onstrated that human blood ex-

pressed inheritable polymorphic structures.

Shortly after the discovery of the ABO sys-

tem, red cells proved to be an easy and ac-

cessible means to test for blood group

polymorphisms in individuals of any age.

As more blood group antigens were de-

scribed, blood group phenotyping pro-

vided a wealth of information about the

polymorphic structures expressed on pro-

teins, glycoproteins and glycolipids on

red cells, and the genetic basis for their

inheritance.

Basic Principles

Inheritance of transmissible characteris-

tics or “traits,” including blood group an-

tigens, forms the basis of the science of

genetics. The genetic material that deter-

mines 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 appear-

ance 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 spe-

cific order along a chromosome with the

precise gene location known as the locus.

Chromosomes

The number of chromosomes and chro-

mosome morphology are specific for each

species. Human somatic cells have 46

chromosomes that exist as 23 pairs (one-

half of each pair inherited from each par-

ent). 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 remain-

ing pair.

223

Each chromosome consists of two arms

joined at a primary constriction, the centro-

mere. 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 indi-

cated 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 chro-

mosome 12). When banded and stained,

each chromosome displays a unique pat-

tern of bands, which are numbered from

the centromere outward (see Fig 10-1).

Chromosomes are identified by the loca-

tion 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 pro-

cess that occurs within days of fertiliza-

tion. Once an X chromosome has become

inactivated, all of that cell’s clonal descen-

dants have the same inactive X. Hence, in-

activation is randomly determined but

once the decision is made, the choice is

permanent. This process is termed lyoni-

zation.

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 pro-

cess whereby the body grows or replaces

dead or injured somatic cells. This process

consists of five stages: prophase, pro-

metaphase, metaphase, anaphase, and

telophase. The end result after cytokinesis

is two complete daughter cells, each with

a nucleus containing all the genetic infor-

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 comple-

ment. Gametes formed following meiosis

are haploid, with a 1N chromosome com-

plement. It is during meiosis that genetic

diversity occurs. One type of diversity is the

224 AABB Technical Manual

Figure 10-1. Diagram of Giemsa-stained normal

human chromosome 7. With increased resolution

(left to right), finer degrees of banding are evi-

dent. Bands are numbered outward from the

centromere (c), which divides the chromosome

intopandqarms.

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 chro-

mosomes. The other type of diversity oc-

curs when homologous pairs of chromo-

somes line up during the first prophase.

The homologous chromosomes genetically

recombine in a process called chromo-

somal crossing over (see below). Therefore,

not only are the chromosomes shuffled

during meiosis, but also portions of chro-

mosomes 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 ho-

mologous chromosomes, are called al-

leles. The ISBT terminology distinguishes

between the alleles for blood group anti-

gens (ie, the genetic polymorphisms) and

the antigens that they encode. For exam-

ple, 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, re-

spectively. Individuals who have identical

alleles at a given locus on both chromo-

somes are homozygous for the allele (eg,

A1/A1or K/K or k/k). In the heterozygous

condition, the alleles present at the par-

ticular locus on each chromosome are

nonidentical (eg, A1/O1or A1/B1or K/k). Ta-

ble 10-1 summarizes genotypes and chro-

mosomal 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

Chapter 10: Blood Group Genetics 225

Figure 10-2. The two types of cell division are mitosis and meiosis.

226 AABB Technical Manual

Table 10-1. Chromosomal Locations of Human Blood Group System Genes*

System ISBT No.

ISBT

Symbol

Gene(s)

Designation

(ISGN) Location

ABO 001 ABO

ABO

9q34.2

MNS 002 MNS

GYPA, GYPB, GYPE

4q28.2-q31.1

P 003 P1

22q11.2-qter

Rh 004 RH

RHD, RHCE

1p36.13-p34.3

Lutheran 005 LU

19q13.2

Kell 006 KEL

KEL

7q33

Lewis 007 LE

FUT3

19p13.3

Duffy 008 FY

DARC

1q22-q23

Kidd 009 JK

SLC14A1

18q11-q12

Diego 010 DI

SLC4A1

17q21-q22

Yt 011 YT

ACHE

7q22.1

Xg 012 XG

Xp22.32

Scianna 013 SC

1p34

Dombrock 014 DO

12p12.3

Colton 015 CO

AQP1

7p14

Landsteiner-Wiener 016 LW

19p13.3

Chido/Rodgers 017 CH/RG

C4A, C4B

6p21.3

H 018 H

FUT1

19q13.3

Kx 019 XK

Xp21.1

Gerbich 020 GE

GYPC

2q14-q21

Cromer 021 CROM

DAF

1q32

Knops 022 KN

CR1

1q32

Indian 023 IN

CD44

11p13

OK 024 OK

CD147

19p13.3

RAPH 025 MER2

MER2

11p15.5

JMH 026 JMH SEMA7A 15q22.3-q23

I 027 I CGNT2 6p24

Globoside 028 P B3GALT3 3q25

GIL 029 GIL AQP3 9q13

*Modified from Zelinski1; Garratty et al2; and Denomme et al.3

person whose phenotype is Jk(a+b–) have a

“double dose” of the Jkaallele and, as a re-

sult, express more Jkaantigenonthered

cell surface than an individual whose phe-

notype is Jk(a+b+) (a single dose of the Jka

allele). The difference in amount of antigen

expressed on the red cell membrane be-

tween a homozygous and a heterozygous

phenotype can often be detected serologi-

cally and is termed the dosage effect. For

example, some anti-Jkasera may give the

following pattern of reactivity:

Phenotype of RBC Donor

Antibody Jk(a+b–) Jk(a+b+)

Anti-Jka3+ 2+

Dosage effect is not seen with all blood

group antigens or even with all antibodies

of a given specificity. Antibodies that typi-

callydemonstratedosageincludethosein

the Rh, MNS, Kidd, and Duffy blood group

systems.

Alleles arise by genetic changes at the

DNA level and may result in a different ex-

pressed phenotype. Some of the changes

found among blood group alleles may re-

sult from:

■Missense mutations (a single nucle-

otide substitution leading to the

coding of a different amino acid)

■Nonsense mutations (a single nucle-

otide substitution leading to the

coding of a stop codon)

■Mutations in motifs involved in tran-

scription

■Mutations leading to alternate RNA

splicing

■Deletion of a gene, exon, or nucleo-

tide(s)

■Insertionofanexonornucleotide(s)

■Alternate transcription initiation site

■Chromosome translocation

■Gene conversion or recombination

■Crossing over

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 fre-

quency) is the proportion that it contrib-

utes to the total pool of alleles at that lo-

cuswithinagivenpopulationatagiven

time. This frequency can be calculated

from phenotype frequencies observed

within a population. The sum of allele fre-

quencies at a given locus must equal 1.

The Hardy-Weinberg Law

The Hardy-Weinberg law is based on the

assumption that genotypes are distrib-

uted in proportion to the frequencies of

individual alleles in a population and will

remain constant from generation to gen-

eration if the processes of mutation, mi-

gration, etc do not occur. For example, the

Kidd blood group system is basically a

two-allele system (Jkaand Jkb; the silent Jk

allele is extremely rare) that can be used

to illustrate the calculation of gene fre-

quencies. This calculation uses the Hardy-

Weinberg equation for a two-allele sys-

tem. If p is the frequency of the Jkaallele

and q is the frequency of the Jkballele,

then the frequencies of the three combi-

nations of alleles can be represented by

the equation p2+2pq+q

2= 1 where:

p = frequency of Jkaallele

q = frequency of Jkballele

p2=frequencyofJka/Jkagenotype

2pq = frequency of Jka/Jkbgenotype

q2=frequencyofJkb/Jkbgenotype

Chapter 10: Blood Group Genetics 227

Using the observation that 77% of indi-

viduals within a population express Jkaanti-

gen on their red cells, then:

p2+2pq = frequency of

persons who

are Jk(a+) and

carry the Jkaallele

=0.77

q2=1–(p

2+2pq)= frequencyof

persons who

are Jk(a–)

(homozygous for

the Jkballele)

q2= 1 – 0.77 = 0.23

0.23

q = 0.48 (allele

frequency of Jkb)

Because the sum of frequencies of both

alleles must equal 1.00,

p+q = 1

p=1–q

p = 1 – 0.48

p = 0.52 (allele frequency of Jka)

Once the allele frequencies have been

calculated, the number of Jk(b+) individu-

als (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-Jkaand anti-Jkbsera are avail-

able, allele frequencies can be determined

more easily by direct counting. As shown in

Table 10-2, the random sample of 100 peo-

ple tested for Jkaand Jkbantigens possess a

total of 200 alleles at the Jk locus (each per-

son inherits two alleles, one from each par-

ent). There are two Jkaalleles inherited by

228 AABB Technical Manual

Figure 10-3. How crossover, recombination, and nucleotide substitution (nt subs) result in variations

of genes producing glycophorin A and B. The changes are associated with the presence of various

low-incidence MNS system antigens. (Modified from Reid.4)

each of the 28 individuals who phenotype

as Jk(a+b–), for a total of 56 alleles. There

are 49 Jkaalleles in the individuals who are

Jk(a+b+), for a total of 105 alleles or a gene

frequency of 0.52 (105 ÷ 200). The fre-

quency of Jkbis 95 ÷ 200 = 0.48.

The Hardy-Weinberg law is generally

used to calculate allele and genotype fre-

quencies in a population when the fre-

quency of one genetic trait (eg, antigen

phenotype) is known. However, it relies on

certain assumptions: no mutation; no mi-

gration (in or out) of the population; lack of

selective advantage/disadvantage of a par-

ticular 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 con-

cept 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 in-

heritance of the ABO alleles (Fig 10-4). In

this example, the members of the paren-

tal generation (P1) are homozygous for an

Aallele and an Oallele. All members of

the first filial generation (F1) will be het-

erozygous (A/O) but will still express the

blood group A antigen (Ois a silent al-

lele). If an F1individual mates with an A/O

genotypic individual, the resulting prog-

eny [termed the second filial generation:

(F2)] will blood group as A (either hetero-

zygous or homozygous) or group O. If the

F1individual 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)andK+k+,

and the other parent is group B (homozy-

gous for B) and K–k+ (homozygous for k),

all the F1children 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,

Chapter 10: Blood Group Genetics 229

Table 10-2. Gene Frequencies in the Kidd Blood Group System Calculated Using

Direct Counting Method*

Phenotype

No. of

Individuals

No. of

Kidd Genes

Gene Frequencies (%)

Jk(a+b–) 28 56 56 0

Jk(a+b+) 49 98 49 49

Jk(a–b+) 23 46 0 46

Totals 100 200 105 95

Gene Frequency 0.525 0.475

*Assumes absence of silent

allele.

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 to-

gether 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 re-

combinations. 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 mea-

surement to estimate the distance between

different loci. This type of analysis can

help in identifying, mapping, and diag-

nosing 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 an-

tigens (Lua,Lu

b) was the first recognized ex-

ample of autosomal linkage in humans.5

Analysis of this relationship also provided

the first evidence in humans of recombina-

tion due to crossing-over and helped dem-

230 AABB Technical Manual

Figure 10-4. Mendel’s law of independent segregation demonstrated by the inheritance of ABO genes.

onstrate that crossing-over occurs more

ofteninfemalesthaninmales.

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 disequi-

librium. The approximate frequencies of

each of the four alleles are:

M=0.53 S=0.33

N=0.47 s=0.67

If the alleles of the M, N, S, and s anti-

gens segregated independently, the ex-

pected frequency of each haplotype would

be the product of the frequencies of the in-

dividual alleles. However, the frequencies

observed are not those expected:

Expected Observed

Frequency Frequency

MS =0.53×0.33 = 0.17 0.24

Ms =0.53×0.67 = 0.36 0.28

NS =0.47×0.33 = 0.16 0.08

Ns =0.47×0.67 = 0.31 0.40

Total 1.00 1.00

This is an example of linkage disequilib-

rium: the tendency of specific combina-

tions 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

Chapter 10: Blood Group Genetics 231

Figure 10-5. Mendel’s law of independent assortment demonstrated by the inheritance of ABO and Kell

genes.

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 disequilib-

rium. Linkage disequilibrium may be posi-

tive or negative, and it may indicate a selec-

tive 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 frequen-

cies that reflect their individual frequencies.

For example, if alleles in the population

have the following frequencies:

Y0.53 Z0.30

y0.47 z0.70

Total 1.00 1.00

then the frequencies of the combination

should be the product of the frequency of

each allele:

YZ 0.53 ×0.3 = 0.16

Yz 0.53 ×0.7 = 0.37

yZ 0.47 ×0.3 = 0.14

yz 0.47 ×0.7 = 0.33

Total 1.00

In such a case, the alleles are in linkage

equilibrium because they are inherited

independently.

Patterns of Inheritance

Dominant and Recessive Traits

Traits are the observed expression of genes.

A trait that is observable when the deter-

mining allele is present is called domi-

232 AABB Technical Manual

Figure 10-6. Very closely linked loci are rarely affected by crossing over so that alleles of those loci

are inherited together (N and S, M and s in the example shown). Loci on the same chromosome that

are not closely linked (the

locus and the

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.

nant; when different alleles on homologous

chromosomes each produce an observable

trait, the term co-dominant is used. A re-

cessive trait is observable only when the

allele is not paired with a dominant allele

(two recessive alleles are present). De-

scribing traits as dominant and recessive

depends on the method used to detect gene

products. Observable traits are called phe-

notypes. 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 geno-

types 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 pos-

sesses the allele. Figure 10-7(A) presents a

pedigree showing the pattern of auto-

somal dominant inheritance. Typically,

each person with the trait has at least one

parent with the trait, continuing back-

ward 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,therecessivetraitwillberareandgen

erally 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

(frequency: <1:10,000) and display the trait,

the parents are often blood relatives [Fig

10-7(B)]. Recessive traits may remain unex-

pressed 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 re-

cessive allele indicates the less likelihood of

consanguinity. Traits inherited in either

autosomal dominant or autosomal reces-

sive fashion typically occur with equal fre-

quency in males and females.

Sex-Linked Dominant or Co-dominant

Trait

A male always receives his single X chro-

mosome from his mother. The predomi-

nant feature of X-linked inheritance, of

either dominant or recessive traits, is ab-

sence of male-to-male (father-to-son)

transmission of the trait. Because a male

passes his X chromosome to all his dau-

ghters, all daughters of a man expressing

adominant 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 chromo-

somes, all her children will express the

trait. X-linked dominant traits tend to ap-

pear 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 car-

rier mothers or, very rarely, from a mother

who is homozygous for the allele and

Chapter 10: Blood Group Genetics 233

therefore expresses the trait. In the mat-

ing 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 daugh-

ters 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 daugh-

ters, half of whom will be homozygous for

the abnormal allele.

Blood Group Co-dominant Traits

Blood group antigens, as a rule, are ex-

pressed as co-dominant traits: heterozy-

gotes 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.

234 AABB Technical Manual

Figure 10-7. Four pedigrees showing different patterns of inheritance.

Figure 10-8 shows the inheritance patterns

of the two active alleles of the Kidd blood

group system (Jkaand Jkb)andtheco-

dominant phenotypic expression of the

two respective antigens Jkaand Jkb.

In the ABO system, the situation is more

complex. The genes of the ABO system do

not code for membrane proteins but con-

trol production of enzymes termed glyco-

syltransferases. These enzymes add specific

sugars to a precursor structure on the red

cell membrane, resulting in specific antigen

expression. In an A1/A2heterozygote, the

phenotype is A1; the presence of the A2al-

lele cannot be inferred. Although the A1al-

lele appears dominant to that of the A2al-

lele by simple cell typing, techniques that

identify the specific transferases reveal that

an A1/A2heterozygote does generate the

products of both alleles, ie, both A1and A2

transferases. Similarly, in an A2/O person, A2

is dominant to O.TheOallele codes for a

specific protein, but this protein (trans-

ferase) is nonfunctional. The presence of

ABO genes can be demonstrated by molec-

ular 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 Ta-

ble 10-1. The Xg and XK loci are the only

bloodgroupgenesmappedtotheXchro

mosome.

Interaction among alleles or the prod-

ucts of different genes may modify the ex-

pression of a trait. The terms “suppressor”

and “modifier” are used to describe genes

that affect the expression of other genes;

Chapter 10: Blood Group Genetics 235

Figure 10-8. Inheritance and co-dominant expression of Kidd blood group antigens.

however, the mechanism of these postu-

lated gene interactions is not always fully

understood. Some observations in blood

group serology have been explained by

gene interaction: weakening of the D anti-

gen expression when the Callele is present

in cis (on the same chromosome) or in

trans (on the paired chromosome),6and 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 develop-

ment 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

Hgene) is an example of epistasis. A muta-

tion database of gene loci encoding com-

mon 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 genet-

ics is essential for parentage testing and

helpful in such clinical situations as pre-

dicting 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 pheno-

types are obtained by testing many ran-

domly selected people of the same race or

ethnic group and observing the propor-

tion of positive and negative reactions

with a specific blood group antibody. In a

blood group system, the sum of pheno-

type 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 anti-

bodies, 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,howmany

ABO-compatible units of blood would

have to be tested to find 4 units of the ap-

propriate phenotype?

Phenotype Frequency (%)

c– 20

K– 91

Jk(a–) 23

To calculate the frequency of the com-

bined phenotype, the individual frequen-

cies 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 individ-

uals 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 deci-

sions 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 sim-

ple Mendelian modes of inheritance, are

useful in parentage analyses. If one as-

236 AABB Technical Manual

sumes maternity and that test results are

accurate, paternity can be excluded in

either of two ways:

1. Direct exclusion of paternity is es-

tablished when a genetic marker is

present in the child but is absent

from the mother and the alleged fa-

ther. Example:

Blood Group Phenotype

Child Mother Alleged Father

BO O

The child has inherited a Bgene, which

could not be inherited from either the

mother or the alleged father, assuming that

neither the mother nor the alleged father is

oftherareO

hphenotype. Based on the phe-

notypes of mother and child, the Bgene

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 pheno-

type)musttransmittohisoffspring.

Example:

Blood Group Phenotype

Child Mother Alleged Father

Jk(a+b–) Jk(a+b–) Jk(a–b+)

In this case, the alleged father is presum-

ably homozygous for Jkbandshouldhave

transmitted Jkbto 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 si-

lent allele. In the example above, the al-

leged father could have one silent allele

(Jk), which was transmitted to the child.

Thechild’sgenotypecouldbeJkaJk instead

of the far more common JkaJka. Interpreta-

tion of phenotypic data must take into ac-

count all biologic and analytic factors

known to influence results.

When the alleged father cannot be ex-

cluded from paternity, it is possible to cal-

culate the probability of paternity. The

probability that the alleged father transmit-

ted the paternal obligatory genes is com-

pared with the probability that any other

randomly selected man from the same eth-

nic/racial population could have transmit-

ted the genes. The result is expressed as a

likelihood ratio (paternity index) or as a

percentage (posterior probability of pater-

nity 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 par-

entage 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 stan-

dards for laboratories that perform parent-

age 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 fer-

tilized zygotes fuse to form one individ-

ual. This condition, although not heredi-

tary, 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 geneti-

cally group O person with implanted A

cells does not produce anti-A. More com-

monly, chimeras are artificial and arise

from the transfer of actively dividing cells,

eg, through hematopoietic transplanta-

tion (see Chapter 25).

Chapter 10: Blood Group Genetics 237

238 AABB Technical Manual

Blood Group Nomenclature

The terminology and notations for blood

group systems embody many inconsis-

tencies because blood group serologists

failed to follow conventions of classic

Mendelian genetics. Listed below are a

few examples of the confusion engen-

dered 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 reces-

sive trait is denoted by both lower-

case letters. The Aand Bco-domi-

nant 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, Kand kof the

Kell blood group system and Cand c

of the Rh system.

3. Some co-dominant traits have iden-

tical base symbols but different su-

perscript symbols, such as Fyaand

Fyb(Duffy system) and Luaand Lub

(Lutheran system).

4. In some allelic pairs, the lower fre-

quency antigen is expressed with an

“a” superscript ( Wrahas a lower fre-

quency than Wrb). In other allelic

pairs, the “a” superscript denotes the

higher incidence antigen (Coahas a

higher frequency than Cob).

5. Some authors have denoted the ab-

sence of a serologic specificity with a

base symbol devoid of superscripts,

and others use a lowercase version

ofthebasesymbol.IntheLutheran

system, the assumed amorphic gene

is called Lu,notlu,whereasthe

amorph in the Lewis system is le.

6. Numeric terminology was intro-

duced for some blood group sys-

tems, resulting in mixtures of letters

and numbers for antigen designa-

tions, eg, K, Kpa, 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, su-

perscripts, 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 informa-

tion for blood group systems. Issitt and

Crookston9and Garratty et al2presented

guidelines for the nomenclature and termi-

nology of blood groups. The International

Society of Blood Transfusion (ISBT) Work-

ing Party on Terminology for Red Cell Sur-

face 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 hemoglo-

bins, immunoglobulin allotypes, histo-

compatibility antigens, clusters of differen-

tiation, STR sequences, and other serum

protein and red cell enzyme systems. Al-

though 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 anti-

gens was devised as a numeric nomencla-

ture suitable for computerization. A six-

digit 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 de-

signed mainly for computer databases and

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 spe-

cific blood group system is dependent on

genetic, serologic, and/or biochemical rela-

tionships. Gene cloning has made the task

of assignment more definitive and has al-

lowed some designations previously un-

proved 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 sys-

tem. 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 to-

gether until genetic information becomes

available. In recent years, the number of

antigens in these three series has dramati-

cally declined as further genetic and bio-

chemical data allow reassignment.

Correct Terminology

The following are accepted conventions

for expressing red cell antigen pheno-

types 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 in-

cludes a subscript (A1), generally the

encoding gene is expressed with a

superscript (A1).

2. Antigen names designated by a su-

perscript or a number (eg, Fya,Fy:1)

arewritteninnormal(Roman)script.

Numeric designations are written on

thesamelineastheletters.Super

script letters are lowercase. (Some

exceptions occur, based on historic

usage: hrS,hr

B.)

3. When antigen phenotypes are ex-

pressed using single letter designa-

tions, results are usually written as +

or –, set on the same line as the let-

ter(s) of the antigen: K+ k–.

4. To express phenotypes of antigens

designated with a superscript letter,

that letter is placed in parentheses

onthesamelineasthesymbolde

fining the antigen: Fy(a+) and Fy(a–).

5. For antigens designated by num-

bers, 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 re-

sults are positive (K:1), but a minus

sign is placed before negative test re-

sults: K:1, K:–1. If tests for several an-

tigens 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 sepa-

rated by commas: K:–1,2,–3,4. Only

antigens tested are listed; if an anti-

body defining a specific antigen was

not tested, the number of the anti-

gen is not listed: K:–1,–3,4.

Although numeric terminology has been

devised for various systems and antigens, it

shouldnotbeassumedthatitmustreplace

conventional terminology. The use of con-

ventional antigen names is also acceptable.

In some systems, notably Rh, multiple ter-

minologies exist and not all antigens within

the system have names in each type.

References

1. Zelinski T. Chromosomal localization of hu-

man blood group genes. In: Silberstein LE, ed.

Molecular and functional aspects of blood

group antigens. Bethesda, MD: AABB, 1995:

41-73.

2. Garratty G, Dzik W, Issitt PD, et al. Terminol-

ogy for blood group antigens and genes—his-

Chapter 10: Blood Group Genetics 239

torical origins and guidelines in the new mil-

lennium. Transfusion 2000;40:477-89.

3. 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 thera-

peutic technologies in transfusion medicine.

Bethesda, MD: AABB Press, 2003: 95-129.

4. 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.

5. Mohr J. A search for linkage between the Lu-

theran blood group and other hereditary

characters. Acta Path Microbiol Scand 1951;

28:207-10.

6. Araszkiewicz P, Szymanski IO. Quantitative

studies on the Rh-antigen D effect of the C

gene. Transfusion 1987;27:257-61.

7. CrawfordNM,GreenwaitTJ,SasakiT.The

phenotype Lu(a–b–) together with unconven-

tional Kidd groups in one family. Transfusion

1961;1:228-32.

8. Gjertson D, ed. Standards for parentage test-

ing laboratories. 6th ed. Bethesda, MD: AABB,

2004.

9. Issitt PD, Crookston MC. Blood group termi-

nology: Current conventions. Transfusion 1984;

24:2-7.

240 AABB Technical Manual

Appendix 10-1. Glossary of Terms in Blood Group Genetics

Allelic: Pairs of genes located at the same site on chromosome pairs.

Centromere: A constricted region of a chromosome that connects the chroma-

tids during cell division.

Chromatid: One of the two potential chromosomes formed by DNA replication

of each chromosome before mitosis and meiosis. They are joined

together at the centromere.

Chromatin: The deeply staining genetic material present in the nucleus of a cell

that is not dividing.

Chromosome: A linear thread made of DNA in the nucleus of the cell.

Co-dominant: A gene that expresses a trait regardless of whether or not an alter-

native allele at the same locus is also expressed on the other paren-

tal chromosome.

Crossing over: 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 ho-

mologous chromosomes.

Dominant: 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.

Gene: The basic unit of heredity, made of DNA. Each gene occupies a spe-

cific location on a chromosome.

Locus: The site of a gene on a chromosome.

Lyonization: TheinactivationofoneofthefemaleXchromosomesduring

embryogenesis. This inactivated chromosome forms the Barr body

in the cell nucleus.

Meiosis: A process of two successive cell divisions producing cells, egg, or

sperm that contain half the number of chromosomes found in so-

matic cells.

Mitosis: Division of somatic cells resulting in daughter cells containing the

same number of chromosomes as the parent cell.

Recessive: 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.

Sex-linked: A gene contained within the X or Y chromosome.

Somatic cell: Nonreproductive cells or tissues.

X-linked: A gene on the X chromosome for which there is no corresponding

gene on the Y chromosome.

Chapter 10: Blood Group Genetics 241

Chapter 11: Immunology

Chapter 11

Immunology

THE IMMUNE RESPONSE is a

highly evolved innate and adap-

tive 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 or-

ganisms. A healthy immune response can

recognize foreign material or pathogens

that invade the body and can initiate a se-

ries of events to eliminate these pathogens

with minimal or no prolonged morbidity

to the host.

The numerous components of the im-

mune 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 pro-

mote healing. If the immune system be-

comes hyperreactive, the body may attack

itsowntissueororgans(autoimmunedis

ease), 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 im-

mune 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. In-

nate 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

cellular phagocytosis, and inflammatory

reactions. It is important to note that in-

nate and adaptive immunity are comple-

mentary—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 immuno-

globulin 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 sur-

face of lymphoid cells. Figure 11-2 illus-

trates the similar structure of some of

these immunology receptors. The overall

structure of these receptors is very simi-

lar, 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: immunoglobu-

lins, T-cell receptor (TCR), major histo-

compatibility complex (MHC) Class I and

Class II molecules, and receptors for growth

factors and cytokines. There are several

hundred members of the IgSF (ie, immu-

noglobulin 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 fac-

tor (TNF), some complement compo-

nents, and some heat shock proteins.3The

genes that encode MHC molecules are lo-

cated 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 al-

most all cells in the body. The molecules

are defined by three major Class I genes

244 AABB Technical Manual

Figure 11-1. Examples of the factors used in innate and adaptive immunity and examples of the two

types of immunity.

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 de-

fined 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 cy-

toplasmic tail, a transmembrane region,

and three extracellular immunoglobu-

lin-like domains. β2-microglobulin is non-

covalently associated with the heavy chain

and is not a transmembrane protein. This

protein is required for Class I MHC expres-

sion and function on the cell surface. The

antigen-binding groove of the Class I mole-

cule is formed by the α1and α2domains of

the heavy chain. The structure of the anti-

gen-binding groove consists of a platform

made up of eight parallel βstrands that is

supported by two αhelices.4The peptides

displayed in the antigen-binding groove are

8to12aminoacidslongandrepresenthy

drolyzed proteins that have been synthe-

sized within the antigen-presenting cell;

hence, they are referred to as endogenous

antigens. The endogenous source of pro-

teins 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.

Chapter 11: Immunology 245

Figure 11-2. Structures of some receptors found on various cells in the immune system.1

MHC Class II Molecules

TherearethreemajorClassIIgeneloci:

HLA-DR, -DQ, and -DP. As with the Class I

molecules, there are many different al-

leles 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 mole-

cules.

ClassIImoleculesareheterodimeric

structures consisting of a heavy αchain (30

to 34 kDa) and a light βchain (26 to 29

kDa). Each chain has a cytoplasmic tail, a

transmembrane region, and an extracellu-

lar portion. There are two immunoglobu-

lin-like domains on each chain (α1and α2

and β1and β2). The α2and β2domains for

each gene have a constant structure; the α1

and β1domains are diverse. The antigen-

bindinggrooveislocatedwithintheα1,β1

domains and is similar in structure to the

ClassImolecules.However,thegrooveis

larger, accommodating peptides that are 12

to 20 amino acids in length. Class II mole-

cules are expressed on monocytes, macro-

phages, 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 anti-

gens 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 sys-

tem 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

date.3Some of the major CD markers on

cells of the immune system are summa-

rized in Table 11-1.

Cell Adhesion Molecules

For normal immune function to occur,

leukocytes must be able to attach to extra-

cellular matrices and each other. Three

families of adhesion molecules facilitate

this attachment process: selectins, inte-

grins, and IgSF adhesion molecules. The

members of the selectin family (L-selec-

tin, E-selectin, and P-selectin) are found

on leukocytes and participate in the pro-

cess of leukocyte rolling along the vascu-

lar endothelium. The integrins (VAL,

LFA-1,andMAC-I)andtheIgSFadhesion

molecules (ICAMs, VCAMs, LFA-2, and

LFA-3) are required to stop leukocyte roll-

ing and mediate leukocyte aggregation

and transendothelial migration.6Most ad-

hesion molecules have CD designations

(eg, LFA-2 is CD2 and LFA-3 is CD58).7

Signal Transduction

Signal transduction is the process of send-

ing signals between or within cells that

results in the initiation or inhibition of

gene transcription. Cell surface activation

signals associated with the immune re-

sponse are initiated by extracellular inter-

action 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 immunodefi-

ciency disease can result from a defi-

ciency of Jak3, which impairs signal

transduction of a specific cytokine recep-

tor subunit.7

246 AABB Technical Manual

Chapter 11: Immunology 247

Table 11-1. Some Major CD Antigens on Cells of the Immune System

Designation

Cell

Population

Other Cells

with Antigen Comments

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 leuke-

mia; 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)

(cont’d)

248 AABB Technical Manual

Table 11-1. Some Major CD Antigens on Cells of the Immune System (cont’d)

Designation

Cell

Population

Other Cells

with Antigen Comments

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 func-

tion 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 dif-

ferent 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; prolifer-

ating cells of other somatic lines;

reticulocytes

This is the transferrin receptor

CD = clusters of differentiation; APCs = antigen-presenting cells; NK = natural killer; TCR = T-cell receptor; MHC = major histocompatibility complex; HIV = human immunodefi-

ciency virus; IL = interleukin; NKT = natural killer T.

Organs of the Immune

System

Numerous organs are involved in the im-

mune system. The central organs include

the marrow, liver, and thymus. Peripheral

organs are the lymph nodes and spleen.

The gastrointestinal tract-associated lym-

phoid 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 in-

volved in the induction of the immune re-

sponse, and both macrophages and poly-

morphonuclear leukocytes participate in

various inflammation responses associated

with the immune response. All of these cells

are of hematopoietic origin (ie, marrow-

derived) and are formed from a single pro-

genitor 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. Granulo-

cytes (neutrophils, basophils, and eosino-

phils), platelets, mast cells, red cells, and

macrophages arise from myeloid progeni-

tors. 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

cells are the precursors of the cells that

make antibody (plasma cells). T cells con-

sist of subpopulations that either help in

antibody formation (helper T cells), kill

target cells (cytotoxic T cells), induce in-

flammation (delayed hypersensitivity T

cells), or inhibit the immune response

(regulatory T cells).

B Lymphocytes

Each B cell can recognize one (or a lim-

ited set) of antigen epitopes through re-

ceptors on the cell surface. These recep-

tors are similar to IgM and IgD molecules,

which are produced and transported to

themembranesurface.Whentheimmu

noglobulin 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 addi-

tional 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 mol-

ecules include Igα,Igβ,MHCClassIImole

cules, complement receptors, and some CD

markers (see Table 11-2). All of these com-

ponents are part of the BCR antigen com-

plex (some of these structures are illustra-

ted in Fig 11-2).

ThechallengeforthebodyistohaveB

lymphocytes that can recognize each of the

thousands of different foreign antigens en-

countered over a lifetime. The immune sys-

tem has a unique approach to ensure this

diversity.Thehumangenomeisknownto

contain approximately 40,000 genes. The B

Chapter 11: Immunology 249

cellsinthebodyprobablymakemorethan

100 million different antibody proteins that

are expressed as immunoglobulin recep-

tors. Because the human body does not

have enough genes to code for the millions

of different foreign proteins that the im-

mune system must have the capability to

recognize, a process called gene rearrange-

ment is used to create the required diver-

sity.9

Several genes code for the heavy chain

and light chain that make up the immuno-

globulin 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 (λlight

chain). Three gene loci contribute to the di-

versity of the immunoglobulin receptor: V

(variable), D (diversity), and J (joining). The

250 AABB Technical Manual

Table 11-2. Receptors/Markers Present on Macrophages, Monocytes, and B

Lymphocytes

Marker Function

Macrophages and Monocytes

Complement receptors

CR1 (C3b receptors, CD35) Binds to cells coated with C3b

CR3 (C3bi receptor, CDIIb) Adhesion and activation

Cytokine receptors (IL-1, IL-4, IFNγ) Receptors that bind cytokines signaling

and migration-inhibition factor activation and other cell functions

Fc receptors

FcγRI (CD64) High affinity for IgG

FcγRII (CD32) Medium affinity for IgG

FcγRIII (CD16) Low affinity for IgG

FcγRII (CD23) Low-affinity receptor for the Fc of IgE

Leukocyte function antigen Adhesion and activation

(LFA1 or CD11a)

Mannose/fucose receptors Binds sugars on microorganisms

P150,95 (cD11c) Adhesion and activation

B Lymphocytes

CD5 Cell marker that identifies a subset of B cells

predisposed to autoantibody production

CD19, 20, and 22 Primary cell markers used to distinguish

B cells

CD72-78 Other cell markers that identify B cells

Complement receptors Play a role in cell activation and “homing”

C3b (CR1, CD35); C3d (CR2, CD21) of cells

IgαTransport and assemble IgM monomers in

the cell membrane

IgβAccessory molecules that interact with the

transmembrane segments of IgM

MHC Class II (DP, DQ, DR) Present on antigen-presenting cells and is

critical for initiating T-cell-dependent

immune responses

Chapter 11: Immunology 251

Figure 11-3. The pluripotent stem cell, in the upper middle part of the diagram, gives rise to the lym-

phoid 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)

fourth locus codes for the C (constant) region

of the immunoglobulin receptor and de-

fines 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-cell-

dependent process that occurs at the DNA

level. The process of isotype switching al-

lows the specificity of antibody molecule to

be maintained regardless of the heavy chain

isotype.9,10 Table 11-3 summarizes the bio-

logic properties of the different immuno-

globulin isotypes.

At the pre-B-cell stage of development,

one heavy chain gene is randomly selected

from each of the four segments (VH,D

H,J

and CH). There are over 104possible combi-

nations because of the large number of

possible genes at each segment. The light

chain gene has only three segments (VL,J

and CL), and one gene is selected from each

of these segments in a process similar to

the heavy chain gene selection. This pro-

cess 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 immu-

noglobulin receptor with unique antigen

specificity. In addition to gene rearrange-

ment, several other processes contribute to

this diversity. These processes include so-

matic 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 combina-

tion of all of these processes ensures that B

cells have immunoglobulin receptors spe-

cific for any foreign antigen that could be

encountered (approximately 1011 antigen

specificities).9-11

Because the formation of the B-cell im-

munoglobulin receptor occurs through

random rearrangement, some of the recep-

tors 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 pro-

cess 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 an-

tigen, 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 (pro-

grammed cell death). Only 25% of the B cells

that mature in the marrow reach the circu-

lation. 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 pe-

ripheral blood circulation, they bind for-

eign antigens that are specific for their im-

munoglobulin receptors. When specific

antigen binds to the immunoglobulin re-

ceptor, a signal occurs causing the recep-

tor/antigen complex to be internalized. In-

side the cell, the antigen is degraded into

small peptides that bind to MHC Class II

molecules within the cell. This MHC-pep-

tide 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 im-

munoglobulin class. However, each time

B-cell proliferation occurs, somatic muta-

tions result in slight differences in the bind-

ing affinity of the immunoglobulin recep-

tor. Because immunoglobulin receptors

with the highest binding affinity will be the

ones most likely to encounter antigen, this

process preferentially results in prolifera-

tion of B cells with the highest affinity for

252 AABB Technical Manual

antigen. This preferential selection is termed

a focused immune response.5,13,14

T Lymphocytes

There are two major types of T cells: cyto-

toxic T cells and helper T cells. These two

cell types can be differentiated by the pre-

senceofspecificCDmarkersonthecell.

Cytotoxic T cells are positive for the sur-

face 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

Chapter 11: Immunology 253

Table 11-3. Characteristics and Biologic Properties of Human Immunoglobulins

Class IgG IgA IgM IgD IgE

Structure

H-chain isotype

Number of subclasses

L-chain, types

Molecular weight (daltons)

κ,λ

150,000

κ,λ

180,000-

500,000

κ,λ

900,000

κ,λ

180,000

κ,λ

200,000

Exists as polymer No Yes Yes No No

Electrophoretic mobility γγbetween γ

and β

between γ

and β

fast γ

Sedimentation constant

(in Svedberg units)

6-7S 7-15S 19S 7S 8S

Gmallotypes(Hchain) +0000

Km allotypes (Kappa L chain:

formerly Inv)

+++??

Amallotypes 0+000

Serum concentration (mg/dL) 1000-1500 200-350 85-205 3 0.01-0.07

Total immunoglobulin (%) 80 15 5 <0.1 <0.1

Synthetic rate (mg/kg/day) 33 24 6-7 <0.4 <0.02

Serum half-life (days) 23 6 5 2-8 1-5

Distribution (% of total

in intravascular space)

45 42 76 75 51

Present in epithelial secretions No Yes No No No

Antibody activity Yes Yes Yes Probably

Yes

Serologic characteristics Usually

nonagglu-

tinating

Usually

nonagglu-

tinating

Usually

agglu-

tinating

Fixes complement Yes No Yes No No

Crosses placenta Yes No No No No

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 dur-

ing T-cell ontogeny. If a T cell is able to rec-

ognize self MHC antigens, it survives (posi-

tive 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 recog-

nize 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 respon-

sible for MHC/peptide recognition is the TCR

(see Fig 11-2). There are two major types of

TCRs: those that express αand βchains or γ

and δchains 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 pro-

cess of gene rearrangement in a manner

similar to MHC and immunoglobulin re-

ceptors. The number of possible TCR αand

βspecificities is estimated at 1015.TheTCRs

bearing γand δare expressed on 5% to 15%

of T cells, predominantly by those T cells in

the mucosal endothelium. These T cells ap-

pear to play an important role in protecting

the mucosal surfaces of the body from for-

eign bacteria.

The TCR is noncovalently associated with

theCD3complex,whichismadeupofthree

pairs of dimers. This CD3 complex is re-

sponsible for signal transduction once pep-

tide is recognized by the TCR.16

Recognition by Cytotoxic T Cells.

Cytotoxic T cells recognize peptides associ-

ated with Class I MHC molecules. As dis-

cussed 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 rec-

ognizes 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 dis-

rupt the integrity of the target cell mem-

brane, resulting in cell death. During this

process, cytokines [TNF-αand 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 effec-

tive mechanism for killing cells infected with

virus, the process can be harmful to the host.

The cytokines produced to prevent viral rep-

lication can cause adverse effects including

the damage or destruction of healthy host

tissue. Liver damage associated with hepa-

titis 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

254 AABB Technical Manual

with CD40 on B cells. This cascade of sig-

nals 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 transform-

ing growth factor β(TGF-β)andIL-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 CD40L-

CD40 interaction is impaired, isotype

switching will not occur and only IgM anti-

body 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 matu-

ration 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 approxi-

mately 10% of the lymphocyte popula-

tion. These cells have the ability to kill

some cells infected with viruses and some

tumor cells. NK cells do this by a mecha-

nism termed antibody-dependent cellular

cytotoxicity or ADCC. ADCC occurs when

viral proteins are expressed on the surface

of an infected cell, usually as a result of

viral budding. The proteins are recog-

nized 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 pres-

ence of antibody bound on the surface of

the cells via their Fcγreceptors. 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 in-

flammation. Eosinophils represent only

2% to 15% of the white cells and play an

important role in regulating the inflam-

matory response by releasing an antihis-

tamine. These cells may also play a role in

phagocytosing and killing microorgan-

isms. 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. Histori-

cally, 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 ac-

complished through the presence of recep-

tors on the cell membrane. There are many

types of receptors including: mannose-fucose

Chapter 11: Immunology 255

receptors that bind to sugars on the surface

of microorganisms, Fc receptors that bind

to IgG, and complement receptors. A sum-

mary of some membrane receptors found

on macrophages and monocytes is found

in Table 11-2. The internalization and pro-

cessing of particulate matter occur through

the production of enzymes (peroxidase and

acid hydrolases).5Under 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

Therearethreemajorsolublecomponents

of the immune response: immunoglobu-

lins, complement, and cytokines.

Immunoglobulins

Immunoglobulins are the proteins that can

be cell bound and serve as antigen recep-

tors on B cells (see section on B lympho-

cytes) or can be secreted in a soluble form

as antibodies. The molecular development

of the immunoglobulin molecule is dis-

cussed in the section on B lymphocytes.

The structures of the different types of

immunoglobulin are discussed below.

Each monomeric immunoglobulin mol-

ecule 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 approxi-

mately 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 (ap-

proximately 210 amino acids; molecular

weight 25 kDa) and can be either kappa (κ)

or lambda (λ). The heavy and light chains

of the immunoglobulin molecule are held

together by disulfide bonds. The polypep-

tide 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 epi-

topes expressed in this region are termed

idiotypes. The remaining domains on both

the heavy and light chains are called con-

stant 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 anti-

gen-binding components to operate inde-

pendently. The biologic functions of the

molecule are associated with the constant

domains on the heavy chain. These func-

tions include: placental transfer, macro-

phage binding, and complement activa-

tion.4

Interchain Bonds

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 CH1andC

H2, in an area of con-

siderable flexibility called the hinge re-

gion. 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 infor-

mation about immunoglobulin structure

and function is derived from the study of

cleavage fragments generated by papain

digestion of Ig molecules. Papain cleaves

theheavychainatapointjustabovethe

hinge, creating three separate fragments.

Two are identical, each consisting of one

256 AABB Technical Manual

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 anti-

body, are called Fab fragments. The joined

C-terminal halves of the heavy chains

constitute a nonantibody protein frag-

ment 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

IgAcanformpolymers.IgG,IgE,andIgD

exist only in the monomeric form; there

are no polymeric forms of these Ig classes.

The IgM synthesized by unstimulated B

cellsandexpressedonthemembraneas

the immunoglobulin receptor is expres-

sed in the monomeric form. As men-

tioned previously, the µheavy 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 recep-

tor. Following clonal expansion and differ-

entiation to a plasma cell, the activated

cell produces µchains with one less con-

stant domain. While still in the plasma-

cell cytoplasm, five IgM monomers are

joined by the formation of disulfide bonds

between the CH3 domains and CH4do

Chapter 11: Immunology 257

Figure 11-4. The basic four-chain immunoglobulin unit. Idiotypic specificity resides in the variable do-

mains of heavy and light chains (VHand VL). Antigen-binding capacity depends on intact linkage be-

tween one light chain (VLand CL) and the amino-terminal half of one heavy chain (VHand CH1), the Fab

fragments of the molecule. Disulfide bonds in the hinge region join carboxy-terminal halves of both

heavy chains (CH1 and CH3, plus CH4for and heavy chains), to form the Fc fragment. (Used with per-

mission from Goldsby et al.8)

mains.5The result is a pentamer that is

secreted to the exterior of the cell and

constitutes the form in which IgM accu-

mulates 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 tri-

mers 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 secre-

tory component appears to protect the bio-

logically 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 penta-

valent.Becauseoftheirlargesizeand

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 macro-

phages.

IgG molecules can be classified into four

subclasses: IgG1, IgG2, IgG3, and IgG4.

Structurally, these subclasses differ primar-

ily in the characteristics of the hinge region

and the number of inter-heavy-chain

disulfide bonds. Biologically, they have sig-

nificantly different properties. IgG3 has the

greatest ability to activate complement, fol-

lowed by IgG1 and, to a much lesser extent,

IgG2. IgG4 is incapable of complement ac-

tivation. 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 ex-

ception that IgG2 has an affinity similar to

IgG1 and IgG3 for an allotype of Fcγrecep-

tor IIa.

258 AABB Technical Manual

IgA. Although there is a large body con-

tent of IgA (10% to 15% of serum immuno-

globulin 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 elimi-

nated as surface secretions are excreted.

This process may be important in control-

ling the development of hypersensitivity.

The heavy chain of IgA has no comple-

ment-binding site; hence, IgA cannot acti-

vate complement through the classical

pathway. IgA can activate complement

through the alternative pathway (see be-

low).

IgE. The concentration of serum IgE is

measured in nanograms, compared with

milligram levels for other immunoglobu-

lins. Even when patients with severe aller-

gies have markedly elevated serum concen-

trations 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 ap-

pears to be involved in reactions to proto-

zoal parasites, no specific protective mecha-

nisms have been identified.

IgD. Serum contains only trace amounts

of IgD. Most IgD exists as membrane im-

munoglobulin 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 sys-

temof25to30serumandmembrane

proteins that act in a cascading manner—

similar to the coagulation, fibrinolytic,

and kinin systems—to produce numerous

biologic effects. The participating pro-

teins remain inactive until an event initi-

ates the process, following which the

product of one reaction becomes the cata-

lyst 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: pro-

motion of acute inflammatory events; alter-

ation of surfaces so that phagocytosis is en-

hanced; and the modification of cell mem-

branes, which leads to cell lysis. These ac-

tions 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.

Different mechanisms17 exist for activat-

ing 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. Combina-

tion 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 inter-

acts with the C1 component of comple-

ment. C1 can combine only with Ig mole-

cules having an appropriate heavy-chain

configuration. This configuration exists in

Chapter 11: Immunology 259

the µheavy chain and in the γchains in

IgGsubclasses1,2,and3.

The C1 component of complement atta-

ches 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 com-

plement cascade. For IgG antibodies to ac-

tivate the sequence, two separate molecules

must attach to antigen sites close together.3

Hence, complement activation by IgG anti-

bodies depends not only on the concentra-

tion and avidity of the antibody, but also on

the topography of the antigen.

Circulating C1 is a macromolecule con-

sisting of three distinct proteins (C1q, C1r,

and C1s). When an antigen-antibody reac-

tion occurs, two or more chains of C1q at-

tach 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 ab-

sence of Ca2+, the complex dissociates.

Thus, chelating agents often used in the

laboratory, such as citrate and oxalate anti-

coagulants, 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 activ-

ity. Most of the C4b generated is inacti-

vated; 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

remainsboundtoC4formsanactivated

260 AABB Technical Manual

Figure 11-5. Diagram illustrating the activation of complement by the classical and alternative path-

ways. (Used with permission from Heddle.1)

Classical Pathway

complex C4b2a,alsoknownasC3con

vertase. Each C3 convertase can cleave more

than 200 native C3 molecules, splitting the

molecule into two fragments. A small frag-

ment (C3a) that has anaphylatoxic activity

is released into the plasma; the larger frag-

ment (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 comple-

ment activation in the absence of an anti-

gen/antibody interaction. This pathway is

a first-line antimicrobial defense for ver-

tebrates and a mechanism whereby pre-

vertebrates can enhance their inflamma-

tory effectiveness. The alternative pathway

is a surface-active phenomenon that can

be triggered by such initiators as dialysis

membranes; the cell wall of many bacte-

ria, yeasts, and viruses; protein com-

plexes, including those containing anti-

bodies that do not bind complement;

anionicpolymerssuchasdextran;and

some tumor cells. The alternative path-

way 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 alterna-

tive pathway: factor B, factor D, properdin

(factor P), and C3. Fluid-phase C3 under-

goes continuous but low-level spontaneous

cleavage, resulting in C3i that is rapidly in-

activated 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-

vated; however, if C3b binds to a foreign

surface such as a bacteria cell wall, the acti-

vation 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, releas-

ing the small Ba fragment leaving cell-

bound C3bBb. This complex will dissociate

unless it is stabilized by properdin, result-

ing in the complex C3bBbP

.LiketheC4b2a

complex of the classical pathway, C3bBbP

iscapableofconvertingmoreC3intoC3b.

In summary, both the classical and alterna-

tive 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 ana-

phylatoxin activity and a larger fragment

(C5b). The C5b fragment binds C6, C7,

C8, and up to 14 monomers of C9, result-

inginalyticholeinthemembrane.Small

amounts of lysis can occur when C8 is

bound; however, the binding of C9 facili-

tates 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 sub-

components of C3 (C3b, iC3b) can facilitate

phagocytosis by acting as opsonins. How-

Chapter 11: Immunology 261

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 identi-

fied 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 lym-

phocytes, it is an opsonic receptor that is

involved in lymphocyte activation. CR1 also

plays a regulatory role in complement acti-

vation by assisting factor I in cleaving C3b

into iC3b and C3dg.

CR2. CR2 is found on B cells, some epi-

thelial cells, and follicular dendritic cells. It

plays an important role in mediating B-cell

activation. It is also the receptor for inter-

feron αand the Epstein-Barr virus.

CR3. CR3 is found on cells of the myeloid

lineage. CR3 mediates phagocytosis of parti-

cles coated with iC3b and is also an impor-

tant 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 op-

sonic activity for iC3b, and it plays a role in

adhesion.

Regulation of Complement Activation

There is a need to control or regulate the

enzyme and activation factors of the com-

plement cascade. The regulatory actions

of these control proteins prevent damage

to host tissue. These control systems

(Table 11-4) act by several different mecha-

nisms: direct inhibition of serine proteases,

decay and destruction of convertases, and

control of membrane attack complexes.19

Physiologic Effects of Complement

Activation

Opsonization. Neutrophils and macro-

phages phagocytose any particle that pro-

trudes from the surface of a cell or micro-

organism with no regard to the nature of

the material. Phagocytosis is more intense

if the particle adheres firmly to the mem-

brane of the phagocytic cell. To achieve

adherence, phagocytic cells have various

receptor molecules such as the Fc recep-

tors 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 ana-

phylatoxins bind to receptors on mast cells

and basophils, causing them to release his-

tamine and other biologic response modifi-

ers that can be associated with anaphylaxis.

More frequently, anaphylatoxins affect vas-

cular permeability, membrane adhesion

properties, and smooth-muscle contraction

that constitute a large part of the acute in-

flammatory response. C5a and C3a also

cause neutrophils and macrophages to mi-

grate to the site of complement activation.

Cytokines

Throughout this chapter, inference has

been made to a number of soluble media-

tors termed cytokines. Cytokines are a

diverse group of intracellular signaling

peptides and glycoproteins that have mo-

lecular weights ranging from 6,000 to

60,000 daltons. Each cytokine is secreted

by particular cell types in response to dif-

262 AABB Technical Manual

ferent stimuli and has been implicated in

a wide variety of important regulatory bi-

ologic functions such as inflammation,

tissue repair, cell activation, cell growth,

fibrosis, and morphogenesis. One cytokine

can have several different functions, de-

pending on the type of cell to which it

binds. Several hundred different cytokines

have been described. Some of the cyto-

kines involved in immune functions are

summarized in Table 11-5.1,5 Cytokines

have been implicated in a variety of clini-

cally important factors of transfusion

medicine (hemolytic and febrile transfu-

sion reactions, immunomodulation, stem

cell collection, etc) (see Chapter 27).

Immunology Relating to

Transfusion Medicine

Several examples of how the immune sys-

tem relates to situations encountered in

transfusion medicine are described below,

including red cell alloimmunization, pla-

telet alloimmunization, immune-medi-

ated red cell destruction, and reagent an-

tibody production.

Chapter 11: Immunology 263

Table 11-4. Summary of the Inhibitors of Complement Activation

Complement Control Proteins Function

Inhibition of Serine Protease

C1 inhibitor

Decay/Destruction of Convertases

A serine protease inhibitor that binds and acti-

vates Clr and Cls

Factor I plus C4 binding protein (C4-bp)

C4-bp

Catabolizes C4 in the fluid phase

Causes dissociation of C2a from C4b2a

Decay accelerating factor

(DAF, CD55)

Inhibits the binding of C2 to C4b

Causes dissociation of C2 from C4b

Complement receptor 1 (CR1, CD35) Accelerates the dissociation of C3bBb

Membrane cofactor protein (MCP) Prompts catabolism of C4b by factor I and is a

cofactor with factor I to cleave C3b

Factor H Causes dissociation of Bb from C3I and C3b and

is a cofactor to factor I for catabolism of C3i

and C3b

Factor I Cleaves and degrades C3b using one of three

cofactors: factor H (plasma), CR1, or MCP

(membrane-bound)

Membrane Attack Complex (MAC)

S protein (Vitronectin) Binds to the C5b67 complex preventing insertion

into the lipid bilayer

C8-binding protein (CD59) This cell-bound protein binds to C8 preventing

C9 from inserting into the membrane

MAC-inhibiting protein (MIP) Inhibitor of C9, also known as homologous

restriction factor (HRF)

264 AABB Technical Manual

Table 11-5. Summary of Some Cytokines Involved in the Immune System, Their

Site of Production, and Function1

Cytokine Produced by Primary Functions

Interleukins

IL-1 Many cells (eg, APCs,

endothelial cells, B cells,

fibroblasts)

T-cell activation, neutrophil

activation, stimulates

marrow, pyrogenic, acute-

phase protein synthesis

IL-2 Activated THcells T-cell growth, chemotaxis,

macrophage activation

IL-4 Activated THcells 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)

Others

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, inhibi-

tory function

Colony-stimulating

factors (GM-CSF,

M-CSF, G-CSF)

Various cells Growth and activation of phagocytic

cells

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.

Red Cell Alloimmunization

The mechanism of red cell alloimmuniza-

tion is not well understood. When allo-

geneic red cells are transfused, some of

the red cells fragment as they age or when

they pass through the spleen, thus releas-

ing membrane-bound red cell proteins

into the bloodstream as the fragments de-

grade. Both formal antigen-presenting

cells and B lymphocytes can present anti-

gen either as a primary immune response

(formal antigen presentation by dendritic

cells) or in the secondary immune re-

sponse by B cells.

HLA Alloimmunization to Platelets

Leukocyte reduction of red cells and pla-

telets to a threshold of 5 × 106per product

has been shown to reduce HLA alloim-

munization, 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 previ-

ously, a small percentage of T cells (<10%)

areabletorecognizeforeign-MHC-carry

ing foreign peptides. When platelets are

transfused, the recipient’s T helper cells

recognize the foreign MHC Class II com-

plex 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 re-

ceptor, resulting in cell proliferation and

MHC Class I (HLA) antibody production

(Fig 11-6). In this case, the donor’s leuko-

cytes serve as the antigen-presenting

cells. This major mechanism is termed

“direct allorecognition” because the recip-

ient’s T cells are directly stimulated by do-

nor antigen-presenting cells.20 Alterna-

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 reduc-

tionappearstobeeffectiveinreducing

HLA alloimmunization because antigen

presentation by donor leukocytes is re-

duced and the amount of HLA Class I an-

tigens 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 predomi-

nantly two mechanisms: intravascular

hemolysis and extravascular red cell de-

struction (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 re-

leased into the plasma. Free hemoglobin

binds to haptoglobin and is excreted in

the urine. The heme portion of hemoglo-

bin binds to albumin, forming methemal-

bumin, which can be visually detected in

plasma by a brownish green discolor-

ation. At times, the inhibitors of comple-

ment 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 trig-

gering phagocytosis. Enzymes can cleave

the cell-bound C3b, leaving a small frag-

ment (C3dg) present on the cells. C3dg-

sensitized red cells survive normally be-

cause phagocytic cells do not have recep-

tors for C3dg. If IgG is present on the red

cells, binding to Fcγreceptors will occur,

resulting in phagocytosis. If both IgG and

Chapter 11: Immunology 265

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 opti-

mal as reagents for use in serologic testing

because they can vary in concentration,

serologic properties, and epitope recogni-

tion and can contain other antibodies of

unwanted specificity. The ideal serum for

serologic testing is a concentrated sus-

pension of highly specific, well-character-

ized, uniformly reactive, immunoglobulin

molecules. Until the 1970s, the only way

to obtain reagents was to immunize ani-

mals or humans with purified antigens and

then perform time-consuming and some-

times unpredictable separation techni-

ques in an attempt to purify the resulting

sera. Monoclonal antibody production

provided an alternative to human and an-

imal sources of these proteins. Using this

approach, a single B-cell clone is propa-

gated 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.

In 1976, Köhler and Milstein21 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, allow-

ing the cytoplasm and the nucleus of two

266 AABB Technical Manual

Figure 11-6. Diagram illustrating the major mechanism of HLA alloimmunization due to leukocytes

present in platelet transfusion. (Used with permission from Heddle.1)

different kinds of cells to fuse into a single

cell. These plasma cell/myeloma cell hy-

brids can be maintained in cell culture for

prolonged periods, producing large quanti-

ties of the selected antibody.

The exquisite specificity of monoclonal

antibodies is both an advantage and a dis-

advantage 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 configura-

tion. Thus, reagent preparations typically

used in the laboratory are blends of several

different monoclonal products, thereby in-

creasing 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 thera-

peutic treatments,22 as well as for use as

typing reagents.23

References

1. Heddle NM. Overview of immunology. In:

ReidME,NanceSJ,eds.Redcelltransfusion.

A practical guide. Totowa, NJ: Humana Press,

1998:13-37.

2. Barclay AN. Membrane proteins with immu-

noglobulin-like domains—a master super-

family of interaction molecules. Semin

Immunol 2003;15:215-3.

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.

3. MarshSG,AlbertED,BodmerWF,etal.No

menclature for factors of the HLA system, 2002.

Hum Immunol 2002;63:1213-68.

4. Santos-Aguado J, Barbosa JA, Biro A, Stro-

minger JL. Molecular characterization of se-

rologic recognition sites in the human HLA-

A2 molecule. J Immunol 1988;141:2811-18.

5. RoittI,BrostoffJ,MaleD.Immunology.5th

ed. St. Louis: Mosby, 1998.

6. Ono SJ, Nakamura T, Miyazaki D, et al.

Chemokines: Roles in leukocyte develop-

ment, trafficking, and effector function. J Al-

lergy Clin Immunol 2003;111:1185-99.

7. Huston DP. The biology of the immune sys-

tem. JAMA 1997;278:1804-14.

8. Goldsby RA, Kindt TJ, Osborne BA. Kuby im-

munology. 4th ed. New York: WH Freeman

and Company, 2000.

9. Tonegawa S. Somatic generation of antibody

diversity. Nature 1983;302:575-81.

10. Alt FW, Backwell TK, Yancopoulos GD. Devel-

opment of the primary antibody repertoire.

Science 1987;238:1079-87.

11. Janeway CA Jr. How the immune system rec-

ognizes invaders. Sci Am 1993;269(3):73-9.

12. Russell DM, Dembic Z, Morahan G, et al. Pe-

ripheral deletion of self-reactive B cells. Na-

ture 1991;354:308-11.

13. Weissman IL, Cooper MD. How the immune

system develops. Sci Am 1993;269(3):64-71.

14. Paul WE. Infectious diseases and the immune

system. When bacteria, viruses and other

pathogens infect the body, they hide in differ-

ent places. Sci Am 1993;269(3):90-7.

15. MarrackP,LoD,BrinsterR,etal.Theeffects

of thymus environment on T cell develop-

ment and tolerance. Cell 1988;53:627-34.

16. 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.

17. Sakamoto M, Fujisawa Y, Nishioka K. Physio-

logic role of the complement system in host

defense, disease, and malnutrition. Nutrition

1998;14:391-8.

18. RoittI.Essentialimmunology.8thed.Oxford:

Blackwell Scientific Publications, 1994.

19. Devine DV. The regulation of complement on

cell surfaces. Transfus Med Rev 1991;5:123-31.

20. Semple JW, Freedman J. Recipient antigen-

processing pathways of allogeneic platelet

antigens: Essential mediators of immunity.

Transfusion 2002;42:958-61.

21. Köhler G, Milstein C. Derivation of specific

antibody-producing tissue culture and tumor

lines by cell fusion. Eur J Immunol 1976;6:

511-19.

22. MarksC,MarksJD.Phagelibraries—anew

route to clinically useful antibodies. N Engl J

Med 1996;335:730-3.

23. Siegel DL. Phage display tools for automated

blood typing (abstract). Transfusion 2004;

44(Suppl):2A.

Suggested Reading

AbbasAK,LichtmanAH,PoberJS,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 leuko-

cyte antigen factsbook. 2nd ed. San Diego, CA: Ac-

ademic Press, 1997.

Carroll MC. The role of complement and comple-

ment receptors in induction and regulation of im-

munity. 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.

PaulWE.Fundamentalimmunology.5thed.Phila

delphia: 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 immuno-

logy. 10th ed. Stamford, CT: Appleton & Lange, 2001.

Vamvakas EC, Blajchman MA, eds. Immunomodu-

latory effects of blood transfusion. Bethesda, MD:

AABB Press, 1999.

268 AABB Technical Manual

Appendix 11-1. Definitions of Some Essential Terms in Immunology

Adhesion molecule:

Any of the many membrane

molecules expressed on white cells and en-

dothelial 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 anti-

body 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. Immu-

noglobulin molecules on the surface of un-

stimulated lymphocytes serve as antigen

receptors.

Antigen:

Any material capable of specific combi-

nation with antibody or with cell-surface re-

ceptors of T lymphocytes. Often used as a

synonym for “immunogen,” although some

antigens that react with products of the im-

mune response are not capable of eliciting

an immune response.

Antigen-presenting cell:

A cell capable of incor-

porating antigenic epitopes into MHC Class II

molecules and displaying the epitope-MHC

complex on its membrane.

Clone:

A population of genetically identical cells

derived from successive divisions of a single

progenitor cell.

Cytokine:

A low-molecular-weight protein, se-

creted from an activated cell, that affects the

function or activity of other cells.

Epitope:

The small portion of an immunogen,

usually5to15aminoacidsor3to5glyco-

sides, that combines specifically with the an-

tigen receptor of a T or B lymphocyte.

Idiotype:

The molecular configuration unique to

the variable portion of an antigen-receptor

molecule, reflecting the DNA rearrangement

occurring in earliest lymphocyte differentia-

tion and conferring upon the cell its specific-

ity of antigen recognition.

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, por-

tions 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 for-

eign.

Ligand:

A molecule, either free in a fluid milieu

or present on a membrane, whose three-di-

mensional 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 αchain

linked noncovalently with the nonpolymorphic

β2-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.

MHC Class II molecules:

Heterodimeric mem-

brane 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 an-

tigens.

Phagocytosis:

The process whereby macro-

phages and granulocytes ingest particulate

material present in the surrounding milieu

and subject it to intracellular alteration.

Receptor:

A cell-membrane protein molecule

whose three-dimensional configuration al-

lows it to form a tightly fitting complex with

another molecule (called its ligand) of com-

plementary shape.

Chapter 11: Immunology 269

Chapter 12: Red Cell Antigen-Antibody Reactions and Their Detection

Chapter 12

Red Cell Antigen-Antibody

Reactions and Their

Detection

DEMONSTRATION OF RED cell an-

tigen-antibody reactions is key to

immunohematology. The combi-

nation of antibody with antigen may pro-

duce a variety of observable results. In

blood group serology, the most com-

monly observed reactions are agglutina-

tion, 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 adja-

cent 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 ad-

ditional 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) neces-

sary 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 ac-

tivates the complement cascade. (The ac-

tions 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,andanti-Jk

a)may

271

cause intravascular hemolysis in a transfu-

sion recipient.

Precipitation is the formation of an in-

soluble, usually visible, complex when solu-

ble 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 proce-

duressuchasimmunodiffusionandim

munoelectrophoresis.

Precipitation may not occur, even when

soluble antigen and its specific antibody

are present. Precipitation of the antigen-an-

tibody complex requires that antigen and

antibody be present in optimal propor-

tions. If antibody is present in excess, too

few antigen sites exist to crosslink the mol-

ecules and a lattice structure is not formed.

Antigen-antibody complexes form but do

not accumulate sufficiently to form a visi-

ble 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. Al-

though a visible precipitate is often not

produced, such inhibition can be useful in

antibody identification procedures by se-

lectively eliminating specific antibodies.

Factors Affecting Red Cell

Agglutination

Agglutination is a reversible chemical re-

action and is thought to occur in two

stages: 1) sensitization, the attachment of

antibody to antigen on the red cell mem-

brane; and 2) formation of bridges be-

tween the sensitized red cells to form the

lattice that constitutes agglutination. Vari-

ous factors affect these two stages and

can be manipulated to enhance (or de-

crease) agglutination. The effects of en-

hancement techniques on the two stages

cannot always be clearly differentiated.1

Stage One: Sensitization

Initially, the antigen and antibody must

come together and interact in a suitable

spatial relationship. The chance of associ-

ation 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.AsshowninFig12-1,antibody

and antigen must complement each other

with both a structural (steric) and a chem-

ical 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-an-

tibody combination is reversible, and ran-

dom bonds are constantly made and bro-

ken until a state of equilibrium is attained.

272 AABB Technical Manual

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)

Chemical Bonding

Various types of chemical bonds are re-

sponsible for the binding of antibody to

antigen, including hydrogen bonds, hy-

drophobic 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 charac-

teristics, 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 con-

trast, hydrophilic bonds formed with pro-

tein antigens are endothermic, so these

bonds are enhanced at higher reaction

temperatures.

Equilibrium (Affinity) Constant of the

Antibody

The equilibrium constant or affinity con-

stant (Ko) of a reaction is determined by

the relative rates of association and disso-

ciation (see Fig 12-2). For each antigen-

antibody reaction, the Kovaries. The Kore-

flects the degree to which antibody and

antigen associate and bind to one another

(“goodness of fit”) and the speed of the re-

action. The higher the Kovalue, the better

the association or “fit.” When the Kois

large, the reaction occurs more readily

and is more difficult to dissociate; such

antibodies may have a greater clinical im-

portance. When the Kois small, a higher

ratioofantibodytoantigenmaybere

quired for detection.

The degree of antigen-antibody “fit” is

influenced by the type of bonds predomi-

nating. Hydrophobic bonds are usually as-

sociated with higher Kothan hydrogen

bonds. The Kois also affected by physical

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 concentra-

tions. In laboratory tests that use agglutina-

tion 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 cate-

gories: those optimally reactive at “cold”

temperatures (eg, 4 to 25 C) and those op-

timally 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-

Chapter 12: Red Cell Antigen-Antibody Reactions and Their Detection 273

All chemical reactions are reversible. Anti-

gen (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).

Ag Ab AgAb

+→

←

By the law of mass action, the speed of

the reaction is proportional to the concen-

trations of the reactants and their prod-

uct. The equilibrium constant (Ko)isa

function of these intrinsic association

constants for the antibody being tested.

[AbAg]

[Ab][Ag]

Figure 12-2. The law of mass action and the equi-

librium constant.

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-re-

active” 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, how-

ever, the temperature of optimal antigen-

antibody reactivity has more to do with

thetypeofreactionandthechemicalna

ture 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.

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 ex-

amples 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 al-

ternativeandmaybeparticularlyhelpful

in solid-phase testing.3

Incubation Time

The time needed to reach equilibrium dif-

fers for different blood group antibodies.

Significant variables include temperature

requirements, immunoglobulin class, and

specific interactions between antigen

configuration and the Fab site of the anti-

body. 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 re-

active antibodies, association may not

reach equilibrium at 30 minutes and ex-

tending the incubation time may increase

sensitivity of the test. Prolonging the incu-

bation time beyond 60 minutes has few dis-

advantages except for the delay before re-

sults are available.

Incubation time at 37 C can usually be

reduced to 10 to 15 minutes if a low-ionic-

strength saline (LISS) solution is used (in-

cluding LISS additive solutions). The use of

water-solublepolymerssuchaspolyethyl

ene glycol (PEG) can also reduce the neces-

sary 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 mole-

cules. This hinders the association of anti-

body with antigen. By lowering the ionic

strength of the reaction medium, how-

ever, this shielding effect can be weak-

ened and electrostatic attractions enhanced.

Reducing the salt concentration of the se-

rum-cell system increases the rate at

which antibody and antigen come into

proximity and may increase the amount

of antibody bound. Extending the incuba-

tion 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 ex-

cess of antigen is desirable. For most red

cell tests, however, antigen excess reduces

the number of antibody molecules bound

274 AABB Technical Manual

per red cell, limiting their ability to agglu-

tinate. Antibody excess is, therefore, de-

sirable 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 anti-

body excess may inhibit direct agglutina-

tion, producing a prozone phenomenon

comparable to what occurs with precipi-

tation reactions. Usually, however, in-

creasing antibody concentration en-

hances the sensitivity of agglutination

tests. Reducing the concentration of red

cells from 5% to 2% or 3% doubles the se-

rum-to-cell ratio, as does adding 4 drops

of serum to the standard cell suspension.

Sometimes, it is useful to increase the vol-

ume of serum to 10 or even 20 drops, par-

ticularly during an investigation of a

hemolytic transfusion reaction in which

routine testing reveals no antibody. Alter-

ations 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 pretrans-

fusion testing.

Stage Two: Agglutination

Once antibody molecules attach to anti-

gens on the red cell surface, the sensitized

cells must be linked into a lattice. This al-

lows visualization of the reaction. The size

and physical properties of the antibody

molecules, the concentration of antigen

sites on each cell, and the distance be-

tween cells all have an effect on the devel-

opment of agglutinates.

The bridges formed between antibodies

interlinked to antigen sites on adjacent red

cells usually result from chance collision of

the sensitized cells. Under isotonic condi-

tions, red cells cannot approach each other

closer than a distance of 50 to 100 Å.1IgG

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 loca-

tion 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 rel-

atively high densities, allowing IgG antibod-

ies to crosslink.

Red cells suspended in saline have a net

negative charge at their surface and there-

fore repel one another. Negatively charged

molecules on the red cell membrane cause

mutual repulsion of red cells. This repul-

sion may be decreased by various labora-

tory manipulations and by inherent or al-

tered red cell membrane characteristics.

Various strategies are used to overcome

this repulsion and to enhance agglutina-

tion. Centrifugation physically forces the

cells closer together. The indirect anti-

globulin test (IAT) uses antiglobulin serum

to crosslink the reaction. Other methods in-

clude reducing the negative charge of sur-

face molecules (eg, proteolytic enzymes),

reducing the hydration layer around the cell

(eg, albumin), and introducing positively

charged macromolecules (eg, Polybrene®)

that aggregate the cells.1

Inhibition of Agglutination

In agglutination inhibition tests, the pres-

ence of either antigen or antibody is de-

tected by its ability to inhibit agglutina-

tion in a system with known reactants

(see Chapter 19). For example, the saliva

Chapter 12: Red Cell Antigen-Antibody Reactions and Their Detection 275

from a secretor contains soluble blood

group antigens that combine with anti-A,

-B, or -H. The indicator system is a stan-

dardized dilution of antibody that aggluti-

nates the corresponding cells to a known

degree. If the saliva contains blood group

substance, incubating saliva with anti-

body will wholly or partially abolish ag-

glutination of cells added to the incu-

bated 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 enhance-

ment effect attributed to albumin may be

due to its attributes as a low-ionic-strength

buffer. Albumin may influence agglutina-

tion by reducing the repulsion between

cells, thus predisposing antibody-coated

cells to agglutinate. Bovine serum albu-

min 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 anti-

bodies, the enzymes destroy certain red

cellantigens,notablyM,N,S,Fy

a,andFy

Proteolytic enzymes reduce the red cell

surfacechargebycleavingpolypeptides

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 en-

zymes often show enhanced agglutination

by IgG molecules.

Polyethylene Glycol

PEG is a water-soluble linear polymer

used as an additive to potentiate anti-

gen-antibody reactions.5It has been sug-

gested that PEG promotes antibody up-

take through steric exclusion of water

molecules in the diluent, such that anti-

gens 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 antibod-

ies and decreases the detection of clini-

cally insignificant antibodies.6

Anti-IgG is usually the antihuman globu-

lin (AHG) reagent of choice with PEG test-

ing, to avoid false-positive reactions with

some polyspecific AHG reagents. Commer-

cially available PEG reagents may be pre-

pared in a LISS solution. PEG can be used

intestswitheluates,aswellaswithserum

or plasma. PEG can enhance warm-reactive

autoantibodies and thus may be advanta-

geous in detecting weak serum autoanti-

bodies for diagnostic purposes. On the

other hand, this enhancement may be dis-

advantageous when trying to detect allo-

antibodies in the presence of autoanti-

bodies. In such cases, testing the serum by

LISS or a saline IAT may allow for the detec-

tion 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

276 AABB Technical Manual

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 prob-

lem appears to be related to elevated serum

globulin levels.6This problem becomes ap-

parent when the IgG-coated red cells are

nonreactive. At least four washes of the red

cells at the antiglobulin phase, with agita-

tion, 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 in-

creases the speed of antibody sensitiza-

tion 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 re-

agent, rather than LISS itself. These com-

mercially available LISS additives may con-

tain albumin in addition to ionic salts and

buffers. LISS solutions increase the rate of

antibody association (Stage 1) when vol-

umeproportionsarecorrect.(SeeMethods

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

testsisproblematic.WhenLISSisusedas

an additive reagent, the manufacturer’s in-

structions must be followed.

The Antiglobulin Test

In 1945, Coombs, Mourant, and Race7de-

scribed procedures for detecting attach-

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 dem-

onstrate 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 sensi-

tized red cells. The direct antiglobulin test

(DAT) is used to demonstrate in-vivo sen-

sitization 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. Ani-

mals injected with human globulins pro-

duce antibody to the foreign protein. Af-

ter the animal serum is adsorbed to remove

unwanted agglutinins, it will react specifi-

cally with human globulins and can be

called AHG serum. AHG sera with varying

specificities can be produced, notably,

anti-IgG and antibodies to several com-

plement components. Hybridoma tech-

niques 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 mole-

cules, 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

Chapter 12: Red Cell Antigen-Antibody Reactions and Their Detection 277

to red cells or are present, free, in serum.

Unbound globulins may react with AHG,

causing false-negative antiglobulin tests.

Unlesstheredcellsarewashedfreeofun-

bound proteins before addition of AHG se-

rum, the unbound globulins may neutralize

AHG and cause a false-negative result.

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 investi-

gating autoimmune hemolytic anemia

(AIHA), drug-induced hemolysis, hemolytic

disease of the fetus and newborn, and

alloimmune reactions to recently trans-

fused red cells.

Indirect Antiglobulin Testing

In indirect antiglobulin procedures, se-

rum (or plasma) is incubated with red

cells, which are then washed to remove

unbound globulins. Agglutination that oc-

curs 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 anti-

gen unknown, as in blood group pheno-

typing 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 obvi-

ate the need to wash coated red cells before

adding antiglobulin reagent. Column ag-

glutination technology, described later in

this chapter, is an example. It uses a micro-

column filled with mixtures of either glass

beads or gel, buffer, and sometimes re-

agents and can be used for direct or indi-

rect antiglobulin procedures. Density barri-

ers allow separation of test serum (or plasma)

from red cells, making a saline washing

phase unnecessary.

Antiglobulin Reagents

Monospecific antibodies to human globu-

lins 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 poly-

clonal in nature. Monospecific mono-

clonal reagents can also effectively be

prepared from hybridomas (see Chapter

11). Monospecific animal or hybridoma-

derived antibodies can be combined into

reagent preparations containing any de-

sired combination of specificities, or dif-

278 AABB Technical Manual

Figure 12-3. The antiglobulin reaction. Anti-

human IgG molecules are shown reacting with

the Fc portion of human IgG coating adjacent red

cells (eg, anti-D coating D-positive red cells).

ferent clones all recognizing the same an-

tigen specificity can be combined into a

single reagent. Thus, reagents may be

polyclonal, monoclonal, blends of mono-

clonal, or blends of monoclonal and poly-

clonal antibodies.

The Food and Drug Administration

(FDA) has established definitions for a vari-

ety of AHG reagents,8as shown in Table 12-1.

Antisera specific for other immunoglobulins

(IgA, IgM) or subclasses (IgG1, IgG3, etc) ex-

ist 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-

tine compatibility tests and antibody de-

tection. These reagents contain antibody

to human IgG and to the C3d component

of human complement. Other comple-

ment antibodies may be present, includ-

ing anti-C3b, -C4b, and -C4d. Currently

available, commercially prepared, poly-

specific antiglobulin sera contain little, if

any, activity against IgA and IgM heavy

chains. However, some reagents may re-

act 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 anti-

bodies are IgG, the most important func-

tion of polyspecific AHG, in most procedures,

is detection of IgG. The anticomplement

Chapter 12: Red Cell Antigen-Antibody Reactions and Their Detection 279

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 neces-

sarily 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 ac-

tivity. Note: The antibody produced in response to immuni-

zation 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 an-

tibody 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.8

component has limited usefulness in cross-

matching and in antibody detection be-

cause 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 com-

ponent 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 immu-

noglobulin 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 anti-

bodies that are not clinically significant.

Anti-C3b, -C3d

Anti-C3b, -C3d reagents prepared by ani-

mal immunization contain no activity

against human immunoglobulins and are

used in situations described for anti-C3d.

This type of anti-C3d characteristically re-

acts 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 spe-

cific for a red cell antigen may cause

attachment of complement to the cell

surface as a consequence of comple-

ment activation by the antigen-anti-

body complex.

2. Immune complexes, not specific for

red cell antigens, may be present in

plasma and may activate comple-

ment 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 reac-

tion is often described as “innocent

bystander” complement coating.

Red cells coated with elements of the

complement cascade may or may not un-

dergo 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 compo-

nent most readily detected is C3 because

several hundred C3 molecules may be

280 AABB Technical Manual

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.

Complement as the Only Coating Globulin

Complement alone, without detectable im-

munoglobulin, may be present on washed

red cells in certain situations.

1. IgM antibodies reacting in vitro oc-

casionally 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 be-

cause IgM molecules tend to disso-

ciateduringthewashingprocess

and partly because polyspecific AHG

contains little if any anti-IgM activ-

ity. 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 pos-

itive DAT due to C3 coating alone.10

No IgG, IgA, or IgM coating is de-

monstrable 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 tempera-

tures up to 32 C.11 Red cells passing

through vessels in the skin at this

temperature become coated with

autoantibody, which activates com-

plement. If the cells escape hemoly-

sis, they return to the central circula-

tion, where the temperature is 37 C,

and the autoantibody dissociates

from the cells, leaving complement

components firmly bound to the red

cell membrane. The component

usually detected by AHG reagents is

C3d.

4. Immune complexes that form in the

plasma and bind weakly and non-

specifically to red cells may cause

complement coating. The activated

complement remains on the red cell

surfaceaftertheimmunecomplexes

dissociate.C3remainsastheonly

detectable surface globulin.

IgG-Coated Cells

The addition of IgG-coated cells to nega-

tive antiglobulin tests (used to detect IgG)

is required for antibody detection and

crossmatching procedures.12(p33) These

sensitized red cells should react with the

antiglobulin sera, verifying that the AHG

reagent was functional. Reactivity with

IgG-sensitized cells demonstrates that, in-

deed, 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, partic-

ularly if the control cells are heavily coated

with IgG. Errors in the original test, such as

omission of test serum, improper centri-

fuge speed, or inappropriate concentra-

tions of test red cells, may yield negative

test results but positive results with control

cells. Oversensitized control cells may ag-

glutinate 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.

Chapter 12: Red Cell Antigen-Antibody Reactions and Their Detection 281

282 AABB Technical Manual

Table 12-2. Sources of Error in Antiglobulin Testing—False-Negative Results

Neutralization of Antihuman Globulin (AHG) Reagent

■Failure to wash cells adequately to remove all serum/plasma. Fill tube at least ¾ full of saline for

each wash. Check dispense volume of automated washers.

■If increased serum volumes are used, routine wash may be inadequate. Wash additional times or

remove serum before washing.

■Contamination of AHG by extraneous protein. Do not use finger or hand to cover tube.

Contaminated droppers or wrong reagent dropper can neutralize entire bottle of AHG.

■High concentration of IgG paraproteins in test serum; protein may remain even after multiple

washes.13

Interruption in Testing

■Bound IgG may dissociate from red cells and either leave too little IgG to detect or may

neutralize AHG reagent.

■Agglutination of IgG-coated cells will weaken. Centrifuge and read immediately.

Improper Reagent Storage

■AHG reagent may lose reactivity if frozen. Reagent may become bacterially contaminated.

■Excess heat or repeated freezing/thawing may cause loss of reactivity of test serum.

■Reagent red cells may lose antigen strength on storage. Other subtle cell changes may cause

loss of reactivity.

Improper Procedures

■Overcentrifugation may pack cells so tightly that agitation required to resuspend cells breaks up

agglutinates. Undercentrifugation may not be optimal for agglutination.

■Failure to add test serum, enhancement medium, or AHG may cause negative test.

■Too heavy a red cell concentration may mask weak agglutination. Too light suspension may be

difficult to read.

■Improper/insufficient serum:cell ratios.

Complement

■Rare antibodies, notably some anti-Jka,-Jk

b, may only be detected when polyspecific AHG is

used and active complement is present.

Saline

■Low pH of saline solution can decrease sensitivity.3Optimal 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.

Use37Cor4Csaline.

Other Methods to Detect

Antigen-Antibody Reactions

The following methods represent alterna-

tives 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 informa-

tion on the phase and temperature of re-

activity 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 di-

rect 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

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

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 pro-

teins. The indicator for attached antibody

is a suspension of anti-IgG-coated red

cells. The reaction is positive if the indica-

tor cells adhere across the sides of the

well. If they pellet to the bottom when

centrifuged, it demonstrates that no anti-

gen-antibody reaction has occurred (see

Fig 12-4).17 In the indirect test, isolated

membrane components (eg, specific pro-

teins), rather than intact cells, can be af-

fixed to the microwell. Solid-phase at-

tachment of antigen or antibody is an

integral part of other tests, such as the

monoclonal antibody-specific immobili-

zation of erythrocyte antigens (MAIEA)

assay, discussed below. Solid-phase sys-

tems have also been devised for use in de-

tecting 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 col-

umn containing a medium that separates

selected red cell populations. As commer-

cially prepared, the systems usually em-

ploy 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 aggre-

gate size.17 Alternatively, in some tests,

specific antisera or proteins can be in-

cluded in the column medium itself; cells

bearing a specific antigen are selectively

captured as they pass through the me-

dium. When the column contains anti-

globulin serum or a protein that specifi-

cally binds immunoglobulin, the selected

284 AABB Technical Manual

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.

cells will be those sensitized with immu-

noglobulin. By using a centrifuge time

and speed that allow red cells to enter the

column but leave serum or plasma above,

theneedforsalinewashingforanti

globulin tests can be eliminated.21

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 ad-

vantage of most such systems is the stabil-

ity of the final reaction phase, which can be

read by several individuals or, in some

cases, documented by photocopying. Col-

umn 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 pro-

cess leading to a technology that uses a col-

umn of gel particles.23 As commercially pre-

pared, the gel test uses six microtubes

instead of test tubes, contained in what is

called a card or strip. The gel particles func-

tion as filters that trap red cell agglutinates

when the cards are centrifuged. Gels con-

taining antiglobulin serum are used to cap-

ture sensitized, but unagglutinated, cells.

Gels with various antisera can be used for

phenotyping cells (see Fig 12-5).

In another column agglutination tech-

nology, a column of glass microbeads in a

diluent is used instead of gel. As with the

gel test, the beads may either entrap agglu-

tinated cells or antisera, such as anti-IgG,

can be added to the diluent.24

Automated Testing Platforms

Several automated devices have been de-

veloped for the detection of antigen-anti-

body reactions. All steps in the testing

process, from sample aliquotting to re-

porting results, are performed by the sys-

tem. These test systems permit the per-

formance of multiple tests, using solid-

phase, gel-column, and/or microtiter

plate technology. The systems are con-

trolled by a computer program and posi-

tive sample identity can be ensured by

barcode technology. The test system’s

computer may be integrated into a labo-

ratory’s information system for results re-

porting. Automated test systems may be

particularly useful in institutions per-

forming large volumes of patient or donor

testing.

Immunofluorescence

Immunofluorescence testing allows iden-

tification and localization of antigens

inside or on the surface of cells. A fluoro-

chrome such as fluorescein or phyco-

erythrin 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 fluores-

cein-labeled antibody is specific for a single

antigen of interest. In an indirect test,

Chapter 12: Red Cell Antigen-Antibody Reactions and Their Detection 285

Figure 12-5. Gel test.

fluorescein-labeled antiglobulin serum is

added to cells that have been incubated

with an unlabeled antibody of known spec-

ificity. Immunofluorescent techniques were

initially used to detect antigens in or on

lymphocytes or in tissue sections. More re-

cently, immunofluorescent antibodies have

been used in flow cytometry. Among their

many applications, they have been used to

quantify fetomaternal hemorrhage, to iden-

tify transfused cells and follow their survival

in recipients, to measure low levels of

cell-bound IgG, and to distinguish homozy-

gous from heterozygous expression of

blood group antigens.25

Enzyme-Linked Immunosorbent Assay

Enzyme-linked immunosorbent assays

(ELISAs) are used to measure either anti-

gen or antibody. Enzymes such as alkaline

phosphatase can be bound to antibody

molecules without destroying either the

antibody specificity or the enzyme activ-

ity. ELISAs have been used to detect and

measure cell-bound IgG and to demon-

strate fetomaternal hemorrhage. When

red cells are examined, the test often is

called an enzyme-linked antiglobulin test

(ELAT).

Monoclonal Antibody-Specific

Immobilization of Erythrocyte Antigens

Assay

IntheMAIEAassay,redcellsareincu

bated with two antibodies. One contains

human alloantibody to a blood group an-

tigen and the other is a nonhuman (usu-

ally mouse monoclonal) antibody that re-

acts 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

antibody also attached). Conjugated anti-

human 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. van Oss CJ. Immunological and physio-

chemical nature of antigen-antibody interac-

tions. In: Garratty G, ed. Immunobiology of

transfusion medicine. New York: Marcel

Dekker, Inc., 1994:327-64.

2. 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.

3. RolihS,ThomasR,FisherE,TalbotJ.Anti-

body detection errors due to acidic or un-

buffered saline. Immunohematology 1993;

9:15-18.

4. 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;36:186-

91.

5. Nance SJ, Garratty G. Polyethylene glycol: A

new potentiator of red blood cell antigen-an-

tibody reactions. Am J Clin Pathol 1987;87:

633-5.

6. Issitt PD, Anstee DJ. Applied blood group se-

rology. 4th ed. Durham, NC: Montgomery

Scientific Publications, 1998:47-8.

7. Coombs RRA, Mourant AE, Race RR. A new

test for the detection of weak and “incom-

plete” Rh agglutinins. Br J Exp Pathol 1945;

26:255-66.

8. Code of federal regulations. Title 21 CFR

660.55. Washington, DC: US Government

Printing Office, 2004 (revised annually).

9. 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.

10. Sokol RJ, Hewitt S, Stamps BK. Autoimmune

haemolysis: An 18-year study of 865 cases re-

ferred to a regional transfusion centre. Br

Med J 1981;282:2023-7.

11. Packman CH. Cryopathic hemolytic syn-

dromes. In: Beutler E, Lichtman MA, Coller

286 AABB Technical Manual

BS, et al, eds. Williams’ hematology. 6th ed.

New York: McGraw-Hill, 2001:649-55.

12. Silva MA, ed. Standards for blood banks and

transfusion services. 23rd ed. Bethesda, MD:

AABB, 2005.

13. Ylagen ES, Curtis BR, Wildgen ME, et al. In-

validation of antiglobulin tests by a high ther-

mal amplitude cryoglobulin. Transfusion

1990;30:154-7.

14. Geisland JR, Milam JD. Spuriously positive di-

rect antiglobulin tests caused by silicone gel.

Transfusion 1980;20:711-13.

15. Grindon AJ, Wilson MJ. False-positive DAT

caused by variables in sample procurement.

Transfusion 1981;21:313-14.

16. Rolih SD, Eisinger RW, Moheng JC, et al. Solid

phase adherence assays: Alternatives to con-

ventional blood bank tests. Lab Med 1985;16:

766-70.

17. Walker P. New technologies in transfusion

medicine. Lab Med 1997;28:258-62.

18. Plapp FV, Sinor LT, Rachel JM, et al. A solid

phase antibody screen. Am J Clin Pathol 1984;

82:719-21.

19. Rachel JM, Sinor LT, Beck ML, Plapp FV. A

solid-phase antiglobulin test. Transfusion

1985;25:24-6.

20. Sinor L. Advances in solid-phase red cell ad-

herence methods and transfusion serology.

Transfus Med Rev 1992;6:26-31.

21. Malyska H, Weiland D. The gel test. Lab Med

1994;25:81-5.

22. Phillips P, Voak D, Knowles S, et al. An expla-

nation and the clinical significance of the

failure of microcolumn tests to detect weak

ABO and other antibodies. Transfus Med

1997;7:47-53.

23. LapierreY,RigalD,AdamJ,etal.Thegeltest:

A new way to detect red cell antigen-antibody

reactions. Transfusion 1990;30:109-13.

24. ReisKJ,ChachowskiR,CupidoA,etal.Col

umn agglutination technology: The antiglo-

bulin test. Transfusion 1993;33:639-43.

25. Garratty G, Arndt P. Applications of flow cyto-

fluorometry to transfusion science. Transfu-

sion 1994;35:157-78.

26. Petty AC. Monoclonal antibody-specific im-

mobilisation of erythrocyte antigens (MAIEA).

A new technique to selectively determine an-

tigenic sites on red cell membranes. J Immunol

Methods 1993;161:91-5.

27. PettyAC,GreenCA,DanielsGL.Themono

clonal antibody-specific antigens assay

(MAIEA) in the investigation of human red-

cell antigens and their associated membrane

proteins. Transfus Med 1997;7:179-88.

Chapter 12: Red Cell Antigen-Antibody Reactions and Their Detection 287

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens

Chapter 13

ABO, H, and Lewis Blood

Groups and Structurally

Related Antigens

THE 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 (oligosaccha-

rides) on common precursor substances. In-

teractions of the ABO, Hh, Sese, and Lele

gene products affect the expression of the

ABO, H, and Lewis antigens. Refer to Ap-

pendix 6 for ISBT numbers and nomencla-

ture 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 indi-

viduals in 19001and, in the following year,

detailed the patterns of reactivity as three

types,nowcalledgroupsA,B,andO.

2,3 He

found that serum from group A individu-

als 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 individu-

als 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

1902 discovered the fourth group, AB.4

TheimportanceofLandsteiner’sdiscov

ery is the recognition that antibodies to A

and B antigens are present when the cor-

responding antigen is missing. Routine ABO

typing procedures developed from these and

later studies.5

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

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-incompati-

ble blood may cause severe intravascular

hemolysis as well as the other manifesta-

tions of an acute hemolytic transfusion re-

action (see Chapter 27). Testing to detect ABO

incompatibility between a recipient and the

donor is the foundation on which all pre-

transfusion testing is based.

Genetics and Biochemistry

The genes for all of the carbohydrate anti-

gens discussed in this chapter encode spe-

cific glycosyltransferases, enzymes that

transfer specific sugars to the appropriate

carbohydrate chain acceptor; thus, the an-

tigens are indirect products of the genes.

Genes at three separate loci (H,Se, and

ABO) control the occurrence and the loca-

tion of the A and B antigens. The Hand 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 consid-

ered 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,isveryrare.

The active allele at the Se locus, Se,pro-

duces a transferase that also acts to form H

antigen, but primarily in secretions such

as saliva.6Eighty percent of individuals are

secretors. The amorphic allele is se.The

enzymes produced by Hand 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,andO.7

The Aand Balleles encode glycosyltrans-

ferases that produce the A and B antigens

respectively; the Oallele does not encode a

functional enzyme.8The red cells of group

O individuals lack A and B antigens but carry

an abundant amount of H antigen, the un-

converted precursor substance on which A

and B antigens are built.

The carbohydrate chains (oligosaccha-

rides) that carry ABH antigens can be at-

tached to either protein (glycoprotein),

sphingolipid (glycosphingolipid), or lipid

(glycolipid) carrier molecules. Glycopro-

teins and glycosphingolipids carrying A or

B antigens are integral parts of the mem-

290 AABB Technical Manual

Table 13-1. Routine ABO Typing

Reaction of Cells

Tested with

Reaction of Serum

Tested Against

Interpre-

tation

Incidence (%) in

US Population

Anti-A Anti-B A1Cells B Cells O Cells

ABO

Group Whites Blacks

+ = agglutination; 0 = no agglutination.

branes of red cells, epithelial cells, and

endothelial cells (Fig 13-1) and are also

present in soluble form in plasma. Glyco-

proteins secreted in body fluids such as sa-

liva contain molecules that may, if the per-

son possesses an Se allele, carry A, B, and H

antigens. A and B antigens that are unat-

tached to carrier protein or lipid molecules

are also found in milk and urine as free oli-

gosaccharides.

The transferases encoded by the A,B,H,

and Se alleles add a specific sugar to a pre-

cursor carbohydrate chain. The sugar that

is added is referred to as immunodominant

because when it is removed from the struc-

ture, the specific blood group activity is

lost. The sugars can be added only in a se-

quential manner. H structure is made first,

then sugars for A and B antigens are added

to H. The Hand Se alleles encode a fuco-

syltransferase that adds fucose (Fuc) to the

precursor chain; thus, fucose is the im-

munodominant sugar for H (see Fig 13-2).

The Aallele encodes N-acetyl-galactosa-

minyltransferase that adds N-acetyl-D-

galactosamine (or GalNAc) to H to make A

antigen on red cells. The Ballele encodes

galactosyltransferase that adds D-galactose

(or Gal) to H to make B antigen. Group AB

individuals have alleles that make transfer-

ases to add both GalNAc and Gal to the pre-

cursor H antigen. Attachment of the A or B

immunodominant sugars diminishes the se-

rologic detection of H antigen so that the ex-

pressions of A or B antigen and of H antigen

are inversely proportional. Rare individuals

who lack both Hand Se alleles (genotype hh

and sese) have no H and, therefore, no A or B

antigens on their red cells or in their secre-

tions (see Ohphenotype 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 mole-

cules linked in linear fashion, or as part of

more complex structures, with many sugar

residues linked in branching chains. Differ-

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens 291

Figure 13-1. Schematic representation of the red cell membrane showing antigen-bearing glycosyla-

tion of proteins and lipids. GPI = glycophosphatidylinositol. (Courtesy of ME Reid, New York Blood

Center.)

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 pre-

dominantly 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 carbo-

hydrate chains, providing additional sites for

conversion to H and then to A and B anti-

gens.

A, B, and H antigens are constructed on

carbohydrate chains that are characterized

by different linkages and composition of the

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(seeFig13-3).Type1A,B,and

H structures are present in secretions, plasma,

andendodermallyderivedtissues.Theyare

not synthesized by red cells but are incor-

porated into the red cell membrane from

the plasma. Type 2 chains are the predomi-

nant ABH-carrying oligosaccharides on red

cells and are also found in secretions. Type

3 chains (repetitive form) are found on red

cells from group A individuals.10 They are

synthesized by the addition of Gal to the

terminal GalNAc Type 2 A chains, thus

formingType3H;Type3Hchainsaresub

sequently 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).

292 AABB Technical Manual

Figure 13-2. Gal added to the subterminal Gal

confers B activity; GalNAc added to the subtermi-

nal Gal confers A activity to the sugar. Unless the

fucose moiety that determines H activity is at-

tached to the number 2 carbon, galactose does

not accept either sugar on the number 3 carbon.

Figure 13-3. Type 1 and 2 oligosaccharide chains

differ only in the linkage between the GlcNAc and

the terminal Gal.

Active alleles Hand Se,oftheFUT1 and

FUT2 genes, encode fucosyltransferases with

a high degree of homology. The enzyme

produced by Hacts 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

Yamamoto et al11 have shown that Aand B

alleles differ from one another by seven

nucleotide differences, four of which re-

sulted in amino acid substitutions at posi-

tions 176, 235, 266, and 268 in the protein

sequence of the A and B transferases. Re-

cent crystallography of A- and B-transfer-

ases demonstrated the role of these criti-

cal amino acids in substrate recognition.12

The initial Oallele 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 Oal-

leles have been identified as well as muta-

tions of Aand Balleles that result in weak-

ened expression of A and B antigens (reviewed

elsewhere).13,14 The A2allele encodes a pro-

tein 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 anti-

gens are not fully developed at birth, pre-

sumably because the branching carbohy-

drate structures develop gradually. By 2 to

4 years of age, A and B antigen expression

is fully developed and remains fairly con-

stant 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 sub-

groups of B. The two principal subgroups

of A are A1and A2. Red cells from A1and A2

persons both react strongly with reagent

anti-A in direct agglutination tests. The

serologic distinction between Aland A2

cells can be determined by testing with

anti-A1lectin (see anti-A1below). There is

both a qualitative and quantitative differ-

ence between A1and A2.15 The A1-trans-

ferase is more efficient at converting H

substance into A antigen and is capable of

makingtherepetitiveType3Astructures.

There are about 10.5 ×105Aantigensites

on adult A1red cells, and about 2.21 ×105

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens 293

Figure 13-4. Type 3 A antigen structure.

A antigen sites on adult A2red cells.9Ap-

proximately 80% of group A or group AB

individuals have red cells that are aggluti-

nated by anti-A1and thus are classified as

A1or A1B. The remaining 20%, whose red

cells are strongly agglutinated by anti-A but

not by anti-A1, are called A2or A2B. Rou-

tine testing with anti-A1is unnecessary for

donors or recipients.

Subgroups weaker than A2occur infre-

quently 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 of-

ten recognized when there is a discrepancy

between the red cell (forward) and serum

(reverse) grouping. Generally, classification

of weak A subgroups (A3,A

x,A

m,A

el)isbased

on the:

1. Degree of red cell agglutination by

anti-A and anti-A1.

2. Degree of red cell agglutination by hu-

man and some monoclonal anti-A,B.

3. Degree of red cell agglutination by

anti-H (Ulex europaeus).

4. Presence or absence of anti-A1in 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 classifi-

cation of A (and B) subgroups was devel-

oped using human polyclonal anti-A,

anti-B, and anti-A,B reagents. These re-

agents 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.

A3red cells give a characteristic mixed-field

pattern when tested with anti-A from group

BorOdonors.A

xred cells are characteristi-

cally not agglutinated by human anti-A

from group B persons but are agglutinated

by anti-A,B from group O persons. Axred

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 adsorp-

tion 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

phenotypehasthesameallele.

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 relation-

ship 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 anti-

genic determinants also exist in other bio-

logic entities, notably bacteria cell walls.

Bacteria are widespread in the environ-

ment, and their presence in intestinal

flora, dust, food, and other widely distrib-

uted agents ensures a constant exposure

of all persons to A-like and B-like antigens.

Immunocompetent persons react to the

environmental antigens by producing an-

tibodies to those that are absent from

their own systems. Thus, anti-A is pro-

duced by group O and group B persons

and anti-B is produced by group O and

group A persons. Group AB people, hav-

ing 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.

294 AABB Technical Manual

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 ma-

ternal origin, acquired by the placental

transfer of maternal IgG; occasionally, in-

fants can be found who produce these

antibodies at the time of birth.17(p124) Thus,

anti-A and anti-B detected in the sera of

newborns or infants younger than 3 to 6

months cannot be considered valid. Anti-

body production increases, reaching the

adult level at 5 to 10 years of age, and de-

clines 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 indi-

viduals 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

Oserum.

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 pref-

erentially agglutinate red cells at room tem-

perature (20-24 C) or below and efficiently

activate complement at 37 C. The comple-

ment-mediated lytic capability of these an-

tibodies 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 tempera-

turesbelow37C.HemolysisduetoABO

antibodies should be suspected when the

supernatant fluid of the serum test is pink

to red or when the cell button is absent or

reduced in size. Hemolysis must be inter-

preted 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 pre-

vent 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 elu-

ate 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, respec-

tively.

Anti-A

Anti-A1occurs as an alloantibody in the

serumof1%to2%ofA

2individuals and

25% of A2B individuals.15 Sometimes,

Anti-A1can also be found in the sera of in-

dividuals with other weak subgroups of A.

Anti-A1can cause discrepancies in ABO

testing and incompatibility in cross-

matches with A1or A1B red cells. Anti-A1

usually reacts better or only at tempera-

tures well below 37 C and is considered

clinically insignificant unless there is re-

activity at 37 C. When reactive at 37 C, only

A2or O red cells should be used for trans-

fusion.

In simple adsorption studies, the anti-A

of group B serum appears to contain sepa-

rable anti-A and anti-A1.NativegroupBse

rum agglutinates A1and A2red cells; after

adsorption with A2red cells, group B serum

reacts only with A1red cells. If further tests

are performed, however, the differences in

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens 295

A antigen expression between A1and A2red

cells appear to be quantitative rather than

qualitative.9

A reliable anti-A1reagent from the lectin

of Dolichos biflorus is commercially avail-

able or may be prepared (see Method 2.10).

The raw plant extract will react with both A1

and A2red 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 re-

quired 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 la-

beled and testing blood of infants less

than 4 months of age; in both of these in-

stances, only ABO testing of red cells is re-

quired.

Anti-A and anti-B typing reagents agglu-

tinate most antigen-positive red cells on di-

rect contact, even without centrifugation.

Anti-A and anti-B in the sera of some pa-

tients and donors are too weak to aggluti-

nate red cells without centrifugation or pro-

longed incubation. Serum tests should be

performed by a method that will adequa-

tely detect the antibodies—eg, tube, micro-

plate, or column agglutination techniques.

Procedures for ABO typing by slide, tube,

and microplate tests are described in Meth-

ods 2.1, 2.2, and 2.3.

Additional reagents, such as anti-A,B for

red cell tests and A2and O red cells for serum

tests, are not necessary for routine testing

but are helpful in resolving typing discrep-

ancies (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 re-

agents have been formulated to detect

some of the weaker subgroups. Manufac-

turers’ inserts should be consulted for spe-

cific reagent characteristics. Special techni-

ques to detect weak subgroups are not

routinely necessary because a typing dis-

crepancy (eg, the absence of expected se-

rum antibodies) usually distinguishes these

specimens from group O specimens.

The A2red 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 interpre-

tations 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 en-

countered, the discrepant results must be

recorded, but interpretation of the ABO

group must be delayed until the discrep-

ancy has been resolved. If the specimen is

from a donor unit, the unit must not be

released for transfusion until the discrep-

ancy has been resolved. When the blood

is from a potential recipient, it may be

necessary to administer group O red cells

oftheappropriateRhtypebeforethein

vestigation has been completed. It is im-

portant to obtain sufficient pretransfusion

blood samples from the patient to com-

plete any additional studies that may be

required.

296 AABB Technical Manual

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 be-

cause negative results are obtained when

positive results are expected, or positive re-

sults are found when tests should have been

negative (see Table 13-2).

Specimen-Related Problems in Testing Red

Cells

ABO testing of red cells may give unex-

pected results for many reasons.

1. Red cells from individuals with vari-

ant Aor Balleles may carry poorly

expressed antigens. Antigen expres-

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens 297

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

sion may also be weakened on the red

cells of some persons with leukemia

or other malignancies.

2. 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 chi-

mera (see Mixed-Field Agglutination

below).

3. Exceptionally high concentrations of

A or B blood group substances in the

serum can combine with and neutra-

lize reagent antibodies to produce an

unexpected negative reaction against

serum- or plasma-suspended red cells.

4. A patient with potent autoagglutinins

may have red cells so heavily coated

with antibody that the red cells ag-

glutinate spontaneously in the pres-

ence of diluent, independent of the

specificity of the reagent antibody.

5. 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 sim-

ulates agglutination.

6. 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 plasma-

suspended red cells may also give

discrepant results due to an antibody

to a reagent dye/constituent21 or to

proteins causing rouleaux.

7. Red cells of some group B individu-

als are agglutinated by a licensed

anti-A reagent that contains a partic-

ular murine monoclonal antibody,

MHO4. These group B individuals had

excessively high levels of Ballele-

specified galactosyltransferase, and

the designation B(A) was given to

this blood group phenotype.22

8. Red cells of individuals with the

acquired B phenotype typically ag-

glutinate 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-determin-

ing sugar (N-acetylgalactosamine)

so that it resembles the B-determin-

ing galactose. The acquired B phe-

nomenon is found most often in in-

dividuals with the A1phenotype.

9. Inherited or acquired abnormalities

of the red cell membrane can lead to

what is called a polyagglutinable state.

The abnormal red cells can be unex-

pectedly agglutinated by human re-

agent anti-A, anti-B, or both because

human reagents will contain anti-

bodies 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 mis-

taken for agglutinates may be seen

in ABO tests with plasma or incom-

pletely 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 anti-

bodies present are generally passively

transferred from the mother.

3. Unexpected absence of ABO aggluti-

ninsmaybeduetothepresenceof

an A or B variant.

4. Patients who are immunodeficient

due to disease or therapy may have

298 AABB Technical Manual

such depressed immunoglobulin levels

that there is little or no ABO aggluti-

nin activity. Samples from elderly

patients whose antibody levels have

declined with age or from patients

whose antibodies have been greatly

diluted by plasma exchange proce-

dures may also have unexpectedly weak

agglutinins.

5. 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 ABO-

mismatched transplants.

6. Cold allo- or autoantibodies that re-

act 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 A1and/or B reagent

red cells.

7. Antibodies to constituents of the dil-

uents used to preserve reagent A1and

B red cells can agglutinate the cells

independent of ABO antigens and

antibodies.

8. Abnormal concentrations of proteins,

altered serum protein ratios, or the

presence of high-molecular-weight

plasma expanders can cause non-

specific red cell aggregation or roule-

aux that is difficult to distinguish from

true agglutination. Rouleaux forma-

tion is easily recognized on micro-

scopic examination if the red cells

assume what has been described as

a “stack of coins” formation.

9. Recent transfusion with plasma com-

ponents containing ABO agglutinins

may cause unexpected reactions.

10. Recent infusion of intravenous im-

mune globulin that may contain ABO

isohemagglutinins can cause unex-

pected reactions.

Mixed-Field Agglutination

Occasional samples are encountered that

contain two distinct, separable populations

of red cells. Usually, this reflects the re-

cent transfusion of group O red cells to a

non-group-O recipient or receipt of a

marrow transplant of an ABO group dif-

ferent from the patient’s own. Red cell

mixtures also occur in a condition called

blood group chimerism, resulting either

from intrauterine exchange of erythro-

poietic 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 agglutina-

tion. Mixed-field reactions due to transfu-

sion last only for the life of the transfused

red cells. After hematopoietic transplanta-

tion, the mixed-field reaction usually dis-

appears when the patient’s own red cells

are no longer produced. Persistent mixed-

cell populations do occur in some marrow

recipients. Mixed-field reactions that arise

through blood group chimerism may per-

sist throughout the life of the individual.

For more information regarding the trans-

fusion and evaluation of hematopoietic

transplant patients, refer to Chapters 21

and 25.

Technical Errors

Technical errors leading to ABO discrep-

ancies 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 in-

structions.

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens 299

6. Under- or overcentrifugation of tests.

7. Incorrect interpretation or recording

of test results.

Resolving ABO Discrepancies

The first step in resolving an apparent se-

rologic 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 re-

peated 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, his-

torical blood group, and history of

previous transfusions, transplanta-

tion, 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 sus-

pected.

In addition to a discrepancy between the

red cell and serum tests, an ABO discrep-

ancy may also exist when the observed re-

activity 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 some-

times 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 per-

son, even though the red cells are not

agglutinated by anti-A or anti-B. The fol-

lowing procedures can be used to en-

hance 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 en-

hance antibody attachment. An inert

(eg, 6% albumin) or autologous con-

trol for room temperature and 4 C

tests is recommended. The manufac-

turer’s directions for any reagent

should be consulted for possible

comments or limitations.

2. Treat the patient’s red cells with a

proteolyticenzymesuchasficin,

papain, or bromelin. Enzyme treat-

ment increases the antigen-antibody

reaction with anti-A or anti-B. In

some instances, reactions between

reagent antibody and red cells ex-

pressing antigens will become de-

tectable 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 correspond-

ing 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 ad-

sorption and elution with any re-

agent to serve as positive and nega-

300 AABB Technical Manual

tive controls. Anti-A1lectin should

not be used for adsorption/elution

studies because in a more concen-

tratedform,suchasaneluate,itmay

react nonspecifically with red cells.

TesttheeluateagainstgroupA

1,B,

and O cells. Unexpected reactivity in

control eluates invalidates the re-

sults obtained with the patient’s red

cells. This indicates that the adsorp-

tion/elution procedure was not per-

formed correctly, another antibody is

present (ie, in a polyclonal reagent),

or the specificity of a monoclonal re-

agent is not distinct enough for the

reagent to be used by this method.

4. TestthesalivaforthepresenceofH

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 A1and B red

cells for 15 to 30 minutes at room

temperature. If there is still no reac-

tion, incubate at 4 C for 15 to 30

minutes. It is recommended to in-

clude an autocontrol and group O

red cells for room temperature and 4

C testing to control for reactivity of

common cold autoagglutinins.

2. Treat the A1and 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 con-

trol for reactivity. The manufacturer’s

directions should be consulted for

possible comments or limitations.

Resolving Discrepancies Due to Unexpected

Red Cell Reactions with Anti-A and Anti-B

Red cell ABO tests sometimes give unex-

pected 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 para-

graphs 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 A1red

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 A1and A2

cells. Except for newborns and immuno-

compromised patients, serum testing should

distinguish this phenomenon from the AB

phenotype in which a subgroup of A is ac-

companied by anti-A1. Testing with an

anti-A without the MHO4 clone should re-

solve the discrepancy. The recipient can be

considered a group B.

Acquired B Phenotype. Red cells aggluti-

nated strongly by anti-A and weakly by

anti-B and a serum containing strong

anti-B suggest the acquired B state. The ac-

quired B phenomenon is found most often

in individuals with the A1phenotype; a few

examples of A2with acquired B have been

found. Most red cells with acquired B anti-

gens react weakly with anti-B, but occa-

sional examples are agglutinated quite

strongly. Behavior with monoclonal anti-B

reagents varies with the particular clone

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens 301

used. Acquired B antigens had been ob-

served 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 fre-

quency of the detection of acquired B is

similar to polyclonal anti-B or have discon-

tinued the use of that clone. To confirm that

group A red cells carry the acquired B struc-

ture:

1. Check the patient’s diagnosis. Ac-

quired B antigens are usually associ-

ated with tissue conditions that al-

low 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 auto-

logous 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 manu-

facturer’s instructions give a detailed

description. Unlike most human poly-

clonal antibodies, some monoclonal

antibodies do not react with the ac-

quired B phenotype; this information

may be included in the manufac-

turer’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 lon-

ger reacts with the acquired B anti-

gen.

Antibody-Coated Red Cells. Red cells

from infants with HDFN or from adults suf-

fering from autoimmune or alloimmune

conditions may be so heavily coated with

IgG antibody molecules that they aggluti-

nate spontaneously in the presence of re-

agent diluents containing high protein con-

centrations. 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 cold-

reactive IgM autoagglutinins may autoag-

glutinate in saline tests. Incubating the cell

suspension briefly at 37 C and then wash-

ing the cells several times with saline

warmed to 37 C can usually remove the an-

tibodies. If the IgM-related agglutination is

not dispersed by this technique, the red cells

can be treated with the sulfhydryl com-

pound dithiothreitol (DTT) (see Method 2.11).

Resolving Discrepancies Due to Unexpected

Serum Reactions

The following paragraphs describe some

events that can cause unexpected or erro-

neous serum test results and the steps that

can be taken to resolve them.

1. Reactivity of the A1reagent cells

when anti-A is strongly reactive with

the red cells suggests the presence of

anti-A1in the serum of an A2or 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 A2red cells.

b. Test the serum against several

examples of each of the follow-

ing: A1,A

2,andOredcells.Only

if the antibody agglutinates all

A1red cells and none of the A2

or O red cell samples can it be

called anti-A1.

2. Strongly reactive cold autoaggluti-

nins, 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 tem-

perature (20-24 C). With few excep-

302 AABB Technical Manual

tions, agglutination caused by the cold

autoagglutinin is weaker than that

caused by anti-A and anti-B. The fol-

lowing 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 aggluti-

nation 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 de-

scribedinMethod4.6.Thead-

sorbed serum can then be tested

against A1and B reagent red cells.

3. 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 anti-

genoflowincidenceontheserum

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 re-

agent A1and B cells to determine

which, if either, carries the cor-

responding antigen. Obtain A1

and B red cells that lack the an-

tigen and use them for serum

testing.

b. Raisethetemperatureto30to

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 allo-

antibody is below the tempera-

ture at which anti-A and anti-B

react, this may resolve the dis-

crepancy.

c. If the antibody detection test is

negative, test the serum against

several examples of A1and B

red cells. The serum may con-

tain an antibody directed against

an antigen of low incidence,

which will be absent from most

randomly selected A1and B red

cells.

4. Sera from patients with abnormal

concentrations of serum proteins, or

with altered serum protein ratios, or

who have received plasma expand-

ers of high molecular weight can ag-

gregate reagent red cells and mimic

agglutination. Some of these sam-

ples 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 re-

placement 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

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens 303

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,A

1B, or (less com-

monly) B individuals have so little uncon-

verted H antigen on their red cells that they

produce anti-H. This form of anti-H is gen-

erally weak, reacts at room temperature or

below, and is not considered clinically sig-

nificant. Individuals of the rare Ohpheno-

type (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).

OhPhenotype

The term Ohor Bombay phenotype has

been used for the very rare individuals

whose red cells and secretions lack H, A,

and B antigens and whose plasma con-

tains 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 ap-

parentwhenserumfromtheO

hindivid-

ual is tested against group O red cells, and

strong immediate-spin agglutination

and/or hemolysis occurs. The anti-H of

an Ohperson reacts over a thermal range

of 4 to 37 C with all red cells except those

of other Ohpeople. Ohpersons must be

transfused only with Ohblood because

their non-red-cell-stimulated antibodies

rapidly destroy cells with A, B, or H anti-

gens. If other examples of Ohred cells are

available, further confirmation can be ob-

tained by demonstrating compatibility of

the serum with Ohred cells. At the geno-

typic level, the Ohphenotype arises from

the inheritance of hh at the Hlocus and

sese at the Se locus. Because the Se allele is

necessary for the formation of Leb,O

hred

cells will be Le(a+b–) or Le(a–b–).

Para-Bombay Phenotype

The para-Bombay phenotype designation,

Ah,B

h,andAB

h, is classically used for indi-

viduals 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 anti-

gen 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 Ohpersons. Indi-

viduals 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 Ahand

Bhpeople 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 molecu-

lar bases for the Bombay and para-Bombay

phenotypes.24 Many mutations at the Hlo-

cus have subsequently been associated

with H-deficiency.

The Lewis System

The Lewis system antigens, Leaand Leb,

result from the action of a glycosyltrans-

ferase encoded by the Le allele that, like

theA,B,andHglycosyltransferases,adds

a sugar to a precursor chain. Leais pro-

duced when Le is inherited with sese and

Lebis 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 Leaor

Lebis produced. Thus, Leaand Lebare not

304 AABB Technical Manual

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 glycosphing-

olipid Type 1 chains adsorbed from plasma

onto red cell membranes. Plasma lipids ex-

change freely with red cell lipids.

Gene Interaction and the Antigens

The synthesis of the Lewis antigens is de-

pendent upon the interaction of two dif-

ferent fucosyltransferases: one from the

Se locusandonefromtheLewislocus.

Both enzymes act upon the same precur-

sorType1substratechains.Thefuco

syltransferase encoded by the Le allele at-

taches fucose in α(1→4) linkage to the

subterminal GlcNAc; in the absence of the

transferase from the Se allele, this config-

uration has Leaactivity. This product can-

not be further glycosylated. Leboccurs

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 Lebconfiguration has two

fucose moieties.9Thus, Lebreflects the

presence of both the Le and Se alleles. Le

without Se results in Leaactivity only; Se

with the amorphic allele le will result in

no secretion of Leaor Lebandtheredcells

will have the Le(a–b–) phenotype.

Table 13-3 shows phenotypes of the Lewis

system and their frequencies in the popula-

tion. 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 en-

coded by a variant secretor allele that com-

petes less efficiently with the Le fuco-

syltransferase.9

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-Leabecause

small amounts of unconverted Leaare

present in their saliva and plasma. It is

most unusual to find anti-Lebin the sera

of Le(a+b–) individuals, but anti-Lebmay

exist along with anti-Leain 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

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens 305

Table 13-3. Phenotypes in the Lewis System and Their Incidence

Reactions with Anti- Adult Phenotype Incidence %

LeaLebPhenotype Whites Blacks

+ 0 Le(a+b−)22 23

0+Le(a−b+) 72 55

00Le(a−b−)6 22

+ + Le(a+b+) Rare Rare

+ = agglutination; 0 = no agglutination.

with HDFN. Lewis antibodies may bind

complement, and fresh serum that con-

tains 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 un-

treated red cells.

Most Lewis antibodies agglutinate sa-

line-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 af-

ter 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,canbe

detected in the antiglobulin phase of test-

ing. Sometimes this reflects complement

bound by the antibody if a polyspecific re-

agent (ie, containing anticomplement) is

used. In other cases, antiglobulin reactivity

results from an IgG component of the anti-

body.

Sera with anti-Lebactivity 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

anti-LebH. Antibodies that react equally well

with the Lebantigen 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 circula-

tion. Lewis antibodies in a recipient’s se-

rum are readily neutralized by Lewis

blood group substance in donor plasma.

For these reasons, it is exceedingly rare for

Lewisantibodiestocausehemolysisof

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 crossmatch-

ing 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

not react with either human anti-Leaor

anti-Leband are considered to be Le(a–b–).

Some can be shown to carry small amounts

of Leawhen tested with potent mono-

clonal anti-Leareagents. 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-spe-

cific transferase in infants; the Se allele-

specific 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 phe-

notype until approximately 2 to 3 years of

age.25

The I/i Antigens and

Antibodies

Cold agglutinins with I specificity are fre-

quently encountered in sera of normal in-

dividuals. 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

306 AABB Technical Manual

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.

ThereisnotrueI–ori–phenotype.

The I and i antigens on red cells are in-

ternal structures carried on the same glyco-

proteins and glycosphingolipids that carry

H, A, or B antigens and in secretions on the

same glycoproteins that carry H, A, B, Lea,

and Lebantigens. 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-acetyl-

galactosamine [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 specific-

ity.27,28 Different examples of anti-I appear to

recognize different portions of the branch-

ed oligosaccharide chain. As branching oc-

cursandasthesugarsforH,A,andBanti-

gens are added, access of anti-i and anti-I

may be restricted.25

The I antigen, together with the i anti-

gen, used to comprise the Ii Blood Group

Collection in the ISBT nomenclature. Re-

cent cloning of the gene that encodes the

transferase responsible for converting i ac-

tive straight chains into I active branched

chains and identification of several muta-

tions responsible for the rare i adult pheno-

type have caused the I antigen to be as-

signed to the new I Blood Group System;

the i antigen remains in the Ii collection.29

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-

nin at 4 C with a titer of <64. Agglutination

with adult red cells and weaker or no ag-

glutination with cord cells is the classic

reactivity. Some stronger examples agglu-

tinate cells at room temperature; others

may react only with the strongest I+ red

cells and give inconsistent reactions. Incu-

bating tests in the cold enhances anti-I re-

activity and helps to confirm its identity;

albumin and testing enzyme-treated red

cells also enhance anti-I reactivity.

Autoanti-I assumes pathologic signifi-

cance in cold agglutinin syndrome (CAS),

in which it behaves as a complement-bind-

ing antibody with a high titer and high ther-

mal amplitude. The specificity of the auto-

antibody 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 pa-

tients with pneumonia due to Mycoplasma

pneumoniae. These patients may experience

transient hemolytic episodes due to the an-

tibody.

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 preferen-

tially 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 be-

havior 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 stud-

ies may be needed to differentiate strong

examples of the antibodies.

Serum containing anti-I or anti-i is some-

times reactive at the antiglobulin phase of

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens 307

testing when polyspecific antihuman glob-

ulin is used. Such reactions rarely indicate

antibody activity at 37 C. Rather, comple-

ment 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 anti-

globulin phase reactivity is usually due to

the anticomplement in a polyspecific anti-

globulin reagent. Usually, avoiding room

temperature testing and using anti-IgG in-

stead of a polyspecific antihuman globulin

help to eliminate detection of cold auto-

antibodies. Cold autoadsorption to remove

the autoantibody from the serum may be

necessary for stronger examples; cold auto-

adsorbed serum or plasma can also be used

in ABO typing (see Method 4.6). The pre-

warmingtechniquecanalsobeusedonce

the reactivity has been confirmed as cold

autoantibody (see Method 3.3).

Complex Reactivity

Some antibodies appear to recognize I de-

terminants with attached H, A, or B im-

munodominant sugars. Anti-IH occurs

quite commonly in the serum of A1indi-

viduals; it reacts stronger with red cells

that have high levels of H as well as I (ie,

group O and A2red cells) and weaker, if at

all, with group A1cells of adults or cord

cells of any group. Anti-IH should be sus-

pected when serum from a group A pa-

tient causes direct agglutination of all

cells used for antibody detection but is

compatible with all or most group A do-

nor blood. Other examples of complex re-

activity include anti-IA, -IP1,-IBH,-ILe

bH,

and -iH.

The P Blood Group and

Related Antigens

The P blood group has traditionally con-

sisted of the P, P1,andP

kantigens, and

later Luke (LKE). However, the biochemis-

try 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 P1anti-

gen is assigned to the P blood group system,

and Pkand LKE remain in the globoside

collection of antigens.29 For simplicity,

these antigens are often referred to as the

P blood group.

308 AABB Technical Manual

Table 13-4. Comparative Serologic Behavior of the I/i Blood Group Antibodies with

Saline Red Cell Suspensions

Temperature Cell Type Anti-I Anti-i

4 C I adult 4+ 0-1+

icord 0-2+ 3+

i adult 0-1+ 4+

22 C I adult 2+ 0

i cord 0 2-3+

i adult 0 3+

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.ThedesignationP

has since been reassigned to an antigen

present on almost all human red cells. The

Pkantigen is also present on almost all hu-

man red cells, but it is not readily detected

unless P is absent, eg, in the rare P1

kor P2

phenotypes. The null phenotype, p, is very

rare. LKE antigen is present on almost all

red cells except those of the rare phenotypes

porP

kand in about 2% of P+ red cells.

Common and Rare Phenotypes

There are two common phenotypes asso-

ciated with the P blood group, P1and P2,

and three rare phenotypes, p, P1

k,andP

asshowninTable13-5.TheP

1phenotype

describes those red cells that react with

anti-P1and anti-P; red cells that do not re-

act with anti-P1, but do react with anti-P,

are of the P2phenotype. When red cells

are tested only with anti-P1and not with

anti-P, the phenotype should be written as

P1+or P1–.

Biochemistry and Genetics

ThePbloodgroupantigens,liketheABH

antigens, are sequentially synthesized by

the addition of sugars to precursor chains.

The different oligosaccharide determi-

nants of the P blood group antigens are

showninFig13-5.Alltheantigensareex

clusively expressed on glycolipids on hu-

man red cells, not on glycoproteins.30 The

precursor of P1can 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

showninFig13-6.Thecommonprecursor

is lactosylceramide, also known as cera-

mide dihexose or CDH. One pathway re-

sults in the formation of paragloboside and

P1. Paragloboside is also the Type 2 precur-

sor for ABH antigens. The other pathway

results in the formation of the globoside se-

ries of antigens: Pk,P,andLKE.

The genes encoding the glycosyltrans-

ferases that are responsible for synthesizing

Pkfrom lactosylceramide and for converting

Pkto P were cloned in 2000. Several muta-

tions that result in the p and Pkphenotypes

have been identified.31-33 The genetic rela-

tionship between P1,PandP

kis still not un-

derstood. Red cells of the p phenotype are

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens 309

Table 13-5. Phenotypes of the P Blood Group and Related Antigens

Reactions with Anti- Phenotype Incidence (%)

P1PP

kP1+P+PkPhenotype Whites Blacks

++ 0 + P

179 94

0+ 0 + P

221 6

00* 00p

+0 + + P

1kAll extremely rare

00 + + P

*Usually negative, occasionally weakly positive.

P1– in addition to being P– and Pk–; the P1–

status of p red cells cannot be explained.

The P1gene is located on chromosome 22

and the Pgene is located on chromosome 3.

Anti-P1

The sera of P1– individuals commonly

contain anti-P1. If sufficiently sensitive

techniques are applied, it is likely that

anti-P1would be detected in the serum of

virtually every person with P1– red cells.25

The antibody reacts optimally at 4 C but

may occasionally be detected at 37 C.

Anti-P1is 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 P1antigen varies widely

among different red cell samples, and anti-

gen strength has been reported to diminish

when red cells are stored. These character-

istics sometimes create difficulties in iden-

tifying 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-P1specific-

ity by incubation at lower temperatures or

by the use of enzyme-treated red cells.

Hydatid cyst fluid or P1substance derived

from pigeon eggs inhibits the activity of

anti-P1. Inhibition may be a useful aid to

310 AABB Technical Manual

Figure 13-6. Biosynthesis of P blood group antigens.

Lactosylceramide (CDH) Galβ(1→4)Glc-Cer

PkGlobotriosylceramide (CTH) Galα(1→4)Galβ(1→4)Glc-Cer

P Globoside GalNAcβ(1→3)Galα(1→4)Galβ(1→4)Glc-Cer

LKE Sialosylgalactosylgloboside NeuAcα(2→3)Galβ(1→3)GalNAcβ(1→3)Galα(1→4)Galβ(1→4)Glc-Cer

Paragloboside Galβ(1→4)GlcnNAcβ(1→3)Galβ(1→4)Glc-Cer

P1Galactosylparagloboside 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.

antibody identification, especially if anti-P1

is present in a serum with antibodies of

other specificities.

Rare Antibodies

Alloanti-P, found as a naturally occurring

potent hemolytic antibody in the sera of

kand P2

kindividuals, reacts with all red

cells except those of the rare p and Pkphe-

notypes. Anti-P can be IgM or a mixture of

IgM and IgG. Anti-PP1Pk, formerly called

anti-Tja, 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-PP1Pkcan be

separated into its components (anti-P,

anti-P1,andanti-P

k) 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. Anti-

PP1Pkhas caused hemolytic transfusion

reactions and, occasionally, HDFN.17(p139)

There is an association between both

anti-P and anti-PP1Pkand spontaneous

abortions occurring early in pregnancy 35,36

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-Land-

steiner test (see Chapter 20).

P Antigens as Receptors for Pathogens

The P blood group antigens are receptors

for several pathogens. P, P1,P

k,andLKE

are receptors for uropathogenic Esche-

richia coli;P

kand P1are receptors for tox-

ins from enterohemorrhagic E. coli37;and

the meningitis-causing bacterium Strep-

tococcus suis binds to Pkantigen.38 The P

antigen (globoside) has also been shown

to serve as a receptor for erythrovirus (par-

vovirus) B19, which causes erythema in-

fectiosum (Fifth disease). Individuals of p

phenotype who lack globoside are natu-

rally resistant to infection with this patho-

gen.39

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system: Biochemical, serological, and clinical

aspects. Transfus Med Rev 1995;9:110-22.

38. Haataja S, Tikkanen K, Liukkonen J, et al.

Characterization of a novel bacterial adhe-

sion specificity of Streptococcus suis recogniz-

ing blood group P receptor oligosaccharides.

J Biol Chem 1993;268:4311-17.

39. Brown KE, Hibbs JR, Gallinella G, et al. Resis-

tance 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 sys-

tem: Isolation and structure of Lewis-c active and

related glycosphingolipids from the plasma of

blood-group O Le(a–b–) nonsecretors. Arch

Biochem Biophys 1986;246:655-72.

Issitt PD, Anstee DJ. Applied blood group serology.

4th ed. Durham, NC: Montgomery Scientific Pub-

lications, 1998.

312 AABB Technical Manual

Judd WJ. Methods in immunohematology. 2nd ed.

Durham, NC: Montgomery Scientific Publications,

1994.

Lee AH, Reid ME. ABO blood group system: A re-

view of molecular aspects. Immunohematology

2000;16:1-6.

Morgan WTJ, Watkins WM. Unraveling the bio-

chemical basis of blood group ABO and Lewis an-

tigenic specificity. Glycoconj J 2000;17:501-30.

Olsson ML, Chester MA. Polymorphism and re-

combination events at the ABO locus: A major

challenge for genomic ABO blood grouping strate-

gies. Transfus Med 2001;11:295-313.

Palcic MM, Seto NOL, Hindsgual O. Natural and

recombinant A and B gene encoded glycosyltrans-

ferases. 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: His-

torical background. Transfus Med 2001;11:243-65.

Yamamoto F. Cloning and regulation of the ABO

genes. Transfus Med 2001;11:281-94.

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens 313

Chapter 14: The Rh System

Chapter 14

The Rh System

THIS CHAPTER USES the DCE no-

menclature—a modification of the

nomenclature originally proposed

by Fisher and Race,1which has been able

to accommodate our present understand-

ing of the genetics and biochemistry of this

complex system. The Rh-Hr terminology

of Wiener is presented only in its histori-

cal 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 new-

born (HDFN) and who experienced a

hemolytic reaction after transfusion of

her husband’s blood. In 1940, Landsteiner

and Wiener3described an antibody ob-

tained by immunizing guinea pigs and

rabbits with the red cells of Rhesus mon-

keys; it agglutinated the red cells of ap-

proximately 85% of humans tested, and

they called the corresponding determi-

nant the Rh factor. In the same year, Le-

vine and Katzin4found 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,

Wiener and Peters5observed 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

animal anti-Rhesus and human anti-D

were not identical, but, by that time, the

Rh blood group system had already re-

ceived its name. Soon after anti-D was

discovered, family studies showed that

the D antigen is genetically determined;

transmission of the trait follows an auto-

somal 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 anti-

gen do not regularly have anti-D. Forma-

tion of anti-D results from exposure,

through transfusion or pregnancy, to red

cells possessing the D antigen. The D an-

tigen has greater immunogenicity than

other red cell antigens; it is estimated that

30% to 85%6,7 of D– persons who receive a

D+ transfusion will develop anti-D. There-

fore, in most countries, the blood of all re-

cipients 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 anti-

gens—C, E, c, and e—had been recog-

nized 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 quan-

titative 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 plate-

lets, 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 in-

dividual but closely linked loci. Tippett’s

prediction15 that two closely linked struc-

tural loci on chromosome 1 determine

the production of Rh antigens has been

shown to be correct.

Genes

Two highly homologous genes on the

short arm of chromosome 1 encode the

nonglycosylated polypeptides that ex-

press the Rh antigens (Fig 14-1).16,17 One

gene, designated RHD, determines the

presence of a membrane-spanning pro-

tein 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 pseudo-

gene (RHD ) responsible for the D– phe-

notype in some Africans has been de-

scribed.19

The RHCE gene determines the C, c, E,

and e antigens; its alleles are RHCe,RHCE,

RHcE,andRHce.20 Evidence derived from

316 AABB Technical Manual

transfection studies21 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

that, modeling studies suggest, traverse

the red cell membrane 12 times and dis-

play only short exterior loops of amino

acids (Fig 14-1). The polypeptides are fatty

acylated and, unlike most blood-group-

associated proteins, carry no carbohy-

drate residues.

Chapter 14: The Rh System 317

Table 14-1. Antigens of the Rh Blood Group System and Their Incidence

Numeric

Designation

Antigen

Name

Incidence (%)*

Numeric

Designation

Antigen

Name

Incidence (%)*

White Black Overall White Black Overall

Rh1 D 85 92 Rh32 Rh32 <0.01 1

Rh2 C 68 27 Rh33 Har <0.01

Rh3 E 29 22 Rh34 Bastiaan >99.9

Rh4 c 80 96 Rh35 Rh35 <0.01

Rh5 e 98 Rh36 Bea<0.1

Rh6 f 65 92 Rh37 Evans <0.01

Rh7 Ce 68 27 Rh39 C-like >99.9

Rh8 Cw2 1 Rh40 Tar <0.01

Rh9 Cx<0.01 Rh41 Ce-like 70

Rh10 V 1 30 Rh42 Ces<0.1 2

Rh11 Ew<0.01 Rh43 Crawford <0.01

Rh12 G 84 92 Rh44 Nou >99.9

Rh17 Hr0>99.9 Rh45 Riv <0.01

Rh18 Hr >99.9 Rh46 Rh46 >99.9

Rh19 hrs98 Rh47 Dav >99.9

Rh20 VS <0.01 32 Rh48 JAL <0.01

Rh21 CG68 Rh49 STEM <0.01 6

Rh22 CE <1 Rh50 FPTT <0.01

Rh23 Dw<0.01 Rh51 MAR >99.9

Rh26 80 96 Rh52 BARC <0.01

Rh27 cE 28 22 Rh53 JAHK <0.01

Rh28 <0.01 Rh54 DAK <0.01

Rh29 total Rh >99.9 Rh55 LOCR <0.01

RH30 Goa0 <0.01 Rh56 CENR <0.01

Rh31 hrB98

*Incidence in White and Black populations where appropriate.8,9

Considerable homology exists between

the products of RHD and RHCE;theprod-

ucts of the different alleles of RHCE are

even more similar.18 C and c differ from one

another in only four amino acids, at posi-

tions 16, 60, 68, and 103, of which only the

difference between serine and proline at

103appearstobecritical.Thepresenceof

proline or alanine at position 226 distin-

guishes 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 Rh-

associated glycoprotein (RhAG), which has

37% sequence homology with the Rh poly-

peptides 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 expres-

sion of other membrane proteins. Rhnull cells

lack LW antigens, are negative for Fy5 of the

Duffy system, and have weakened expres-

sion of the antigens carried on glycophorin

B(S,s,andU).

23 Although Rh/RhAG pro-

teins 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 ammo-

nium transport.24,25

Rh Terminology

Three systems of nomenclature were de-

veloped 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 prod-

uct to be a single entity he called an

agglutinogen. An agglutinogen was char-

acterized by numerous individual speci-

ficities, called factors, that were identified

by specific antibodies. This theory was in-

correct, but for the designation of pheno-

type, particularly in conversation, many

serologists use a shorthand system based

on Wiener’s Rh-Hr notation. The pheno-

type notations convey haplotypes with the

single letters R and r. R is used for haplo-

318 AABB Technical Manual

Figure 14-1. Schematic representation of

RHD, RHCE,

and

RHAG

genes and RhD, RhCE, and RhAG pro-

teins. 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.

types that produce D, r for haplotypes that

do not produce D. Subscripts or, occa-

sionally, superscripts indicate the combi-

nations of other antigens present. For ex-

ample, R1indicates DCe haplotype; R2

indicates DcE;rindicatesdce;R

0indicates

Dce; and so on (Table 14-2).

CDE terminology was introduced by

Fisher and Race,1who postulated three sets

of closely linked genes (Cand c,Dand d,

and Eand e). Both gene and gene product

have the same letter designation, with ital-

icsusedforthenameofthegene.Amodi

fied CDE terminology is now commonly

used to communicate research and sero-

logic findings. Rosenfield and coworkers26

proposed a system of nomenclature based

on serologic observations. Symbols were

not intended to convey genetic informa-

tion, merely to facilitate communication of

phenotypic data. Each antigen is given a

number, generally in the order of its discov-

ery or its assignment to the Rh system. Ta-

ble 14-1 lists the Rh system antigens by

number designation, name, and incidence.

Determining Phenotype

In clinical practice, five blood typing re-

agents are readily available: anti-D, -C, -E,

-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

thefiveantigensandtheprobableRh

phenotype in modified Wiener terminol-

ogy.

Serologic Testing for Rh

Antigen Expression

Expression of D

D– persons either lack RHD, which en-

codes for the D antigen, or have a non-

functional RHD gene. Most D– persons

are homozygous for RHce, the gene en-

coding c and e; less often they may have

RHCe or RHcE, which encode C and e or c

and E, respectively. The RHCE gene,

whichproducesbothCandE,isquite

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 in-

dividual 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 so-

called “position effect.” If the interaction is

between genes or the product of genes on

the same chromosome, it is called a cis ef-

fect. If a gene or its product interacts with

one on the opposite chromosome, it is

called a trans effect. Examples of both ef-

fects 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

Chapter 14: The Rh System 319

Table 14-2. The Principal

Gene

Complexes and the Antigens Encoded

Haplotype

Genes

Present

Antigens

Present

Pheno-

type

RHD,RHCe

D,C,e R1

RHD,RHcE

D,c,E R2

RHD,RHce

D,c,e Ro

RHD,RHCE

D,C,E Rz

′

RHCe

C,e r′

″

RHcE

c,E r″

r RHce

c,e r

RHCE

C,E r

weaker than E produced by cE.Theynoted

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 anti-

gens. 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

genes differs from one geographic group

to another. For example, a White person

with the phenotype Dce would probably

be Dce/ce,but,inaBlackperson,thege

notype 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 geno-

types. In any population, the most proba-

ble genotype is DCe/DcE.Boththese

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 Dlocus (for exam-

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

Antigens

Present

Probable

Phenotype

Anti-D Anti-C Anti-E Anti-c Anti-e

++0++D,C,c,eR

++00+D,C,eR

1R1

+++++D,C,c,E,eR

1R2

+00++D,c,eR

0R0/R0r

+0+++D,c,E,eR

+0++0D,c,ER

2R2

+++0+D,C,E,eR

1Rz

++++0D,C,c,ER

2Rz

+++00D,C,ER

zRz

000++c,err

0+0++C,c,er′r

00+++c,E,er″r

0++++C,c,E,er′r″

ple, DCe/cE,DcE/Ce,orDCE/ce), but these

are uncommon in all populations. Table

14-4 gives the incidence of the more com-

mon genotypes in D+ persons. The figures

given are for Whites and Blacks. The ab-

sence of the RHD gene is uncommon in

other ethnic groups.

Determining Genotype

Identifying antigens does not always al-

low confident deduction of genotype. Pre-

sumptions regarding the most probable ge-

notype rest on the incidence of antigenic

combinations determined from population

studies in different ethnic groups. Infer-

ences about genotype are useful in popu-

lation studies, paternity tests, and in pre-

dicting the Rh genes transmitted by the

husband/partner of a woman with Rh an-

tibodies (Table 14-4).

Molecular techniques are now available

that can determine Rh genotype. Determi-

Chapter 14: The Rh System 321

Table 14-4. Incidence of the More Common Genotypes in D+ Persons*

Likelihood of Zygosity for D (%)

Antigens

Present

Genotype Incidence (%) Homo- Hetero- Homo- Hetero-

DCE

Mod.

Rh-hr Whites Blacks Whites Blacks

D,C,c,e

DCe/ce R1r

31.1 8.8

DCe/Dce R1R0

3.4 15.0 10 90 59 41

Dce/Ce R0r

0.2 1.8

D,C,e

DCe/DCe R1R1

17.6 2.9 91 9 81 19

DCe/Ce R1r

1.7 0.7

D,c,E,e

DcE/ce R2r

10.4 5.7 10 90 63 37

DcE/Dce R2R0

1.1 9.7

D,c,E

DcE/DcE R2R2

2.0 1.3 87 13 99 1

DcE/cE R2r

0.3 <<0.1

D,C,c,E,e

DCe/DcE R1R2

11.8 3.7

DCe/cE R1r

0.8 <0.1 89 11 90 10

DcE/Ce R2r

0.6 0.4

D,c,e

Dce/ce R0r

3.0 22.9 6 94 46 54

Dce/Dce R0R0

0.2 19.4

*For the rare phenotypes and genotypes not shown in this table, consult the Suggested Readings listed at the end of this

chapter.

nations of genotype with polymerase chain

reaction methods can be made using DNA

harvested from white cells or amnio-

cytes19,27,28 or from noncellular fetal DNA in

maternal plasma.30 Rarely, DNA genotype

results will disagree with serologic findings.

Weak D

Most D+ red cells show clear-cut macro-

scopic agglutination after centrifugation

with reagent anti-D and can be readily

classified as D+. Red cells that are not im-

mediately or directly agglutinated cannot

as easily be classified. For some D+ red

cells, demonstration of the D antigen re-

quires incubation with the anti-D reagent

or addition of antihuman globulin (AHG)

serum after incubation with anti-D [indi-

rect antiglobulin test (IAT)]. These cells are

considered D+, even if an additional step

in testing is required.

In the past, red cells that required addi-

tional steps for the demonstration of D

were classified as Du.ThetermD

uis no lon-

ger considered appropriate; red cells that

carry weak forms of D are classified as D+

and should be described as “weak D.” Im-

provement of polyclonal reagents and the

more widespread use of monoclonal anti-D

reagents have resulted in the routine detec-

tion of some D+ red cells that would have

been considered weak D when tested with

less sensitive polyclonal reagents. Addition-

ally, 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. Con-

versely, 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 character-

istics 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 associ-

ated with reduced D antigen expression on

the red cell membrane. The transmemb-

rane or cytoplasmic location of the amino

acid changes in the altered D protein does

notresultinthelossofDepitopes;thus,

the production of alloanti-D as in the par-

tial 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 ex-

pression 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

reactstronglybyanIAT.

Red cells from some persons of the ge-

notype 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 accom-

panied by RHCe in trans position. Many of

the weak D phenotypes due to position ef-

fect that were reported in the early litera-

ture appear as normal D.

Partial D

The concept that the D antigen consists of

multiple constituents arose from observa-

tions 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 re-

act strongly when tested with anti-D. But

some, especially those of the DVI pheno-

322 AABB Technical Manual

type, give weaker reactions than normal

D+ red cells or react only in the AHG test.

Red cells lacking components of the D an-

tigen have been referred to in the past as

“D mosaic” or “D variant.” Current termi-

nology 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 com-

prises numerous epitopes. Partial D pheno-

types can be defined in terms of their D

epitopes. Tippett et al33 established at least

10 epitopes but point out that the D anti-

gen 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 anti-

body 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 ex-

change between the RHCE gene and the RHD

gene or from single-point mutations.9,18

Significance of Weak/Partial D in Blood

Donors

AABB Standards for Blood Banks and

Transfusion Services36(p32) requires donor

blood specimens to be tested for weak ex-

pression of D and to be labeled as D+ if

the test is positive. Transfusion of blood

with weak expression of the D antigen to

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, how-

ever, seem to be less immunogenic than

normal D+ blood; transfusion of a total of

68 units of blood with weak D to 45 D– re-

cipients 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 re-

sponsible 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 de-

bate. Most of these patients can almost al-

ways receive D+ blood without risk of im-

munization, but if the weak D expression

reflects the absence of one or more D epi-

topes, the possibility exists that transfu-

sion of D+ blood could elicit the produc-

tion 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. Cur-

rently 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– blood without problems. Some

serologists consider this practice wasteful

of D– blood and prefer to test potential re-

cipients for weak D, then issue D+ blood

when indicated.

Chapter 14: The Rh System 323

If D+ blood is given to recipients of the

weak D phenotype, it is important to safe-

guard against careless or incorrect interpre-

tation of tests. D– recipients erroneously

classified as D+, possibly due to a positive di-

rect antiglobulin test (DAT) causing a false-

positive test for weak D, run the risk of im-

munization to D if given D+ blood. Individu-

als whose weakly expressed D antigen is de-

tectable 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 sta-

tus by the IAT resolves the apparent discrep-

ancy 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 reas-

signed. Of the currently included 49, most

beyond D, C, c, Cw,E,andeandtheircor

responding antibodies are encountered

much less frequently in routine blood

transfusion therapy.

Cis

Product Antigens

Themembranecomponentsthatexhibit

Rh activity have numerous possible anti-

genic subdivisions. Each gene or gene

complex determines a series of interre-

lated surface structures, of which some

portions are more likely than others to

elicit an immune response. The poly-

peptides 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 differ-

ent haplotypes, for example, in a person

of the genotype DCE/ce. Similar cis prod-

uct 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 prod-

uct 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 pheno-

types 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 phe-

notype would also be f–.

Anti-Ce is frequently the true specificity

of the apparent anti-C that a DcE/DcE per-

son produces after immunization with C+

blood. This knowledge can be helpful in es-

tablishing 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 posi-

tion 103 of the Rh polypeptides and is en-

324 AABB Technical Manual

coded by either RHD or RHCE.41 As a re-

sult, the G antigen is almost invariably

present on red cells possessing either C or

D. Antibodies against G appear superfi-

cially to be anti-C+D, but the anti-G activ-

ity 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– per-

sons 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 pa-

tients, however, some serologists believe

it is essential to distinguish the antibody

specificities to determine the need for Rh

immune globulin prophylaxis.42 Differen-

tial adsorption and elution studies to dis-

tinguish anti-D, -C, and -G are outlined by

Issitt and Anstee.43

Rare red cells have been described that

possess G but lack D and C. The rGpheno-

type is found mostly in Blacks; generally,

the G antigen is weakly expressed and is as-

sociated with the presence of the VS anti-

gen. The rGphenotype has been described

in Whites but is not the same as rGin

Blacks. Red cells also exist that express par-

tial D but lack G entirely, for example, per-

sons 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 com-

mon Rh phenotypes. It has been conve-

nient to consider C and c, and E and e as

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

most common is Cw,buttherelationship

is phenotypic only because Cwand Cxare

antithetical to the high-incidence antigen,

MAR. Variant forms of the e antigen have

been identified, for example, hrSor hrBan-

tigens (Rh19 and Rh31, respectively). Per-

sons who are e+ and hrS–and/orhr

B–are

found more frequently in Black popula-

tions.9The absence of hrBis 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 pheno-

type, described as Rhnull, is produced by at

least two different genetic mechanisms.

Inthemorecommonregulatortypeof

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 off-

spring.

The other form of Rhnull, the amorph

type, has a normal RHAG gene; however,

there is a mutation in each RHCE gene to-

gether with the common deletion of RHD

(as in D– individuals).18 Theamorphtypeof

Rhnull is considerably rarer than the regula-

tor type. Parents and offspring of this type

of Rhnull are obligate heterozygotes for the

amorph.

Chapter 14: The Rh System 325

Red Cell Abnormalities

Whatever the genetic origin, red cells lack-

ing Rh/RhAG proteins have membrane

abnormalities and a compensated hemoly-

tic 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

AfewRh

null individuals were recognized

because their sera contained Rh antibod-

ies. 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 pa-

tients 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 peo-

ple. This antibody, considered to be “anti-

total Rh,” has been given the numerical

designation anti-Rh29.

Rhmod

The Rhmod phenotype represents less com-

plete suppression of Rh antigen expres-

sion. Unlike Rhnull red cells, those classi-

fied as Rhmod do not completely lack Rh

and LW antigens. Rhmod red cells show

much reduced and sometimes varied ac-

tivity, depending on the Rh system genes

the individual possesses and on the po-

tency 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 de-

gree. As in the regulator type of Rhnull,a

mutation in RHAG was shown to result in

the Rhmod phenotype.18

Deleted Phenotypes

Rare RH haplotypes encode D antigen but

fail to encode some or all of the CE anti-

gens. Some examples of deleted phenotypes

includeD––,D••,DC

w–, 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 rec-

ognized during routine testing with anti-D.

TheD––phenotypemaybeidentifiedin

the course of studies to investigate an un-

expected antibody. Such persons may have

alloantibody of complex specificity be-

cause the person’s red cells lack all the

epitopes expressed on the RhCE poly-

peptide. Antibody with Rh17 (Hro) speci-

ficity is often made by persons of this rare

phenotype, although some sera have been

reported to contain apparently separable

specificities, such as anti-e.

TheD••phenotypeissimilartoD––,ex

cept that the D antigen is not elevated to

the same degree. D•• red cells may be ag-

glutinated weakly by some examples of sera

from immunized Rh-deletion persons if the

serum sample contains anti-Rh47 in addi-

tion to anti-Rh17.39 Rh47 is a high-inci-

dence antigen encoded by the RHCE gene

and is absent from other deleted pheno-

types (eg, D– –, DCw–,Dc–).Anotherdistin

guishing characteristic of D•• red cells is

that they possess the low-incidence antigen

Rh37 (Evans).

326 AABB Technical Manual

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-occur-

ring. D is the most potent immunogen,

followed by c and E. Although a few exam-

ples of Rh antibodies behave as saline ag-

glutinins, most react best in high-protein,

antiglobulin, or enzyme test systems. Even

sera containing potent saline-reactive

anti-D are usually reactive at higher dilu-

tions in antiglobulin testing. Some work-

ers find enzyme techniques especially use-

ful for detecting weak or developing Rh

antibodies.

Antibody usually persists for many years.

If serum antibody levels fall below detect-

able thresholds, subsequent exposure to the

antigen characteristically produces a rapid

secondary immune response. With exceed-

ingly rare exceptions, Rh antibodies do not

bind complement, at least to the extent rec-

ognizable by techniques currently used. As

a result, primarily extravascular hemolysis,

instead of intravascular hemolysis, occurs

in transfusion reactions involving Rh anti-

bodies.

Dosage Effect

Anti-D seldom shows any difference in re-

activity between red cells from individu-

als homozygous or heterozygous for RHD,

but D expression seems to vary somewhat

with the accompanying alleles of the ge-

notype. For example, red cells from a

DcE/DcE individual carry more D antigen

sites than red cells from a DCe/DCe per-

son and may show higher titration scores

with anti-D. Dosage effects can some-

times be demonstrated with some anti-

bodies directed at the E, c, and e antigens

and, occasionally, at the C antigen.

Anti-D in D+ Individuals

Alloanti-D may be produced in D+ indi-

viduals with partial D phenotype (see Par-

tial D), although not all persons who are

D+ and produce what appears to be anti-D

should be assumed to have epitope-defi-

cient red cells. Weakly reactive anti-LWab

or anti-LWamay react with D+ cells but

not with D– cells. A D+ person whose an-

tibody is a weakly reactive anti-LWamay

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 sys-

tem in Chapter 15.) Anti-LW can be differ-

entiated from anti-D by testing the anti-

body with 0.02M DTT-treated red cells

(Method 3.10); the LW antigen is de-

stroyed by sulfhydryl reagents, whereas D

is unaffected.

Concomitant Antibodies

Some Rh antibodies tend to occur in con-

cert. For example, the DCe/DCe person

manifesting immune anti-E almost cer-

tainly 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 seem-

ingly 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 anti-

body has not been detected, but some

practitioners feel that the DCe/DCe recipi-

ent with anti-E is a case that merits avoid-

ance of c+ blood as well.45 Anti-E less con-

sistently 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

Chapter 14: The Rh System 327

majority of c– donor blood will be nega-

tive 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, ob-

taining compatible blood for a patient

with an Rh antibody, investigating parent-

age or other family studies, selecting a

panel of phenotyped cells for antibody

identification, or evaluating whether a

person is likely to be homozygous or het-

erozygous for RHD.

In finding compatible blood for a recipi-

ent with a comparatively weak Rh antibody,

tests with potent blood typing reagents

more reliably confirm the absence of anti-

gen than mere demonstration of a compat-

ible crossmatch. Determination of the pa-

tient’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 re-

agents of human polyclonal origin that

were suitable for slide, tube, or microplate

tests were used for most routine testing.

More recently, monoclonal anti-D re-

agents have become widely available.

Tests may employ red cells suspended in

saline, serum, or plasma, but test condi-

tions should be confirmed by reading the

manufacturer’s directions before use. Pro-

cedures for microplate tests are similar

to those for tube tests, but very light sus-

pensions of red cells are used.

Slidetestsproduceoptimalresultsonly

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. Represen-

tative procedures for tube, slide, and micro-

plate 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 addi-

tives. Such reagents are nearly always pre-

pared from pools of human sera and give

rapid reliable results when used in accor-

dance with manufacturers’ directions.

High-protein levels and macromolecular

additives may cause false-positive reac-

tions. A false-positive result could cause a

D– patient to receive D+ blood and be-

come immunized. An appropriate control

tested according to the manufacturer’s di-

rections must be performed.

Control for High-Protein Reagents

Manufacturers offer their individual dilu-

ent formulations for use as control re-

agents. The nature and concentration of

additives differ significantly among re-

agents 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

328 AABB Technical Manual

valid. In most cases the presence or ab-

sence of D can be determined with other

reagents, as detailed later in this chapter.

Misleading Results with High-Protein

Reagents

False Positives. The following circum-

stances can produce false-positive red cell

typing results.

1. Cellular aggregation resulting from

immunoglobulin coating of the pa-

tient’s red cells or serum factors that

induce rouleaux will give positive re-

sultsinboththetestandthecontrol

tubes. Serum factors can be elimi-

nated by thoroughly washing the red

cells (with warm saline if cold agglu-

tinins 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 immunoglobu-

lin coating of the red cells, which

should then be tested with low-pro-

tein reagents.

2. Red cell aggregation, simulating ag-

glutination, may occur if red cells and

anti-D are incubated too long and

excessive evaporation occurs during

the slide test. It is important to fol-

low the manufacturer’s recommen-

dations to interpret the test within

the recommended period.

False Negatives. The following circum-

stances can produce false-negative red

cell typing results.

1. Too heavy a red cell suspension in

thetubetestortooweakasuspen

sion in the slide test may weaken ag-

glutination.

2. Saline-suspended red cells must not

be used for slide testing.

3. Red cells possessing weakly expres-

sed D antigen may not react well

within the 2-minute limit of the slide

test or upon immediate centrifuga-

tion in the tube test.

Low-Protein Reagents

The low-protein Rh reagents in current

use are formulated predominantly with

monoclonal antibodies. Immunoglobu-

lin-coated red cells can usually be suc-

cessfully typed with low-protein Rh re-

agents that contain saline-agglutinating

antibodies.

Monoclonal Source Anti-D

Monoclonal anti-D reagents are made

predominantly from human IgM antibod-

ies, which require no potentiators and ag-

glutinate 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 par-

tial D red cells in antiglobulin tests. Cer-

tain rare D+ red cells (DHAR, DVa, DVc,

Rh43+)mayreactatimmediatespinwith

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 mono-

clonal reagents are incubated in excess of a

manufacturer’s product directions. These re-

agents, prepared in a low-protein medium,

can be used to test red cells with a positive

Chapter 14: The Rh System 329

DAT, provided those tests are not subjected

to an IAT.

Control for Low-Protein Reagents

Most monoclonal blended reagents have

a total protein concentration approximat-

ing that of human serum. False-positive

reactions due to spontaneous aggregation

of immunoglobulin-coated red cells occur

nomoreoftenwiththiskindofreagent

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 observ-

ing absence of agglutination by anti-A

and/or anti-B in the cell tests for ABO. For

red cell specimens that show agglutina-

tion in all tubes (ie, give the reactions of

group AB, D+), a control should be per-

formed as described by the reagent man-

ufacturer; this is not required when do-

nors’ cells are tested. In most cases, a

suitable control is a suspension of the pa-

tient’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 re-

sult serves as an adequate control. For ex-

ample, 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 immunoglob-

ulin, a low protein reagent is usually nec-

essary for Rh testing. Occasionally, the in-

fant’s red cells may be so heavily coated

with antibody that all antigen sites are oc-

cupied, leaving none available to react

with a low protein antibody of appropri-

ate specificity. This “blocking” phenome-

non 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 an-

tibody.

Anti-D is the specificity responsible for

nearly all cases of blocking by maternal an-

tibody. It is usually possible to obtain cor-

rect 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 per-

formed 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 low-

protein (usually monoclonal or mono-

clonal/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 nega-

tive 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 immedi-

ate 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-

330 AABB Technical Manual

tively common.9It is impossible to obtain

anti-e reagents that react strongly and con-

sistently 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 ex-

pression of C on red cells. Variant E and c

antigens have been reported but are con-

siderably less common.

Whatever reagents are used, the manu-

facturer’s directions must be carefully fol-

lowed. 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 (nonmono-

clonal) used to prepare reagents for the

other Rh antigens may contain anti-

globulin-reactive, “contaminating” speci-

ficities. 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 anti-

gens having an incidence of less than

1% in the population may occasion-

ally be present and cause false-posi-

tive reactions, even when the manu-

facturer’s directions are followed.

For crucial determinations, many

workers routinely perform replicate

tests using reagents from different

sources. Replicate testing is not an

absolute safeguard, however, because

reagents from different manufactur-

ers may not be derived from different

sources.

3. Polyagglutinable red cells may be ag-

glutinated by any reagent containing

human serum. Although antibodies

that agglutinate these surface-altered

red cells are present in most adult

human sera, polyagglutinins in re-

agents very rarely cause problems.

Aging, dilution, and various steps in

the manufacturing process tend to

eliminate these predominantly IgM

antibodies.

4. Autoagglutinins and abnormal pro-

teins in the patient’s serum may cause

false-positive reactions when un-

washed red cells are tested.

5. Reagent vials may become contami-

nated with bacteria, with foreign

substances, or with reagent from an-

other vial. This can be prevented by

the use of careful technique and the

periodic inspection of the vials’ con-

tents. However, bacterial contamina-

tion may not cause recognizable tur-

bidity because the refractive index of

bacteria is similar to that of high-

protein 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 di-

rected predominantly at a cis-prod-

Chapter 14: The Rh System 331

uct Rh antigen failed to give a reli-

ably detectable reaction with red

cells carrying the individual antigens

as separate gene products. This oc-

cursmostoftenwithanti-Csera.

5. The manufacturer’s directions were

not followed.

6. The red cell button was shaken so

roughly during resuspension that

small agglutinates were dispersed.

7. Contamination, improper storage,

or outdating cause antibody activity

to deteriorate. Chemically modified

IgG antibody appears to be particu-

larly susceptible to destruction by

proteolytic enzymes produced by cer-

tain bacteria.

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2. Levine P, Stetson RE. An unusual case of

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3. Landsteiner K, Wiener AS. An agglutinable

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4. Levine P, Katzin EM. Isoimmunization in

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5. Wiener AS, Peters HR. Hemolytic reactions

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22. Ridgwell K, Spurr NK, Laguda B, et al. Isola-

tion of cDNA clones for a 50 kDa glycoprotein

of the human erythrocyte membrane associ-

ated with Rh (rhesus) blood-group antigen

expression. Biochem J 1992;287:223-8.

23. Tippett P. Regulator genes affecting red cell

antigens. Transfus Med Rev 1990;4:56-68.

24. HemkerMB,GoedelC,vanZwietenR,etal.

The Rh complex exports ammonium from

human red blood cells. Br J Haematol 2003;

122:333-40.

25. Westhoff CM, Seigel D, Burd C, Foskett JK.

Mechanism of genetic complementation of

ammonium transport in yeast by human

332 AABB Technical Manual

erythrocyte Rh-associated glycoprotein. J

Biol Chem 2003;279:17443-8.

26. Rosenfield RE, Allen FH Jr, Swisher SN,

Kochwa S. A review of Rh serology and pre-

sentation of a new terminology. Transfusion

1962;2:287-312.

27. Wagner FF, Flegel WA. RHD gene deletion oc-

curred in the Rhesus box. Blood 2000;95:

3662-8.

28. Chiu RW, Murphy MF, Fidler C, et al. Determi-

nation 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.

29. Lawler SD, Race RR. Quantitative aspects of

Rh antigens. Proceedings of the International

Society of Hematology 1950:168-70.

30. 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.

31. Wagner F, Gassner C, Müller T, et al. Molecu-

lar basis of weak D phenotypes. Blood 1999;

93:385-93.

32. Wagner FF, Frohmajer A, Ladewig B, et al.

Weak D alleles express distinct phenotypes.

Blood 2000;95:2699-708.

33. Tippett P, Lomas-Francis C, Wallace M. The

Rh antigen D: Partial D antigens and associ-

ated low incidence antigens. Vox Sang 1996;

70:123-31.

34. Scott ML. Section 1A: Rh serology. Coordina-

tor’s report. 4th International Workshop on

Monoclonal Antibodies Against Human Red

Cell Surface Antigens, Paris. Transfus Clin Biol

2002;9:23-9.

35. Chang TY, Siegel DL. Genetic and immuno-

logical properties of phage-displayed human

anti-Rh(D) antibodies: Implications for Rh(D)

epitope topology. Blood 1998;91:3066-78.

36. Silva MA, ed. Standards for blood banks and

transfusion services. 23rd ed. Bethesda, MD:

AABB, 2005.

37. Flegel WA, Khull SR, Wagner FF. Primary

anti-D immunization by weak D type 2 RBCs.

Transfusion 2000;40:428-33.

38. Schmidt PJ, Morrison EG, Shohl J. The antige-

nicity of the RhoDublood factor. Blood 1962;

20:196-202.

39. DanielsG.Humanbloodgroups.2nded.Ox

ford: Blackwell Scientific Publications, 2002.

40. 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.

41. FaasBHW,BeckersEAM,SimsekS,etal.In

volvement of Ser103 of the Rh polypeptides

in G epitope formation. Transfusion 1996;36:

506-11.

42. ShireyRS,MirabellaDC,LumadueJA.Differ

entiation of anti-D, -C, and -G: Clinical rele-

vance in alloimmunized pregnancies. Trans-

fusion 1997;37:493-6.

43. Issitt PD, Anstee DJ. Applied blood group se-

rology. 4th ed. Durham, NC: Montgomery

Scientific Press, 1998:350-3.

44. ReidME,StorryJR,IssittPD,etal.Rhhaplo

typesthatmakeebutnothr

Busually make

VS. Vox Sang 1997;72:41-4.

45. Shirey RS, Edwards RE, Ness PM. The risk of

alloimmunization to c (Rh4) in R1R1patients

who present with anti-E. Transfusion 1994;34:

756-8.

46. Rodberg K, Tsuneta R, Garratty G. Discrepant

Rh phenotyping results when testing IgG-

sensitized rbcs with monoclonal Rh reagents

(abstract). Transfusion 1995;35(Suppl):67S.

Suggested Reading

Agre P, Cartron JP. Molecular biology of the Rh an-

tigens. 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.

IssittPD.Aninvitedreview:TheRhantigene,its

variants, and some closely related serological ob-

servations. 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. Immuno-

hematology 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.

Chapter 14: The Rh System 333

Reid ME, Lomas-Francis C. The blood group anti-

gen factsbook. 2nd ed. London: Academic Press,

2004.

Sonneborn H-H, Voak D, eds. A review of 50 years

of the Rh blood group system. Biotest Bulletin 1997;

5(4):389-528.

Vengelen-Tyler V, Pierce S, eds. Blood group sys-

tems: Rh. Arlington, VA: American Association of

Blood Banks, 1987.

White WD, Issitt CH, McGuire D. Evaluation of the

use of albumin controls in Rh typing. Transfusion

1974;14:67-71.

334 AABB Technical Manual

Chapter 15: Other Blood Groups

Chapter 15

Other Blood Groups

THERE ARE MANY antigens on red

cells in addition to the ones men-

tioned in previous chapters. These

antigens are grouped into blood group

systems, collections, and a series of inde-

pendent 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 geneti-

cally 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 ge-

netic 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

terminology, and their gene location.1-3

Additional information on ISBT terminol-

ogy 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 independ-

ent high-incidence and low-incidence an-

tigens. 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, where-

as 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

Antibody

In-Vitro

Hemolysis

Saline Albumin Papain/Ficin

Associated

with

4C 22C 37C AHG 37C AHG HDFN HTR

Anti-M 0 Most Some Few Few 0 0 Few Few

Anti-N 0 Most Few Occ. Occ. 0 0 Rare No

Anti-S 0 Few Some Some Most See text Yes Yes

Anti-s 0 No Few Few Most See text Yes Yes

Anti-U 0 No Occ. Some Most Most Most Yes Yes

Anti-Lua0 Some Most Few Few Few Few No No

Anti-Lub0 Few Few Few Most Few Few Mild Yes

Anti-K 0 Few Some Most Some Most Yes Yes

Anti-k 0 Few Few Most Some Most Yes Yes

Anti-Kpa0 Some Some Most Some Most Yes Yes

Anti-Kpb0 Few Few Most Some Most Yes Yes

Anti-Jsa0 Few Few Most Few Most Yes Yes

Anti-Jsb0 0 0 Most Few Most Yes Yes

Anti-LeaSome Most Most Some Some Some Most No Rare

Anti-LebSome Most Most Some Some Some Most No No

Anti-Fya0 Rare Rare Most 0 0 Yes Yes

Anti-Fyb0 Rare Rare Most 0 0 Mild Yes

Anti-JkaSome Few Few Most Some Most Mild Yes

Anti-JkbSome Few Few Most Some Most Mild Yes

Anti-Xga0 Few Few Most 0 0 No report

Anti-Dia0 Some Some Most Some Some Yes Yes

Anti-Dib0 Most Some Some Yes Yes

Anti-Yta000Most0SomeNoYes

Anti-Ytb0 All No report

Anti-Doa0 0 0 Some Some Most No Yes

Anti-Dob0 All All All No Yes

Anti-Coa0 0 0 Some Some Most Yes Yes

Anti-Cob0 0 0 Some Some Most Mild Yes

Anti-Sc1 0 All No No

Anti-Sc2 0 Some Some Most Most Most 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.

Antigens that occur as codominant traits,

such as Jkaand Jkb, may have a variable in-

cidence 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 anti-

body 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 be-

tween groups of diverse Asian, South Ameri-

can, or Native American origins make gen-

eralizations about phenotypes inappropriate.

MNS System

M, N, S, s, and U Antigens

The MNS system is a complex system of

over 40 antigens carried on two glycophorin

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 medi-

cine. They have also been important to

our understanding of biochemistry and

genetics. The M and N antigens are lo-

cated 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 dis-

equilibrium 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 proxim-

ity on chromosome 4.4See the section be-

low on Genes Encoding Glycophorins and

Chapters 9 and 10 for more information

about gene interactions.

Red cells that lack S and s may be nega-

tive for a high-incidence antigen called U;

persons who lack U may make anti-U when

exposed to U+ red cells.

Low-Incidence Antigens of the MNS System

The MNS system includes several low-inci-

dence antigens. Recent biochemical data

Chapter 15: Other Blood Groups 337

Table 15-2. Phenotypes and Frequencies in the MNS System

Reactions with Anti- Phenotype Frequency (%)

M N S s U Phenotype Whites Blacks

+ 0 M+N– 28 26

+ + M+N+ 50 44

0 + M–N+ 22 30

+ 0 + S+s–U+ 11 3

+ + + S+s+U+ 44 28

0 + + S–s+U+ 45 69

0 0 0 S–s–U– 0 Less than 1

0 0 (+) S–s–U+w 0 Rare*

*May not be detected by some antisera and are listed as U–.

attribute the reactivity of various low-in-

cidence 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 anti-

gens 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 re-

sults 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 ho-

mologous sequences, GYPA and GYPB dif-

fer significantly in the 3′end sequences.

Hybrid Molecules

Pronounced SGP modifications occur in

hybrid molecules that may arise from un-

equal crossing over or gene conversion

between GYPA and GYPB.HybridSGPs

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 low-

incidence antigens Hil (MNS20), Sta

(MNS15), Dantu (MNS25), and Mur

(MNS10), among others, are associated with

hybrid SGPs. Some variants are found in

specificethnicgroups.Forexample,the

Dantu antigen occurs predominantly in

Blacks, although the antigen is of low inci-

dence.

Many of the MNS low-incidence anti-

gens were categorized into a subsystem

called the Miltenberger system, based on

reactivity with selected sera. As more anti-

gens 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 trans-

membrane glycoproteins. The carboxy (C)

terminal of each glycophorin extends into

the cytoplasm of the red cell, and a hydro-

phobic segment is embedded within the

lipid bilayer. An amino (N) terminal seg-

ment extends into the extracellular envi-

ronment. The molecules are sensitive to

cleavage at varying positions by certain

proteases (see Fig 15-1).

There are approximately 1,000,000 cop-

ies of GPA per red cell. M and N blood

group antigen activity resides on the extra-

cellular segment, a sequence of 72 amino

acids with carbohydrate side chains at-

tached within the first 50 residues of the

amino terminal. When GPA carries M anti-

gen activity (GPAM), the first amino acid res-

idue is serine and the fifth is glycine. When

it carries N antigen activity (GPAN), leucine

and glutamic acid replace serine and gly-

cine 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–) indi-

viduals may produce antibodies (collec-

tively called anti-Ena) that react with vari-

ous portions of the extracellular part of the

glycoprotein. Some En(a–) persons may

produce an antibody against an antigen

called Wrbthat is part of the Diego blood

group system. Wrbarises from an interac-

tion between GPA and the anion exchange

molecule, AE-1 (also known as band 3).5

338 AABB Technical Manual

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 ac-

tivity has threonine at that position (see Fig

15-1). The N-terminal 26 amino acids of

GPB are identical to the sequence of GPAN,

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 well-

defined 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 sys-

tem antigens reflects the point at which

the particular enzyme cleaves the anti-

gen-bearing SGP and the position of the

antigen relative to the cleavage site (see

Fig 15-1). Sensitivity of the antigens to

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

-linked oligosaccharide side chain, indicates

-linked polysaccharide side chain. Approximate locations of protease cleavage sites are indi-

cated. (Courtesy New York Blood Center.)

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 encoun-

teredaredirectedattheM,N,S,andsan

tigens.

Anti-M

Anti-M is detected frequently as a saline

agglutinin if testing is done at room tem-

perature. 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 sig-

nificant. 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 per-

formed by a strictly prewarmed method

(see Method 3.3) should eliminate the re-

activity 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.

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

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, investigators7have 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 anti-

body to a high-incidence antigen.

Antibodies to Low-Incidence Antigens

There are many examples of antibodies to

low-incidence antigens, such as anti-Mg

or anti-VW, in the MNS blood group sys-

tem. 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 de-

tected if present.

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

340 AABB Technical Manual

reducing agents and with acid. The anti-

gens are carried on one protein and en-

coded 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.

ThealleleresponsiblefortheKantigenis

present in 9% of Whites and approxi-

mately 2% of Blacks. The existence of the

expected allele for k was confirmed when

an antithetical relationship was estab-

lished between K and the antigen de-

tected by anti-k. Anti-k reacts with the red

cells of over 99% of all individuals.

Other Kell Blood Group Antigens

Other antithetical antigens of the Kell sys-

tem include Kpa,Kp

b,andKp

c;Js

aand Jsb;

K11 and K17; and K14 and K24. Not all

theoretically possible genotype combina-

tions have been recognized in the Kell

system. For example, Kpaand Jsahave

never been found to be produced by the

same chromosome. Kpais an antigen

found predominantly in Whites, and Jsais

found predominantly in Blacks. The

haplotype producing K and Kpahas also

not been found. Table 15-3 shows some

phenotypes of the Kell system. The table

also includes Ko,anullphenotypein

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 Kored cells.

For simplicity, various Kell antigens of

high and low incidence have not been

included in the table.

Phenotypes with Depressed Kell Antigens

Kmod is an umbrella term used to describe

phenotypes characterized by weak ex-

pression of Kell system antigens. Adsorp-

tion/elution tests are often necessary for

their detection. The Kmod phenotype is

thought to arise through several different

point mutations of the KEL gene. Red cells

of persons with some Gerbich negative

Chapter 15: Other Blood Groups 341

Table 15-3. Some Phenotypes and Frequencies in the Kell System

Reactions with Anti- Frequency (%)

KkKp

aKpbJsaJsbPhenotype Whites Blacks

+ 0 K+k– 0.2 Rare

++ K+k+ 8.8 2

0 + K–k+ 91.0 98

+ 0 Kp(a+b–) Rare 0

+ + Kp(a+b+) 2.3 Rare

0 + Kp(a–b+) 97.7 100

+ 0 Js(a+b–) 0.0 1

+ + Js(a+b+) Rare 19

0 + Js(a–b+) 100.0 80

000000 K

0Exceedingly rare

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.

ThepresenceoftheKp

aallele 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 ex-

amples 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 pro-

tein. Kell system antigens are easily inac-

tivated by treating red cells with sulfhy-

dryl reagents such as 2-mercaptoethanol

(2-ME), dithiothreitol (DTT), or 2-amino-

ethylisothiouronium bromide (AET).

Such treatment is useful in preparing red

cells that artificially lack Kell system anti-

gens to aid in the identification of Kell-re-

lated antibodies. Treatment with sulfhydryl

reagents may impair the reactivity of

other antigens (LWa,Do

a,Do

b,Yt

a, and oth-

ers). Thus, although treatment with these

reagents may be used in antibody prob-

lem solving, specificity must be proven by

other means. As expected, Kell system an-

tigens are also destroyed by ZZAP, a mix-

ture 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 un-

known,butithasstructuralsimilaritiestoa

family of zinc-binding neutral endopep-

tidases. It has most similarity with the

common acute lymphoblastic leukemia

antigen (CALLA or CD10), a neutral endo-

peptidase 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 lo-

cus (XK) is on the Xp21 region of the X

chromosome, evidence suggests that the

Kell and Kx proteins form a covalently

linked complex on normal red cells.8On

red cells that carry normal expressions of

Kell antigens, Kx appears to be poorly ex-

pressed. 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 Koindividuals re-

act strongly with anti-Kx. Similarly, the re-

moval or denaturation of Kell glycopro-

teins 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 es-

sential 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 sys-

tem antigens but also shortened survival,

reduced deformability, decreased perme-

ability to water, and acanthocytic morphol-

ogy. This combination or group of red cell

abnormalities is called the McLeod pheno-

type, after the first person in whom these

observations were made. Persons with McLeod

red cells also have a poorly defined abnor-

mality of the neuromuscular system, char-

342 AABB Technical Manual

acterized by persistently elevated serum

levels of the enzyme creatine phospho-

kinase 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 pheno-

type 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 pheno-

type associated with CGD appears to result

from deletion of a part of the X chromo-

some that includes the XK locusaswellas

the gene responsible for X-linked CGD.

Kell System Antibodies

Anti-K and Anti-k

Because the K antigen is strongly immuno-

genic, anti-K is frequently found in sera

from transfused patients. Rare examples

of anti-K have appeared as a saline agglu-

tinin in sera from subjects never exposed

to human red cells. Most examples are of

immune origin and are reactive in anti-

globulin testing; some bind complement.

Some workers have observed that exam-

ples of anti-K react less well in tests that in-

corporate low-ionic-strength-saline (LISS)

solutions (notably the Polybrene test) than

in saline tests or tests that include albumin.

Others, however, have not shown differ-

ences in antibody reactivity, testing many

examples of anti-K in low ionic systems.

Anti-KhascausedHTRsonnumerousoc

casions, both immediate and delayed.

Anti-K can cause severe HDFN and fetal

anemia may be caused by the immune de-

struction of K+ erythroid progenitor cells by

macrophages in the fetal liver.11

Becauseover90%ofdonorsareK–,itis

not difficult to find compatible blood for

patients with anti-K. Anti-k has clinical and

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 correspond-

ingly more difficult.

Other Kell System Antibodies

Anti-Kpa,anti-Kp

b,anti-Js

a,andanti-Js

bare

allmuchlesscommonthananti-Kbut

show similar serologic characteristics and

are considered clinically significant. Any of

them may occur following transfusion or

fetomaternal immunization. Antibody fre-

quency is influenced by the immunogeni-

city of the particular antigen and by the

distribution of the relevant negative phe-

notypes among transfusion recipients and

positive phenotypes among donors. In

Black patients frequently transfused with

phenotypically matched blood, usually

from other Black donors, anti-Jsais rela-

tively common. This is due to the approx-

imate 20% incidence of the Jsaantigen in

the Black population (see Table 15-3). Ordi-

narily, 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

Kpband Jsb. Anti-Ku is the antibody char-

acteristically seen in immunized Koper-

sons. It has been reported to cause a fatal

HTR,12 and it appears to be directed at a

singledeterminantbecauseithasnotbeen

separable into other Kell specificities.

However, antibodies to other Kell system

antigens may be present in serum contain-

inganti-Ku.SomepeopleoftheK

mod pheno-

type have made a Ku-like antibody.

Duffy System

Duffy System Antigens

The antigens Fyaand Fybare encoded by a

pair of codominant alleles at the Duffy

Chapter 15: Other Blood Groups 343

(FY) locus on chromosome 1. Anti-Fyaand

anti-Fybdefine 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–) in-

dividuals 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

thebrain,kidney,spleen,heart,andlung.

The Fy(a–b–) individual can be the result of

the FyFy genotype or null phenotype. How-

ever, in many Black Fy(a–b–) individuals,

the transcription in the marrow is pre-

vented and Duffy protein is absent from the

red cells. These individuals have an allele

that is the same in the structural region as

the Fybgene that prevents the transcrip-

tion.8(p439) However, the Duffy protein is ex-

pressed 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 Fybcalled

Fyxhas been described and is probably due

to a point mutation. The Fyxantigen may go

undetected unless potent anti-Fybis used in

testing. The Fy5 antigen appears to be de-

fined 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 anti-

bodies 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 multi-

pass membrane glycoprotein. The antigens

Fya,Fy

b, and Fy6 are located on the N-ter-

minal of the Duffy glycoprotein and are

sensitive to denaturation by proteases such

as ficin, papain, and α-chymotrypsin, un-

like Fy3 or Fy5. Fy3 has been located on

the last external loop of the Duffy glyco-

protein. It is unaffected by protease treat-

ment (reviewed in Pierce and Mac-

pherson).14 The glycoprotein is the recep-

tor for the malarial parasite Plasmodium

vivax, and persons whose red cells lack

Fyaand Fybare resistant to that form of

the disease. In sub-Saharan Africa, nota-

bly West Africa, the resistance to P. v i v a x

malaria conferred by the Fy(a–b–) pheno-

type may have favored its natural selec-

tion, and most individuals are Fy(a–b–).

The Duffy gene has been cloned13 and

the Duffy glycoprotein has been identified

as an erythrocyte receptor for a number of

344 AABB Technical Manual

Table 15-4. Phenotypes and Frequencies in the Duffy System

Reactions with Anti- Adult Phenotype Frequency (%)

FyaFybPhenotype Whites Blacks

+ 0 Fy(a+b–) 17 9

+ + Fy(a+b+) 49 1

0 + Fy(a–b+) 34 22

0 0 Fy(a–b–) Very rare 68

chemokines, notably interleukin-8.15 Be-

cause 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

Anti-Fyais quite common and may cause

HDFN and HTRs. Anti-Fybis rare and gen-

erally is weakly reactive. Anti-Fybcan 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-Fyaand

anti-Fybare usually nonreactive in en-

zyme test procedures.

Weak examples of anti-Fyaor anti-Fyb

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 anti-

gens 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–)

may be FyaFy.

Anti-Fy3 was first described in the serum

of a White person of the Fy(a–b–) pheno-

type and is directed at the high-incidence

antigen Fy3. The only cells with which it is

nonreactive are Fy(a–b–). Unlike Fyaand

Fyb, the Fy3 antigen is unaffected by prote-

ase treatment, and anti-Fy3 reacts well with

enzyme-treated cells positive for either Fya

or Fyb. 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 de-

scribed, both reactive with papain-treated

red cells. One example of anti-Fy4 has been

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 reactiv-

ity 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 pro-

vided a previously unrecognized distinction

between the Fy(a–b–) phenotype so com-

mon in Blacks and the one that occurs, but

very rarely, in Whites.8(p447)

Anti-Fy6 is a murine monoclonal anti-

body that describes a high-incidence anti-

gen in the same region as Fyaand 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 nonreac-

tive with enzyme-treated red cells.

Kidd System

Jkaand JkbAntigens

The Jkaand Jkbantigens 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 resis-

tant to lysis by 2M urea.16 Red cells of nor-

malJkphenotypeswellandlyserapidlyin

asolutionof2Murea.

The four phenotypes identified in the Kidd

system are shown in Table 15-5. The

Jk(a–b–) phenotype is extremely rare, ex-

cept in some populations of Pacific Island

origin. Two mechanisms have been shown

to produce the Jk(a–b–) phenotype.17 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

Chapter 15: Other Blood Groups 345

the In(Lu) suppression of the Lutheran sys-

tem.

Kidd System Antibodies

Anti-Jk

and Anti-Jk

Anti-Jkawas first recognized in 1951 in the

serum of a woman who had given birth

to a child with HDFN. Two years later,

anti-Jkbwas found in the serum of a pa-

tient who had suffered a transfusion reac-

tion. Both antibodies react best in anti-

globulin testing, but saline reactivity is

sometimes observed in freshly drawn

specimens or when antibodies are newly

forming. Both anti-Jkaand anti-Jkbare of-

ten weakly reactive, perhaps because,

sometimes, they are detected more readily

through the complement they bind to red

cells. Some examples may become unde-

tectable on storage. Other examples may

react preferentially with red cells from

homozygotes.

Some workers report no difficulties in

detecting anti-Jkaand anti-Jkbin low ionic

tests that incorporate anti-IgG. Others find

that an antiglobulin reagent containing an

anticomplement component may be im-

portant for the reliable detection of these

inconsistently reactive antibodies. Stronger

reactions may be obtained with the use of

polyethylene glycol (PEG) or enzyme-

treated red cells in antiglobulin testing.

Kidd system antibodies occasionally

causeHDFN,butitisusuallymild.These

antibodies are notorious, however, for their

involvement in severe HTRs, especially de-

layed hemolytic transfusion reactions

(DHTRs). DHTRs occur when antibody de-

velops so rapidly in an anamnestic re-

sponse to antigens on transfused red cells

that it destroys the still-circulating red cells.

In many cases, retesting the patient’s pre-

transfusion serum confirms that the anti-

body 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. Al-

though a minor anti-Jkaor anti-Jkbcom-

ponent is sometimes separable, most of

the reactivity has been directed at an anti-

gen 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-

346 AABB Technical Manual

Table 15-5. Phenotypes and Frequencies in the Kidd System

Reactions with Anti- Phenotype Frequency (%)

JkaJkbPhenotype Whites Blacks

+ 0 Jk(a+b–) 28 57

+ + Jk(a+b+) 49 34

0 + Jk(a–b+) 23 9

0 0 Jk(a–b–) Exceedingly rare

viewed here briefly; the interested reader

should refer to other texts and reviews for

greater detail.

Lutheran System

and Lu

Antigens

The phenotypes of the Lutheran system,

as defined by anti-Luaand anti-Lub,are

showninTable15-6.TheLu(a–b–)pheno

type is very rare and may arise from one

of three distinct genetic circumstances

(reviewed in Pierce and Macpherson14). In

the first, a presumably amorphic Lu-

theran gene (Lu) is inherited from both

parents. In the second and most com-

mon, the negative phenotype is inherited

as a dominant trait attributed to the inde-

pendently segregating inhibitor gene,

In(Lu), which prevents the normal expres-

sion of Lutheran and certain other blood

group antigens (notably P1,I,AnWj,In

and Inb). 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,

Lu16, Lu17, and Lu20) has been assigned

to the Lutheran system because the corre-

sponding antibodies do not react with

Lu(a–b–) red cells of any of the three ge-

netic backgrounds. Two low-incidence

antigens, Lu9 and Lu14, have gained ad-

mission 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 glyco-

protein 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 de-

stroyed by trypsin, α-chymotrypsin, and

sulfhydryl-reducing agents.20 These re-

sults and results of immunoblotting ex-

periments suggest the existence of inter-

chain or intrachain disulfide bonds. Tests

performed with monoclonal anti-Lubsug-

gest that the number of Lubantigen sites

per red cell is low, approximately 600-

1600 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 ex-

ample of autosomal linkage in humans.

The two loci have been assigned to chro-

mosome19.ThegeneencodingtheLu

glycoproteins has been cloned.22

Lutheran System Antibodies

Thefirstexampleofanti-Lu

a(-Lu1) was

found in 1945 in a serum that contained

several other antibodies. Anti-Luaand

anti-Lubare not often encountered. They

Chapter 15: Other Blood Groups 347

Table 15-6. Phenotypes and Frequencies

in the Lutheran System in Whites*

Reactions with

Anti- Phenotype

Frequency

(%)

LuaLubPhenotype

+ 0 Lu(a+b–) 0.15

+ + Lu(a+b+) 7.5

0 + Lu(a–b+) 92.35

0 0 Lu(a–b–) Very rare

*Insufficient data exist for the reliable calculation of fre-

quencies in Blacks.

are most often produced in response to

pregnancy or transfusion but have oc-

curred in the absence of obvious red cell

stimulation. Lutheran antigens are poorly

developed at birth. It is not surprising that

anti-Luahas not been reported to cause

HDFN; neither has it been associated with

HTRs. Anti-Lubhas been reported to shor-

ten the survival of transfused red cells but

causes no, or at most very mild, HDFN.

Most examples of anti-Luaand some anti-

Lubwill agglutinate saline-suspended red

cells possessing the relevant antigen, char-

acteristically producing a mixed-field ap-

pearance 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 inde-

pendent pairs of antithetical antigens,

called Dia/Diband Wra/Wrb. The system

also contains a large number of low-inci-

dence antigens as seen in Appendix 6.

The antigens are located on AE-1 (band

3), which is encoded by a gene on chro-

mosome 17. The Diaand Dibantigens are

useful as anthropologic markers because

the Diaantigen is almost entirely confined

to populations of Asian origin and Native

NorthandSouthAmericans,inwhichthe

incidence of Diacanbeashighas54%.

8(p583)

The Wraand Wrbantigens are located on

AE-1 in close association with GPA. Wrbex-

pression is dependent upon the presence of

GPA (see MNS Blood Group System).

Diego System Antibodies

Anti-Diamay cause HDFN or destruction

of transfused Di(a+) red cells. Anti-Dibis

rare but clinically significant. Anti-Wrais

fairly common and can occur without red

cell alloimmunization. It is a rare cause of

HTR or HDFN. Anti-Wrbis a rarely en-

countered antibody that may be formed

by rare Wr(a+b–) and some En(a–) indi-

viduals. Anti-Wrbmay recognize an en-

zyme-resistant or enzyme-sensitive anti-

gen.23 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

consists of two antigens, Ytaand Ytb(see

Table 15-7). A gene on chromosome 7 en-

codes the antigens. The Yt antigens are lo-

cated on red cell acetylcholinesterase

(AChE),24 an enzyme important in neural

transmission, but the function of which is

unknown on red cells. Enzymes have a

variable effect on the Ytaantigen but 0.2M

DTT appears to destroy the Ytaantigen ex-

pression.

Cartwright Antibodies

Some examples of anti-Ytaare benign. A

few cases of anti-Ytahave shown acceler-

ated destruction of transfused Yt(a+) red

cells. Prediction of the clinical outcome by

the monocyte monolayer assay has proved

successful.25,26 Anti-Ytais not known to cause

HDFN. Anti-Ytbis rare and has not been

implicated in HTR or HDFN.

Xg System

Antigen

In 1962, an antibody was discovered that

identified an antigen more common among

womenthanamongmen.Thiswouldbe

expected of an X-borne characteristic be-

cause females inherit an X chromosome

from each parent, whereas males inherit

X only from their mother. The antigen was

named Xgain recognition of its X-borne

manner of inheritance. Table 15-8 gives

the phenotype frequencies among White

348 AABB Technical Manual

males and females. Enzymes, such as pa-

pain and ficin, denature the antigen. The

gene encoding Xgahas been cloned.27,28

Antibody

Anti-Xgais an uncommon antibody that

usually reacts only in antiglobulin testing.

Anti-Xgahas not been implicated in HDFN

or HTRs. Anti-Xgamay be useful for trac-

ing the transmission of genetic traits asso-

ciated with the X chromosome, although

linkage with the Xg locus has been dem-

onstrated for few traits to date.

Scianna System

Scianna Antigens

Five antigens—Sc1, Sc2, Sc3, Rd, and

STAR—are recognized as belonging to the

Scianna blood group system. Scianna an-

tigens are expressed by the red cell adhe-

sion protein ERMAP.3Sc1 is a high-inci-

Chapter 15: Other Blood Groups 349

Table 15-7. Phenotype Frequencies in Other Blood Group Systems with

Co-Dominant Antithetical Antigens

System Reactions with Anti- Phenotype

Phenotype

Frequency in

Whites (%)*

Yt YtaYtb

+ 0 Yt(a+b–) 91.9

+ + Yt(a+b+) 7.9

0 + Yt(a–b+) 0.2

Dombrock DoaDob

+ 0 Do(a+b–) 17.2

+ + Do(a+b+) 49.5

0 + Do(a–b+) 33.3

Colton CoaCob

+ 0 Co(a+b–) 89.3

+ + Co(a+b+) 10.4

0 + Co(a–b+) 0.3

0 0 Co(a–b–) Very rare

Scianna Sc1 Sc2

+ 0 Sc:1,–2 99.7

+ + Sc:1,2 0.3

0 + Sc:–1,2 Very rare

0 0 Sc:–1,–2 Very rare

Indian InaInb

+ 0 In(a+b–) Very rare

+ + In(a+b+) <1

0 + In(a–b+) >99

Diego DiaDib

+ 0 Di(a+b–) Rare

+ + Di(a+b+) Rare

0 + Di(a–b+) >99.9

*There are insufficient data for the reliable calculation of frequencies in Blacks.

dence antigen, whereas Sc2 occurs very

infrequently. Sc1 and Sc2 behave as prod-

ucts 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 Sc2gene, analogous to Fy3. Rd and

STAR are low-incidence antigens recently

assigned to the Scianna system. The anti-

gens 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

of Doaand Dob, with the phenotype fre-

quency as shown in Table 15-7. The dis-

covery that red cells negative for the high-

incidence antigen Gyawere Do(a–b–)29 has

led to the recent expansion of the Dombrock

blood group system. Three high-incidence

antigens are Gya,Hy,andJo

a.Theinterre

lationship 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 exclu-

sively in Blacks. The Dombrock antigens

are located on a glycoprotein of 46 to 58

kD, the function of which is unknown.

Dombrock Antibodies

Anti-Doaand anti-Dobare uncommon an-

tibodies and are usually found in sera

containing multiple red cell antibodies.

This can make the detection and identifi-

cation of anti-Doaand anti-Dobdifficult.

Anti-Doahas caused HDFN and HTR.31

HDFN due to anti-Dobhas not been re-

ported, but examples have caused HTR.

Antibodies to Gya,Hy,andJo

amay cause

shortened survival of transfused anti-

gen-positive red cells or mild HDFN. Re-

activity of Dombrock antibodies may be

enhanced by papain or ficin treatment of

350 AABB Technical Manual

Table 15-8. Frequencies of the Xg(a+)

and Xg(a–) Phenotypes in White Males

and Females

Phenotype Frequency (%)

Phenotype Males Females

Xg(a+) 65.6 88.7

Xg(a–) 34.4 11.3

Frequencies are based on the combined results of testing

nearly 7000 random blood samples from populations of

Northern European origin. There are insufficient data for

reliable calculation of frequencies in Blacks.

Table 15-9. Relationship of the Dombrock Blood Group Antigens

Phenotype DoaDobGyaHy Joa

Normal Do(a+b–) +0+++

Normal Do(a+b+) +++++

Normal Do(a–b+) 0++++

Gy(a–) 00000

Hy– 0 (+) (+) 0 (+)

Jo(a–) (+) (+) + (+) 0

(+) = weak antigen expression.

red cells but is weakened or destroyed by

sulfhydryl reagents.

Colton System

Colton Antigens

The Colton system consists of Coa,ahigh-

incidence antigen; Cob, a low-incidence

antigen; and Co3, an antigen (like Fy3)

considered to be the product of either the

Coaor Cobgene. The antigens are products

of a gene on chromosome 7. The pheno-

type 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

Anti-Coahas been implicated in HTRs and

HDFN.8Anti-Cobhas caused an HTR and

mild HDFN. Enzyme treatment of red

cells enhances reactions with the Colton

antibodies.

LW System

LW Antigens

Table 15-10 shows the LW phenotypes and

their frequencies. The antigens are dena-

tured 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

LWais more strongly expressed on D+

than D– red cells. However, the gene en-

coding the LW antigens is independent of

the genes encoding the Rh proteins. Ge-

netic independence was originally estab-

lished 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-

mosome 19 and has been cloned. The

glycoprotein it encodes has homology

with cell adhesion molecules.33

Red cells from persons of the Rhnull phe-

notype 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

Anti-LWahas 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 con-

tained anti-LWa. Reduced expression of

LW antigens can occur in pregnancy and

some hematologic diseases. LW antibod-

ies may occur as an autoantibody or as an

apparent alloantibody in the serum of

such individuals. Anti-LWab has been re-

ported 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-

Chapter 15: Other Blood Groups 351

Table 15-10. Phenotypes and

Frequencies in the LW System in

Whites

Reactions with

Anti-

Phenotype

Frequency

(%)

LWaLWb

+ 0 LW(a+b–) >99%

+ + LW(a+b+) <1%

0 + LW(a–b+) Very rare

0 0 LW(a–b–) Very rare

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 mecha-

nism that remains unclear.35 Ch has been

subdivided into six antigens and Rg into

two antigens. A ninth antigen, WH, re-

quires the interaction of Rg1 and Ch6 for

expression. C4 is encoded by two linked

genes, C4A and C4B, on chromosome 6.

Existing sera are poorly classified, but

the phenotype frequency may be consid-

ered as in Table 15-11. The antigens are de-

stroyed by papain/ficin treatment but unaf-

fected by DTT/AET treatment.

Chido/Rodgers Antibodies

Antibodies to Ch and Rg are generally be-

nign but may be a great nuisance in sero-

logic investigations. Rapid identification

is possible using red cells coated with C4

or by inhibition with pooled plasma from

antigen-positive individuals. (See Method

3.9.) The antibodies are nonreactive with

enzyme-treated red cells.

Gerbich System

Gerbich Antigens

The Gerbich system includes eight anti-

gens, of which three (Ge2, Ge3, and Ge4)

are of high incidence and five (Wb, Lsa,

Ana,Dh

a,andGEIS)areoflowincidence.

Several phenotypes that lack one or more

of the high-incidence antigens are shown

in Table 15-12; all are rare. Red cells with

the Gerbich or Leach phenotype have a

weakened expression of some Kell system

antigens.Ge2,Ge4,Wb,Ls

a,An

a,Dh

a,and

GEIS are destroyed by papain and ficin,

but Ge3 resists protease treatment.

352 AABB Technical Manual

Table 15-11. Some Blood Group Antigens with Phenotypic Relationships

Approximate Frequency (%)

Antigens Phenotypes Whites Blacks

Chido (Ch) and Rodgers (Rg) Ch+,Rg+ 95.0

Ch–,Rg+ 2.0

Ch+,Rg– 3.0

Ch–,Rg– Very rare

Cost (Csa)* and York (Yka) Cs(a+),Yk(a+) 82.5 95.6

Cs(a+),Yk(a–) 13.5 3.2

Cs(a–),Yk(a+) 2.1 0.6

Cs(a–),Yk(a–) 1.9 0.6

Knops-Helgeson (Kna) and McCoy (McCa) Kn(a+),McC(a+) 97.0 95.0

Kn(a+),McC(a–) 2.0 4.0

Kn(a–),McC(a+) 1.0 1.0

Kn(a–),McC(a–) Rare Rare

*Although Csais not part of the Knops blood group system, there is a phenotypic association between Ykaand Csa.

The antigens of the Gerbich blood group

system are carried on glycophorin C (GPC)

and glycophorin D (GPD). GPC carries Ge3

and Ge4, whereas GPD carries Ge2 and

Ge3. Anais carried on an altered form of

GPD. DhaandWbarelocatedonaltered

forms of GPC. Lsaisfoundonanaltered

form of GPC and GPD.36 The proteins are

the product of a single gene, GYPC,on

chromosome 2. GPC is approximately four

times more abundant than GPD. The

mechanism whereby these two proteins are

derived from a single gene involves an al-

ternative initiation site in the gene. GPC

and GPD interact directly with protein

band 4.1 in the membrane skeleton. It is

clear that the interaction is important in

maintaining cell shape because deficien-

cies of either band 4.1 or GPC/D cause

elliptocytosis.36

Gerbich Antibodies

The antibodies that Ge-negative individu-

als may produce are shown in Table 15-12;

they may be immune or occur without red

cell stimulation. Anti-Ge is usually IgG

but may have an IgM component. The

clinical significance of the antibodies is

variable. Antibodies to the Gerbich anti-

gensmaybeararecauseofHDFN.

Cromer System

Cromer Antigens

A total of 13 antigens have been assigned

to the Cromer blood group system: 10 high-

incidence antigens and three low-inci-

dence antigens (see Table 15-13). Tcais

antithetical to the low-incidence antigen

Tcbin Blacks and to Tccin Whites. WESbis

the high-incidence antigen antithetical to

WESa.Cr

a,Dr

a,Es

a,UMC,GUTI,SERF,and

ZENA are not associated with low-inci-

dence antigens. IFC is absent only in the

null phenotype (Inab).

The antigens are located on the comple-

ment regulatory protein called decay-accel-

erating factor (DAF). The protein is en-

coded by DAF, one gene of the regulators of

complement activation (RCA) complex, on

chromosome 1. The antigens are on leuko-

cytes, platelets, and trophoblasts of the pla-

centa as well as in soluble form in the se-

rum/plasma and urine.37 The antigens are

not affected by ficin or papain. DTT or AET

may weaken the antigens but do not com-

pletely destroy them.37

Cromer Antibodies

Antibodies to antigens of the Cromer sys-

tem are immune-mediated and extremely

uncommon. Most examples of anti-Cra,

-WESb,and-Tc

ahave been found in the

sera of Black individuals. Anti-GUTI was

found in a Canadian of Chilean ancestry.37

The clinical significance of the antibodies

is variable, and some examples cause de-

creased survival of transfused red cells.

The antibodies will not cause HDFN be-

cause the placenta tissue is a rich source

of DAF, which is thought to adsorb the

maternal antibodies.37

Chapter 15: Other Blood Groups 353

Table 15-12. Ge– Phenotypes

Phenotype Antibody Produced

Ge: –2,3,4 (Yus type) Anti-Ge2

Ge:–2,–3,4 (Gerbich type) Anti-Ge2 or -Ge3

Ge:–2,–3,–4 (Leach type) Anti-Ge2, -Ge3, or -Ge4

Knops System

Knops Antigens

Most of the eight Knops system antigens

(Kna,Kn

b,McC

a,McC

b,Sl

a,Yk

a, Vil, and Sl3)

have been located on the C3b/C4b recep-

tor (CR1), the primary complement re-

ceptor on red cells. A gene on chromo-

some 1 encodes CR1. Kna,McC

a,Sl

a,Yk

and Sl3 are high-incidence antigens. Knb,

McCb, and Vil are low-incidence antigens.

Some variation in frequency is observed

between the red cells of Whites and Blacks

(see Table 15-11).

The Knops system antigens are not de-

stroyed by ficin or papain but may be weak-

ened or destroyed by DTT or AET.

Knops Antibodies

The antibodies commonly show variable

weak reactivity in the antiglobulin phase

of testing but may continue to react even

at high dilutions. Moulds et al38 have shown

that the variable reactivity of anti-CR1-re-

lated sera is a direct reflection of the

number of CR1 sites that exhibit both size

and expression polymorphisms and vary

widely among individuals. The antibodies

are of no clinical significance.

Indian System

Inaand Inbare located on CD44, a protein

of wide tissue distribution with the char-

acteristics of a cell adhesion molecule. Ina

is a low-incidence antigen, and Inbis of

high incidence (see Table 15-7). Inbshows

reduced expression on Lu(a–b–) red cells

of the In(Lu) type but is normally ex-

pressed on Lu(a–b–) red cells from per-

sons homozygous for the amorph or pos-

sessing the X-borne suppressor gene. The

antigens are destroyed by papain and ficin

as well as by reducing agents such as 0.2

M DTT. There are few data on the clinical

significance of the corresponding antibod-

ies.

Other Blood Group Systems

Ok System

The Ok system consists of a single high-

incidence antigen, Oka.Thefewrare

Ok(a–) individuals to date have been Jap-

anese. Proteases do not seem to weaken

expression of Okain routine agglutination

tests. Anti-Okareacts optimally by an indi-

rect antiglobulin test and appears to be

clinically significant in transfusion ther-

apy, causing rapid destruction of Ok(a+)

red cells.

Raph System

The Raph system consists of a single anti-

gen, MER2. Anti-MER2 has been reported

in three Israeli Jews. All three were on re-

nal dialysis, raising the possibility that an-

tibody production may be associated with

kidney disease. The MER2 antigen has

354 AABB Technical Manual

Table 15-13. Antigens of High and Low

Incidence in the Cromer Blood Group

System

Antigen Incidence (%)

Cra>99

Tca>99

Tcb<1

Tcc<1

Dra>99

Esa>99

IFC >99

WESa<1

WESb>99

UMC >99

GUTI >99

SERF >99

ZENA >99

been detected on the red cells of 92% of

those tested. The MER2 antigen is sensi-

tive to DTT but is not affected by treat-

ment with ficin, papain, or chloroquine.

The antibodies have been IgG and some

have bound complement. To date, there

has been no information on whether these

antibodies are capable of causing HTR or

HDFN.

John Milton Hagen System

The John Milton Hagen (JMH) antigen is

carried on a GPI-linked CD108 glycopro-

tein. JMH antigen decreases over time. The

JMH– phenotype can be transient. The

JMH– phenotype can be acquired or in-

herited. The antigen is destroyed or al-

tered by ficin or papain treatment and by

AET or DTT treatment.

Antibodies that react with JMH show

variation in reactivity and are usually weak.

Autoanti-JMH can often be found in older

people, along with an acquired absent or

weak JMH antigen expression. The anti-

body is not routinely considered capable of

HTR or HDFN.

GIL System

There is one antigen of high frequency, GIL,

in this system. GIL is located on aquaporin

3 (AQP3), which is a glycerol transporter.

The antibody has not been reported to

cause HDFN or HTR.

Blood Group Collections

In addition to the blood group systems,

there are collections of antigens that ex-

hibit shared characteristics but do not as

yet meet the criteria for blood group sys-

tem status defined by the ISBT. They in-

clude Cost (ISBT 205), Er (ISBT 208), and

Vel (ISBT 211).

Cost

Csaand Csbare all that remain of this col-

lection after the Knops antigens were

identified on CR1. Csaoccurs in a fre-

quency greater that 98% in most popula-

tions, whereas Csbappears in about 34%

of the population.19 There is, however, an

unexplained connection between the Yka

and Csaantigens, such that red cells nega-

tive for one antigen are often weak or neg-

ativefortheother.(SeeTable15-11.)The

antigens are not destroyed by ficin, papain,

or DTT. Anti-Csabehaves similarly to anti-

bodies produced to the Knops system an-

tigens and is not considered clinically sig-

nificant.

The Er collection consists of two antigens,

which give rise to four phenotypes: Er(a+b–),

Er(a+b+), Er(a–b+), and Er(a–b–). Erais a

high-incidence antigen present on the red

cells of >99% of all individuals, but Erbhas

a prevalence of less than 1%. The presence

of a silent third allele, Er, is thought to ac-

countfortheEr(a–b–)phenotype,asde-

monstrated by family studies. The antigens

are not destroyed by ficin, papain, or DTT

but are destroyed by EDTA-glycine acid.

Vel

The Vel collection was recently created to

include two serologically related antigens

of high incidence, Vel and ABTI.

Vel is a high-incidence antigen that is

unaffected by protease and sulfhydryl treat-

ment. It is well developed at birth, but anti-

gen expression is variable.8Despite its oc-

currence after known immunizing stimuli,

anti-Vel is most commonly IgM. It has been

reported to range from causing only a posi-

tive DAT in the neonate to causing severe

HDFN.19 It has been implicated in HTRs.

Anti-Vel binds complement, and in-vitro

hemolysis of incompatible red cells is often

Chapter 15: Other Blood Groups 355

seen when testing freshly drawn serum

containing this antibody. Reactivity of

anti-Vel is usually enhanced by enzyme

treatment of red cells expressing the anti-

gen.

High-Incidence Red Cell

Antigens Not Assigned to a

Blood Group System or

Collection

Table 15-14 lists the antigens of high inci-

dence that are independent of a blood

group system or collection. Persons who

make alloantibody to a specific blood

group antigen necessarily have red cells

lacking that antigen. For this reason, anti-

bodies directed at high-incidence antigens

are rarely encountered. The antibodies

corresponding to these antigens usually

react best by antiglobulin testing.

Lan Antigen

Lan is a high-incidence antigen that is re-

sistant to enzyme-treatment and to 0.2 M

DTT treatment. A weak form of the Lan

antigen has been reported.19 Anti-Lan is

characteristically IgG, may bind comple-

ment, and may cause HTRs. Cases of

HDFN due to anti-Lan have been mild

even though the Lan antigen is present on

cord red cells.19

AtaAntigen

Atais a high-incidence antigen that is re-

sistant to enzyme treatment and to 0.2 M

DTT treatment. The At(a–) phenotype has

been found only in Black individuals.19

Anti-Atais characteristically IgG. The anti-

body appears to cause only moderate

HTRs and no clinical HDFN.19

JraAntigen

Jrais a high-incidence antigen that is re-

sistant to enzyme treatment and to 0.2 M

DTT treatment. The Jr(a–) phenotype is more

commonly found in Japanese individuals

but has been found in other populations

as well.8(pp805-806) Anti-Jrahas been shown to

cause reduced red cell survival.8(pp805-806)

Other examples of anti-Jrahave shown lit-

tle or no clinical significance in HDFN or

HTR.8(p806),39

AnWj Antigen

AnWj is a high-incidence antigen that is

resistant to enzyme treatment but weak-

ened by 0.2 M DTT treatment.19 The anti-

geniscarriedonCD44,whichalsocarries

the Indian blood group system antigens.

The AnWj antigen is weakened on the red

cells from individuals with the In(Lu)

gene (see section on Lutheran System).

Some patients with Hodgkin’s disease

may experience a long-term suppression

of the AnWj antigen.8(pp783-784) The AnWj an-

tigen is the receptor for Haemophilus

influenzae.8(pp784-785) Anti-AnWj has been

356 AABB Technical Manual

Table 15-14. Some Antigens of High

Incidence Not Assigned to a Blood

Group System or Collection

Name Symbol

August Ata

Langereis Lan

Sid Sda

Duclos

Jra

Emm

AnWj

PEL

MAM

implicated in severe HTRs but not in HDFN

because the antigen is not present on cord

red cells.

SdaAntigen

Sdais an antigen of fairly high incidence,

widely distributed in mammalian tissues

and body fluids. The antigen is variably

expressed on the red cells of Sd(a+) indi-

viduals. Sdaexpression may diminish dur-

ing pregnancy and the Sd(a–) phenotype

is observed in 30% to 75% of pregnant

women. The antigen is not present on

cord cells. The strongest expression of Sda

has been observed on polyagglutinable

red cells of the Cad phenotype. The fre-

quency of Sd(a–) blood is considered to

be around 9%, but weakly positive reac-

tions are often difficult to distinguish from

negative ones.

Anti-Sdacan be reactive by antiglobulin

testing. Microscopic examination of posi-

tive reactions generally shows mixed-field

agglutination, with relatively small, tightly

agglutinated clumps of red cells present

against a background of free red cells.

These agglutinates are refractile and may

have a shiny appearance. Because the ma-

jority of the examples of anti-Sdaare IgM,

thewideuseofanti-IgGmeansthatmany

examples of anti-Sdaare no longer de-

tected. Anti-Sdais not considered to be clin-

ically significant. However, there have been

reported cases of HTRs with red cells with

strong Sdaexpression.8(pp816-817)

The immunodominant sugar of Sdais

N-acetylgalactosamine (GalNAc), also the

immunodominant sugar of the A blood

group antigen and of the Tamm-Horsfall

glycoprotein, found in human and guinea

pig urine. Anti-Sdaactivity can be inhibited

by incubation with urine from guinea pigs

or from Sd(a+) humans. See Method 3.11

for the performance of a urine neutraliza-

tion of anti-Sda.

Low-Incidence Red Cell

Antigens Not Assigned to a

Blood Group System or

Collection

Many independent low-incidence red cell

antigens have been recognized in addition

to a growing number that have been as-

signed to the MNS, Rh, and Diego systems.

Table 15-15 lists those that have been

studied and shown to be inherited in a

dominant manner. Antibodies specific for

these low-incidence antigens react with

so few random blood samples that they

virtually never cause difficulties in select-

ing blood for transfusion but may be im-

plicated in some rare cases of HDFN. The

antibodies are of interest to the serologist,

however, because of the unexpectedly

high incidence with which they occur,

often without an identifiable antigenic

stimulus.

Chapter 15: Other Blood Groups 357

Table 15-15. Antigens of Low Incidence

Not Assigned to a Blood Group System

or Collection

Batty (By) Livesay (Lia)

Biles (Bi) Milne

Box (Bxa)Oldeide(Ol

Christiansen (Chra) Peters (Pta)

HJK Rasmussen (RASM)

HOFM Reid (Rea)

JFV REIT

JONES SARA

Jensen (Jea) Torkildsen (Toa)

Katagiri (Kg)

The antigens occur with a frequency of 1 in 500 or less.

Antibodies to Low-Incidence

Antigens

Antibodies to low-incidence antigens have

sometimes been implicated in transfusion

reactions and HDFN. These antibodies

are usually encountered by chance, when

the red cells used for antibody detection

or selected for crossmatching happen to

carry the corresponding antigen.

Antibodies to low-incidence antigens may

also be present as unsuspected contaminants

in blood typing reagents prepared from hu-

man serum and may cause false-positive

test results if the red cells tested carry the

antigen. Testing with reagents from differ-

ent manufacturers may not eliminate this

error because it is not uncommon for a sin-

gle individual with an uncommon antibody

to provide the serum used for reagent prep-

aration by different manufacturers.

Some antibodies to low-incidence anti-

gens react as saline agglutinins. They can

also occur as IgG antibodies reactive only

by antiglobulin testing, even if there has

been no exposure to red cell immunization.

It is common for several low-incidence an-

tibodies to occur together in a single serum;

multiple specificities are especially likely in

sera from patients with autoimmune con-

ditions.

Bg (Bennett-Goodspeed) Antigens

Antibodies directed at certain leukocyte

antigens sometimes cause confusing re-

actions in serologic tests with red cells. At

least three separate specificities have been

given names as Bg antigens: Bgacorresponds

to HLA-B7; Bgbcorresponds to HLA-B17;

and Bgccorresponds to HLA-A28. A fourth

antibody in some antileukocyte sera re-

acts with red cells of persons who express

HLA-A10.Theso-calledBgantigensare

expressed to variable degrees on red cells,

with the result that reactions of differing

strengths are observed when a single se-

rum containing “anti-Bg” is tested with

different Bg+ red cells. Reactivity is most

commonly observed in antiglobulin test-

ing, but highly potent anti-Bg sera may di-

rectly agglutinate red cells with an unusu-

ally strong expression of the Bg antigens.

Confident and precise classification of

reactivity is made difficult by the weak ex-

pression of these antigens on some red cells

and by multiple specificities among differ-

ent examples of the Bg antibodies. These

antibodies may also occur as unsuspected

contaminants in human source blood typ-

ing sera, where they may cause false-posi-

tive reactions with cells having unusually

strong expression of the corresponding Bg

antigen. Bg-related antigens are denatured

by chloroquine diphosphate or a solution

of glycine-HCl/EDTA.

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3. Daniels GL, Cartron JP, Fletcher A, et al. Inter-

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6. Rygiel SA, Issitt CH, Fruitstone MJ. Destruc-

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8. Issitt PD, Anstee DJ. Applied blood group se-

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13. ChaudhuriA,PolyakovaJ,ZbrezeznaV,etal.

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Chapter 15: Other Blood Groups 359

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Akane A, Mizukami H, Shiono H. Classification of

the standard alleles of the MN blood group sys-

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Liu M, Jiang D, Liu S, et al. Frequencies of the ma-

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group systems in the Chinese, Han, Hui, and Ti-

betan nationalities. Immunohematology 2003;19:

22-5.

Lögdberg L, Reid M, Miller J. Cloning and genetic

characterization of blood group carrier molecules

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360 AABB Technical Manual

Chapter 16: Platelet and Granulocyte Antigens and Antibodies

Chapter 16

Platelet and Granulocyte

Antigens and Antibodies

ANTIBODIES REACTIVE WITH an-

tigens expressed on platelets and

leukocytes are assuming increas-

ing importance. Some blood group anti-

gens are shared by red cells, white cells,

and platelets; others are specific to cer-

tain cell types. This chapter discusses an-

tibodies directed at antigens expressed on

platelets and neutrophils, with an empha-

sis on those specific for these cells. HLA

antibodies and antigens are covered more

fully in Chapter 17.

Platelet Antigens

Antigens Shared with Other Tissues

Platelets express a variety of antigenic

markers on their surface. Some of these

antigens are shared with other cell types,

as in the case of ABH antigens and HLA

antigens, which are shared with virtually

all nucleated cells in the body. Others are

observed to be essentially platelet spe-

cific.

ABH Antigens

The ABH antigens expressed on platelets

are a combination of structures intrinsic

totheplasmamembraneandthosead

sorbed from the plasma. The amount of

ABH antigen present on platelets is quite

variable from individual to individual,

with from 5% to 10% of non-group-O in-

dividuals expressing extremely elevated

amounts of A or B substance on their

platelets. These people appear to have a

“high-expresser” form of glycosyltrans-

ferase in their sera. Although platelets are

often transfused without regard to ABO

compatibility, in some cases, ABO anti-

bodies (particularly IgG antibodies of

high titer in group O recipients) may react

with platelets carrying large amounts of A

or B antigen.1High-expresser platelets are

particularly vulnerable to this type of im-

361

mune destruction. ABO antibodies in re-

cipients may also cause reduced survival

of ABO-incompatible platelets from nor-

mal-expresser phenotype donors, causing

occasional patients to exhibit refractori-

ness to platelet transfusions on this basis.

Other red cell antigens—including Lea,

Leb,Ii,andP

2as well as the Cromer antigens

associated with decay accelerating fac-

tor3—are also found on platelets, but there

is no evidence that antibodies to these anti-

gens significantly reduce platelet survival in

vivo.

HLA Antigens

HLA antigens are found on the surfaces of

both platelets and white cells (see Chap-

ter 17). In fact, platelets are the major

source of Class I HLA antigens in whole

blood.2Recent evidence indicates that

most Class I HLA molecules on platelets

are integral membrane proteins, and

smaller amounts may be absorbed from

surrounding plasma.3

HLA alloantibodies do not occur natu-

rally, arising only after sensitization by

pregnancy or blood transfusion. Studies of

HLA alloimmunization in patients trans-

fused with platelets document the develop-

ment of antibodies within 3 to 4 weeks after

primary exposure and as early as 4 days af-

ter secondary exposure in patients previ-

ously transfused or pregnant.4The likeli-

hood of HLA alloimmunization by

transfusion in patients not previously sensi-

tized is variable,4,5 and the risk of HLA

alloimmunization appears to be related to

the underlying disease as well as to the

immunosuppressive effects of treatment

regimens. Platelets carry Class I HLA anti-

gens but lack Class II antigens, which are

necessary for primary sensitization. There-

fore, exposure to leukocytes expressing

HLA antigens during transfusion is the

principal cause of primary HLA alloim-

munization.

Platelet Transfusion Refractoriness

A less-than-expected increase in platelet

count occurs in about 20% to 70% of

multitransfused thrombocytopenic pa-

tients,6and patients treated for malignant

hematopoietic disorders are particularly

likely to become refractory to platelet

transfusions. A widely accepted definition

of refractoriness was used in a random-

ized controlled clinical trial of platelet

transfusion therapy, sponsored by the Na-

tional Institutes of Health (NIH). In this

study, two consecutive 1-hour posttrans-

fusion platelet corrected count increments

(CCI) of less than 5000 platelets ×m2body

surface area/µL indicated refractoriness.7

Others have used less stringent criteria

(eg, three platelet transfusions over a

2-week period that yield inadequate post-

transfusion platelet counts).8,9 Response is

often determined by calculating either a

CCI or a posttransfusion platelet recovery

(PPR) between 10 and 60 minutes after

transfusion (see Table 16-1). Responses of

7500 platelets ×m2body surface area/µL

or 20% can be considered acceptable from

the CCI or the PPR calculation, respec-

tively.

Alloimmune platelet refractoriness is

most often the result of HLA sensitization

and can be diagnosed by demonstration of

significant levels of HLA antibodies. Patient

serum is tested against a panel of lympho-

cytes (or synthetic beads bearing Class I an-

tigens) that represent most of the Class I

HLA specificities in the population. A

panel-reactive antibody (PRA) score of 20%

or higher is evidence that HLA sensitization

may be contributing to the platelet refrac-

toriness (see Chapter 17).

Although platelet alloimmunization is

one cause of refractoriness, there are multi-

362 AABB Technical Manual

ple, nonimmune reasons why transfused

platelets may not yield the expected in-

crease in platelet count [eg, sepsis, dissemi-

nated intravascular coagulation (DIC), or

the administration of certain drugs]. Some

of the most commonly cited nonimmune

causes of platelet refractoriness are listed in

Table 16-2. A study of patients undergoing

marrow transplant suggested that pa-

tient-related variables such as total body ir-

radiation, advanced disease status, and liver

dysfunction are important predictors of

poor platelet count increments as well.10,11

Even when possible immune causes of re-

fractoriness are identified, nonimmune fac-

tors are often simultaneously present.

Several strategies may be considered

when selecting platelets for patients with

immune-mediated refractoriness. When

antibodies to HLA antigens are demon-

strated, a widely used approach is to supply

apheresis platelets matched to the patient’s

HLA type.12 A disadvantage is that a pool of

several thousand HLA-typed potential

apheresis donors is necessary to find suffi-

cient HLA-compatible matches.13 Moreover,

donor selection on the basis of HLA type

can lead to the exclusion of donors with

HLA types different from that of the recipi-

ent but potentially effective if the recipient

is alloimmunized to other antigenic deter-

minants.14 For patients who are likely to re-

quiremultipleplatelettransfusions,HLA

typing should be performed in advance of a

planned course of treatment.

It is important to understand the degree

of match that may be provided (see Table

16-3). Platelets received following a request

for “HLA-matched” platelets are typically

the closest match obtainable within the

constraints of time and donor availability.

In one study,15 43% of platelets provided as

Chapter 16: Platelet and Granulocyte Antigens and Antibodies 363

Table 16-1. Determination of Response to Transfused Platelets

Calculation of Corrected Count Increment (CCI)

CCI = Body Surface Area (m2)×Platelet Count Increment ×1011

No. of Platelets Transfused

EXAMPLE: If 4 ×1011 platelets are transfused to a patient whose body surface area is 1.8 m2and

the increase in posttransfusion platelet count is 25,000/µL, then:

CCI 1.8 m 25,000 / L 10

410 11,250 plat

211

=××

×=

µelets m / L

×µ

Calculation of Posttransfusion Platelet Recovery (PPR)

PPR(%) Estimated Total Blood Volume* Platelet

=×Count Increment

No. of Platelets Transfused

*Total blood volume can be estimated in adult patients as 75 mL/kg

EXAMPLE: If 4 ×1011 platelets are transfused to a 70-kg patient and the increase in

posttransfusion platelet count is 25,000/µL, then:

PPR 70 kg 70 mL / kg 25,000 plts / L 10

=×× ×

0 platelets 30.6%

11 =

HLA-matched were relatively poor grade B

or C matches. The most successful re-

sponses occur with the subset of grade A

and B1U or B2U HLA matches, but mis-

matches for some antigens (B44, 45) that

are poorly expressed on platelets can be

useful. According to AABB Standards for

Blood Banks and Transfusion Services,16(p43)

HLA-matched platelets should be irradi-

ated to prevent transfusion-associated

graft-vs-host disease.

A second approach to provide effective

platelets is to use a pretransfusion platelet

crossmatching assay. This approach can be

used to predict and, therefore, avoid subse-

quent platelet transfusion failures.17 The

solid-phase red cell adherence test (SPRCA)

is the most widely used method for platelet

crossmatching, and test results are reason-

ably predictive of posttransfusion platelet

counts.18-20 Compared with HLA matching,

crossmatching can prove both more conve-

nient and economically advantageous. It

avoids exclusion of HLA-mismatched but

compatible donors and has the added ad-

vantage of selecting platelets when the an-

tibody (-ies) involved is (are) directed at a

platelet-specific antigen. Platelet cross-

matching, however, will not always be suc-

cessful, particularly when patients are

highly alloimmunized (PRA >50%). In these

instances, finding sufficient compatible

units may be problematic, and selection of

HLA-matched platelets may be more prac-

tical. Although the incidence of platelet-

specific antibodies causing patients to be

refractory to most or all attempted platelet

transfusions is very small, this possibility

should be investigated when most of the at-

tempted crossmatches are positive. If pla-

telet-specific antibodies are present, donors

of known platelet antigen phenotype or

364 AABB Technical Manual

Table 16-2. Some Nonimmune Causes

of Platelet Refractoriness

Massive bleeding

Fever

Sepsis

Splenomegaly (splenic sequestration)

Disseminated intravascular coagulation

Allogeneic transplantation

Poor storage of platelets before transfusion

Effects of drugs (may include immune

mechanisms)

Intravenous amphotericin B

Thrombotic thrombocytopenic purpura

Table 16-3. Degree of Matching for HLA-Matched Platelets

Match

Grade Description

Examples of Donor

Phenotypes for a Recipient

Who Is A1,3;B8,27

A 4-antigen match A1,3;B8,27

B1U 1 antigen unknown or blank A1,-;B8,27

B1X 1 cross-reactive group A1,3;B8,7

B2UX 1 antigen blank and 1 cross-reactive A1,-;B8,7

C 1 mismatched antigen present A1,3;B8,35

D 2 or more mismatched antigens present A1,32;B8,35

R Random A2,28;B7,35

family members, who are more likely to

share the patient’s phenotype, should be

tested.

An alternative approach to supplying

HLA-compatible transfusions is to deter-

mine the specificity of the patient’s HLA an-

tibodies and select donors whose platelets

lack the antigens with which the antibodies

react. This is termed the antibody specific-

ity prediction (ASP) method.8One study

compared the effectiveness of transfused

platelets selected by the ASP method with

those selected on the basis of HLA match-

ing, platelet crossmatching, or on a random

basis.8Platelets selected by the ASP method

were equally effective as those selected by

HLA matching or by crossmatching, and

superior to randomly selected platelets. In

addition, from a file of HLA-typed donors,

many more potential donors were identi-

fied by the ASP method than were available

using traditional HLA matching criteria,

making the acquisition of compatible

platelets for alloimmunized refractory pa-

tients much more feasible.

A further refinement of HLA matching

was proposed by Duquesnoy.21 Acomputer-

ized algorithm—HLA Matchmaker, available

at http://tpis.upmc.edu/tpis/HLAMatchmaker—

is employed for evaluation of the molecular

similarities and differences between HLA

Class I epitopes. First developed to aid in

locating compatible organs for alloim-

munized prospective renal transplant pa-

tients, the strategy is based on the concept

that immunogenic epitopes are repre-

sented by amino acid triplets on exposed

partsofproteinsequencesoftheClassI

alloantigens that are accessible to allo-

antibodies. Using this scheme, many Class I

HLA antigens classified as mismatches to a

patient’s HLA type have no incompatible

exposed amino acid triplets and, therefore,

would not be expected to elicit an antibody

response. The pool of potentially compati-

ble HLA-selected donors is thereby greatly

expanded. Although this strategy may

prove useful in selecting platelet donors for

refractory patients, it has not yet been eval-

uated in a clinical trial for this purpose and

remains to be validated for this indication.

Prevention of Platelet Alloimmunization

Once refractoriness resulting from plate-

let alloimmunization is established, it is

very difficult, if not impossible, to reverse.

Therefore, in addition to developing meth-

ods of selecting compatible platelet do-

nors for the refractory patient, several

strategies have been evaluated to prevent

alloimmunization to platelets from occur-

ring in the first place. They include reduc-

tion in the number of leukocytes in the

platelet products and ultraviolet B (UVB)

irradiation. The report of the Trial to Re-

duce Alloimmunization to Platelets (TRAP)

Study Group7indicated that use of either

leukocyte-filtered or UVB-irradiated blood

components reduced the incidence of HLA

antibody generation from 45% to between

17% and 21%. The incidence of platelet

refractoriness was reduced from 16% to

between 7% and 10%. Although a rela-

tionship had been reported between allo-

immunization and the number of donor

exposures in one report,22 a second study23

found no relationship between the num-

ber of donor exposures and the rate or se-

verity of alloimmunization. The TRAP

study found that leukocyte reduction, not

the number of donor exposures, was sig-

nificant in modifying the rate of allo-

immunization. Thus, leukocyte-reduced,

pooled, whole-blood-derived platelets

appear to be clinically equivalent to

apheresis platelets, at least in terms of re-

ducing primary alloimmunization.24

Antibodies to HLA antigens may be de-

tected by lymphocytotoxicity tests or by

many of the platelet antibody tests dis-

cussed below. Lymphocytotoxicity tests de-

Chapter 16: Platelet and Granulocyte Antigens and Antibodies 365

tect complement-binding antibodies capa-

ble of killing lymphocytes. One strategy for

managing patients who are receiving multi-

ple platelet transfusions and who have de-

veloped clinical refractoriness is to test for

thepresenceofHLAantibodiesusinga

lymphocytotoxicity antibody screen against

a panel of lymphocytes representing most

of the Class I HLA antigens present in the

donor population. Reactivity to greater

than 20% of the cells in the panel (PRA

>20%) indicates that HLA sensitization may

be at least a contributing cause of the

platelet refractoriness. Newer, more sensi-

tive HLA antibody detection techniques

such as the Flow PRA25 (One Lambda,

Canoga Park, CA) have been adapted to a

PRA result format, and some HLA testing

laboratories use these methods instead of

the traditional lymphocytotoxicity test.

The laboratory detection of lymphocyto-

toxic antibodies does not necessarily indi-

cate that the patient will experience re-

duced survival of transfused platelets.

Moreover, HLA antibodies may disappear

from the patient’s plasma despite contin-

ued exposure through transfusions.26 A

fuller discussion of HLA antibody and anti-

gen testing can be found in Chapter 17.

Platelet-Specific Alloantigens

To date, 22 platelet-specific alloantigens

have been characterized as to their local-

ization to platelet surface glycoprotein

structures, quantification of their density

on the platelet surface, and determination

of DNA polymorphisms in genes encod-

ing for them (see Table 16-4).27 Several

others have been described serologically,

but genetic polymorphisms underlying

them have not yet been determined.28 The

term “platelet specific,” is a misnomer for

some of these markers because they may

be found on other types of cells as well

(especially endothelial cells). However,

their chief clinical importance remains

linked to their presence on platelets. Of

the dozens of recognized platelet mem-

brane glycoproteins, at least five [GPIa, Ib

(alpha and beta), IIb, IIIa, and CD109] are

polymorphic and have been demon-

strated to be alloimmunogenic.28 In addi-

tion, rare individuals who lack a sixth

membrane glycoprotein, GPIV (CD36),

may become sensitized to this antigen.29

Approximately 3% to 5% of individuals of

Asian or African ethnicity lack GPIV on

their platelets30 and can become immu-

nized by transfusion or pregnancy.31 Al-

though antibodies to these various mem-

brane glycoproteins may be associated, in

rare instances, with refractoriness to pla-

telet transfusions, alloantibodies to plate-

let-specific antigens are more often asso-

ciated with the alloimmune syndromes

posttransfusion purpura (PTP) and neo-

natal alloimmune thrombocytopenia

(NAIT).

Several antigen systems on platelets are

now recognized32 (Table 16-4).27,33 The no-

menclature adopted by the International

Society of Blood Transfusion classifies the

systems numerically according to the date

of publication and alphabetically to reflect

their frequency in the population.34 As with

red cells, different terminologies for platelet

antigens often coexist. The first recognized

antigen,35 Zwa, is now designated HPA-1a of

the HPA-1 system. The HPA-1a antigen is

often better known as PlA1.HPA-1aispres

ent on the platelets of about 98% of persons

of European ethnicity and anti-HPA-1a

(anti-PlA1) is the most frequently encoun-

tered clinically significant platelet-specific

antibody in this population. Its antithetical

antigen, HPA-1b (PlA2), occurs in 27% of this

population.

The HPA-1a and HPA-1b alleles reside on

the platelet membrane glycoprotein GPIIIa.

Patients with Glanzmann’s thrombasthenia

Type I, a disorder of platelet function, lack

366 AABB Technical Manual

Chapter 16: Platelet and Granulocyte Antigens and Antibodies 367

Table 16-4. Alloantigenic Polymorphisms of Platelet Glycoproteins that Have Been Implicated in Alloimmune Syndromes*

HPA System

Name

Antigens (Familiar

Names)

Phenotypic

Frequencies GP Location

Amino Acid

Substitution

Alloimmune

Syndromes

POLYMORPHISMS OF GLYCOPROTEIN IIIa

HPA-1 HPA-1a, (PlA1,Zw

a)98% IIIa Leu↔Pro33 NAIT, PTP

HPA-1b, (PlA2,Zw

b)27% IIIa

HPA-4 HPA-4a (Pena,Yuk

b)†99.9% IIIa Arg↔Gln143 NAIT, PTP

HPA-4b (Penb,Yuk

a)<1% IIIa

HPA-6 HPA-6bw (Caa,Tu

a)<1% IIIa Arg↔Gln489 NAIT

HPA-7 HPA-7bw (Mo) <1% IIIa Pro↔Ala407 NAIT

HPA-8 HPA-8bw (Sra) <1% IIIa Arg↔Cys636 NAIT

HPA-10 HPA-10bw (Laa) <1% IIIa Arg↔Gln62 NAIT

HPA-11 HPA-11bw (Groa) <1% IIIa Arg↔His633 NAIT

HPA-14 HPA-14bw (Oea) <1% IIIa Lys611 Deleted NAIT

HPA-16 HPA-16bw (Duva) <1% IIIa Ile↔Thr140 NAIT

POLYMORPHISMS OF GLYCOPROTEIN IIb

HPA-3 HPA-3a (Baka,Lek

a) 85% IIb IIe↔Ser843 NAIT, PTP

HPA-3b (Bakb, Lekb)63% IIb

HPA-9 HPA-9bw (Maxa) 0.6% IIb Val↔Met837 NAIT

(cont’d)

368 AABB Technical Manual

HPA System

Name

Antigens (Familiar

Names)

Phenotypic

Frequencies GP Location

Amino Acid

Substitution

Alloimmune

Syndromes

POLYMORPHISMS OF GLYCOPROTEIN Ia

HPA-5 HPA-5a (Brb,Zav

b) 99% Ia glu↔Lys505 NAIT, PTP

HPA-5b (Bra,Zav

a) 20% Ia

HPA-13 HPA-13bw (Sita) <0.2% Ia Thr↔Met799 NAIT

POLYMORPHISMS OF GLYCOPROTEIN Ib

HPA-2 HPA-2a (Kob,Sib

b) 99% Ib alpha Thr↔Met145 NAIT

HPA-2b (Koa,Sib

a) 15% Ib alpha

HPA-12 HPA-12bw (Iya)0.3%IbbetaGly↔Glu15 NAIT

OTHER PROBABLE PLATELET ALLOANTIGEN SPECIFICITIES

HPA-15 HPA-15a (Govb) 80% CD109 Tyr↔Ser703 NAIT, PTP

HPA-15b (Gova) 60% CD109

*Modified from Kroll.27 GP = glyprotein; NAIT = neonatal alloimmune thrombocytopenia; PTP = posttransfusion purpurpa.

Table 16-4. Alloantigenic Polymorphisms of Platelet Glycoproteins that Have Been Implicated in Alloimmune Syndromes*

(cont’d)

this glycoprotein and, therefore, do not ex-

press HPA-1 antigens. The HPA-1 polymor-

phism arises by the substitution of a single

base pair (leucine in HPA-1a and proline in

HPA-1b)ataminoacidposition33ofthe

protein’s DNA coding sequence. GPIIIa is

also the carrier of HPA-4, -6, -7, -8, -10, -11,

-14, and -16 antigens. Alleles in each of

these systems also arise as a result of single

amino acid substitutions at different posi-

tions. The HPA-2 antigen system is situated

on GPIb alpha; the HPA-3 system on GPIIb;

and the HPA-5 system on GPIa.36,37

On the platelet membrane, most of the

glycoproteins that carry these “platelet-spe-

cific” antigens are present as heterodimeric

compounds, ie, each consists of two differ-

ent glycoprotein molecules (see Fig 16-138).

Therefore, platelet glycoprotein names are

often paired (eg, Ia/IIa, IIb/IIIa, or Ib/IX),

referring to the alpha and beta chains in

each complex. GPIb/IX is a leucine-rich

membrane glycoprotein that serves as a re-

ceptor for von Willebrand factor on plate-

lets. The Ia/IIa and IIb/IIIa complexes are

members of a broadly distributed family of

adhesion molecules called integrins. Inte-

grins are essential for platelet adhesion and

aggregation because the molecules serve as

receptors for ligands such as fibrinogen

(IIb/IIIa), von Willebrand factor (Ib/IX), and

collagen (Ia/IIa). When present on other

cells, the glycoprotein pairings may differ.

For example, on platelets, GPIIIa is nor-

mally paired with GPIIb. On endothelial

cells,fibroblasts,andsmoothmuscle,how

ever, GPIIIa is paired with a different

glycoprotein. Thus, these cells share the

Chapter 16: Platelet and Granulocyte Antigens and Antibodies 369

Figure 16-1. Schematic diagram of platelet glycoprotein complex IIb/IIIa. Dots and letters (Yuk, Oe,

Pla, Ca, Gro, Sr, Mo, Bak, Max) designate positions and names of recognized allotypic epitopes. The

molecular regions where autoepitopes have been recognized are indicated by brackets.38

HPA alloantigens found on the GPIIIa mol-

ecule, but not those found on the GPIIb

molecule.

CD109 is an exception to the hetero-

dimeric rule, occurring as a monomeric

structure on the platelet membrane. This

GPI-linked protein is found on activated T

cells, cultured endothelial cells, several tu-

mor cell lines, as well as platelets.39 Two

alloantigens designated HPA-15wb (Gova)

and HPA-15wa (Govb) have been localized

to platelet CD109.39 Unlike most platelet-

specific alloantigens, both alleles are highly

expressed [0.53% (Govb) and 0.47% (Gova)]

in persons of European ethnicity (Table

16-4). Sensitization to Gov alloantigens has

been associated with platelet refractoriness,

NAIT, and PTP, albeit usually together with

other alloantibodies to platelet antigens.40

Clinical Importance of Platelet-Specific

Antigens and Antibodies

Neonatal Alloimmune Thrombocytopenia

Neonatal alloimmune thrombocytopenia

(variously abbreviated NAIT, NATP, etc) is

described in Chapter 23.

Posttransfusion Purpura

Posttransfusion purpura (PTP) is charac-

terized by the development of dramatic,

sudden, and self-limiting thrombocytopenia

5 to 10 days after a blood transfusion in a

patient with a history of sensitization by

pregnancy or transfusion. Coincident with

the thrombocytopenia is the development

of a potent platelet-specific alloantibody

in the patient’s serum, usually anti-HPA-

1a. Other specificities have been implicated,

almost always associated with antigens

on GPIIb/IIIa.37,41 PTP differs from transfu-

sion reactions caused by red cell antibod-

ies because the patient’s own antigen-

negative (usually HPA-1a-negative) plate-

lets as well as any transfused antigen-pos-

itive platelets (which may be accompa-

nied by clinically severe reactions) are de-

stroyed. Transfusion of antigen-negative

platelets may be of value during the acute

phaseofPTP;however,suchplatelets

have a reduced in-vivo survival.42 Plasma-

pheresis—once the treatment of

choice—has largely been supplanted by

the use of intravenous immune globulin

(IGIV). The mechanism by which these

treatments are efficacious is unknown.

Platelet antibody assays usually reveal a

serum antibody with HPA-1a specificity.

Typing of the patient’s platelets after re-

covery will document a HPA-1a-negative

phenotype or analogous typing for other

platelet-specific antigen systems. Follow-

ing recovery, future transfusions should

be provided using washed antigen-nega-

tive RBC units if possible. Washed RBC

units may offer some protection against

recurrence, although at least one case of

PTPcausedbyantibodytoHPA-5bwas

precipitated by transfusion of a washed

RBC unit.43

Testing for Platelet-Specific Antigens and

Antibodies

Clinically useful platelet antibody assays

emerged later than serologic assays to di-

agnose immunologic disorders involving

redcells.Thisismainlybecauseitisdiffi

cult to separate platelets from whole blood

specimens and to distinguish antibody-

dependent endpoints from nonspecific

changes that occur in platelets under as-

say conditions. Three types of platelet an-

tibody detection methods have been de-

veloped44 (Table 16-5). The earliest were

Phase I assays that involved mixing pa-

tient serum with normal platelets and

used platelet function-dependent end-

points such as alpha granule release, ag-

gregation, or agglutination. Phase II tests

measured either surface or total platelet-

associated immunoglobulin on patient

370 AABB Technical Manual

platelets or on normal platelets after sen-

sitization with patient serum. Phase III

solid-phase assays were developed in

which the binding of antibodies to iso-

lated platelet surface glycoproteins is de-

tected. The test methods are examples of

Phase I, II, and III assays; variations of each

test method are also used. Lymphocyto-

toxicity tests are discussed in Chapter 17.

Mixed Passive Hemagglutination Assay

(MPHA). A Phase II assay used for the de-

tection of platelet-specific antibodies as

well as for platelet crossmatching is the

MPHA. Shibata et al were the first to use

this method to detect and identify clinically

significant platelet alloantibodies.45 Amodi

fication of MPHA, the SPRCA, is widely

used.18 In this assay, intact platelets are im-

mobilized in the round-bottom wells of a

microtiter tray and are sensitized with anti-

body to be detected. After washing, detec-

tor red cells previously coated with an anti-

body specific for human immunoglobulin

are added. After incubation from several

hours to overnight, the tray is subjected to a

slow centrifugation and examined visually.

If antibody is bound to the immobilized

platelets, the indicator red cells fail to form

a compact button in the center of the well

because they are evenly distributed like a

Chapter 16: Platelet and Granulocyte Antigens and Antibodies 371

Table 16-5. Platelet Antibody Assays

Phase I Assays

Platelet aggregation

Inhibition of platelet aggregation

Inhibition of clot retraction

Inhibition of platelet migration

Complement fixation

Platelet factor 3 release

51Chromium release

14C-Serotonin release

Phase II Assays

Detection of platelet surface-associated immunoglobulin

■Platelet suspension immunofluorescence test (PSIFT)

■Flow cytometry

■Radioimmunoassay (125I staphylococcal Protein A,125I antihuman immunoglobulin, polyclonal or

monoclonal)

■Antiglobulin consumption (two-stage assay)

■Solid-phase red cell adherence

Detection of total platelet-associated immunoglobulin

■Nephelometry

■Electroimmunoassay

■Radial immunodiffusion

Phase III Assays

Monoclonal antibody immobilization of platelet antigens (MAIPA)

Antigen capture ELISA (ACE)

Modified antigen capture ELISA (MACE)

Immunobead assay

Immunoblotting

“carpet” over the antibody-coated platelets.

In a negative reaction, a red cell button forms

in the center of the well. A limitation of the

MPHA assay is that it fails to distinguish

platelet-specific from non-platelet-specific

antibodies. A modification of the MPHA as-

say, the Capture-P (Imucor Gamma, Norcross,

GA), is available as a commercial kit and is

most often marketed for platelet cross-

matching.18 SPRCA testing may be modified

by treatment of target platelets with chloro-

quine or acid,46,47 which disrupts the Class I

HLA heavy chain-peptide-β2-microglobulin

trimolecular complex. This modifies anti-

genic epitopes, reducing the binding of

specific antibodies directed against HLA on

platelets. However, strong HLA antibodies

may still bind, giving the impression that

the antibody is directed to non-HLA anti-

gens.

Flow Cytometry. Another example of a

Phase II assay is platelet antibody detection

using immunofluorescence. Originally a

slide-based method,48 thetechniquenow

uses flow cytometry to detect platelet-reac-

tive antibody in patient sera that binds to

intact platelets.49 In the assay, washed plate-

lets are sensitized with patient or control

serum for up to 60 minutes, usually at room

temperature. The platelets are then washed

repeatedly to remove nonspecific immuno-

globulins, and platelet-bound antibodies

are detected with a fluorescent-labeled

(usually fluorescein isothiocyanate, FITC)

polyclonal or monoclonal antibody specific

for human immunoglobulin. The platelets

are analyzed in the flow cytometer and re-

sults can be expressed as a ratio of the

mean or peak channel fluorescence of nor-

mal platelets sensitized with patient serum

over that of normal platelets incubated in

normal serum. In order to prevent nonspe-

cific binding of the immunoglobulin probe

via Fc receptors on the target platelets, the

probe antibodies are enzyme treated to re-

move the Fc end of the molecule. There-

fore, binding of the labeled probe can be

assumed to be via its F(ab’)2or antigen-spe-

cific end. A second fluorescent label [eg,

phycoerythrin (PE)] can be attached to an

antihuman IgM probe to detect IgM plate-

let antibodies. Because FITC and PE fluo-

resce with peak light intensities at different

wavelengths when exposed to the mono-

chromatic argon laser in the flow cytometer

(520-nm green light and 580-nm reddish-

orange light, respectively), cells labeled

with FITC can be distinguished from those

labeled with PE. Both anti-IgG and anti-

IgM labeled with different fluorochromes

can be added to the same tube with washed

sensitized platelets for the simultaneous

detection of antiplatelet IgG and IgM.

Flow cytometry has proven to be a very

sensitive method for detection of alloanti-

bodies. The assay is capable of detecting

very small numbers of antibody molecules

bound to platelets as is the case with allo-

antibodies specific for antigens of the

HPA-5 (Br) system having only 1000 to 2000

sites per platelet. Moreover, some alloanti-

bodies that are specific for labile epitopes

that are unreliably detected in Phase III as-

says can be detected on intact platelets

using flow cytometry.

Because the target platelets used in the

assay are intact, flow cytometry does not

differentiate between platelet-specific (ie,

platelet glycoprotein directed) and non-

platelet-specific antibodies. Examples of

the latter are HLA and ABO antibodies. This

is an advantage when the method is used to

detect antibodies that will affect the success

of a platelet transfusion, and, for this rea-

son, flow cytometry has been advocated as

a platelet crossmatching method. However,

when used to investigate cases of suspected

NAIT or PTP, the method has a potential

drawback—the more relevant platelet-spe-

cific antibodies characteristic in these dis-

eases can be obscured by non-platelet-spe-

cific reactivity.

372 AABB Technical Manual

Monoclonal Antibody-Specific Immobi-

lization of Platelet Antigen (MAIPA). An ex-

ample of a Phase III assay is the MAIPA,50-52

perhaps the most widely used assay to de-

tect platelet-specific antibodies. The assay

requires the use of murine monoclonal an-

tibodies (MoAbs) that recognize the target

antigens of interest but do not compete

with the human antibody being detected.

In the assay, target platelets are simulta-

neously sensitized with patient serum and

a murine MoAb recognizing the desired tar-

get molecule on the platelet surface. After

the initial sensitization step, platelets are

washed and solubilized in a nonionic de-

tergent. After centrifugation to remove

cytoskeletal fragments, an aliquot of the

supernatant lysate is added to wells of a

microtiter tray containing immobilized

goat antibody specific for mouse IgG. The

MoAb is thereby captured and the platelet

surface glycoprotein with its bound human

antibody is immobilized. After a wash step,

the human antibody is detected with an

enzyme-labeled goat antihuman immuno-

globulin probe.

There are several other versions of Phase

III assays in use today, including the anti-

gen capture enzyme-linked immunosorbent

assay (ACE), the modified antigen capture

ELISA (MACE),53,54 and the commercially

available GTI PAKPLUS55 (GTI, Waukesha,

WI). Each relies on MoAbs to immobilize

only the glycoproteins of interest, thereby

reducing or eliminating interfering reactions

due to non-platelet-specific antibodies, es-

peciallyanti-HLA,which,ifpresent,isde

tected only in wells containing pools of im-

mobilized HLA Class I antigens.

Platelet Typing Using Molecular Meth-

ods. Molecular typing by polymerase chain

reaction (PCR) is available for many platelet

antigens. Because immunophenotyping is

limited by the shortage of characterized

typing antisera and by low platelet counts,

several DNA-based HPA typing techniques,

such as restriction fragment length poly-

morphism (RFLP) analysis and sequence-

specific oligonucleotide hybridization, have

been developed.56,57 All of these techniques

are reliable, but they are also laborious and

time-consuming. For this reason, PCR

genotyping with sequence-specific primers

(SSP) appears to be much more practical to

use.58,59 In a recent workshop, SSP-PCR was

the most common and reliable method of

determining platelet antigens,60 making it

feasible for genotyping HPAs independent

of the patient’s platelet count and of rare

typing sera.61

Autoimmune Platelet Disorders

Idiopathic (Autoimmune)

Thrombocytopenic Purpura

Autoantibodies directed against platelet

antigens may result in thrombocytopenia.

Chronic idiopathic thrombocytopenic

purpura (ITP), most often a disease in

adults, is characterized by an insidious

onset and moderate thrombocytopenia

that may exist for months to years before

diagnosis. Females are twice as likely to

be affected as males. Spontaneous remis-

sions are rare, and treatment is usually

required to raise the platelet count. First-

line therapy consists of steroids, high-

dose IGIV or Rh immunoglobulin (RhIG),

followed by splenectomy in nonresponders.

Many other therapies have been used in

patients who fail to respond to splenec-

tomy. Results have varied. Chronic auto-

immune thrombocytopenia may be idio-

pathic or associated with other diseases

(eg, HIV infection, malignancy, other au-

toimmune conditions). Acute ITP is mainly

a childhood disease characterized by abrupt

onset of severe thrombocytopenia and

bleeding symptomatology, often after a

viral infection. The majority of cases re-

solve spontaneously over a 2- to 6-month

Chapter 16: Platelet and Granulocyte Antigens and Antibodies 373

period. If treatment is required, IGIV or

RhIG infusions are usually effective in

raising the platelet count. Steroids are

used less often because of serious side ef-

fects in children. Splenectomy, if used, is

reserved for those children whose disease

is severe and lasts longer than 6 months,

similar to chronic ITP in adults.

Testing for Platelet Autoantibodies

Numerous Phase I, II, and III platelet anti-

body assays have been developed to de-

tect relevant autoantibodies in ITP pa-

tients. Although many tests have been

demonstrated to be quite sensitive, par-

ticularly in detecting total or cell surface

platelet-associated immunoglobulins (Phase

II assays),62 none has been sufficiently

specific to be particularly useful in either

the diagnosis or management of ITP. The

American Society of Hematology’s prac-

tice guidelines for ITP state that serologic

testing is unnecessary, assuming the clini-

cal findings are compatible with the diag-

nosis.63 However, platelet antibody tests

may be helpful in the evaluation of patients

suspected of having ITP when other, non-

immune causes may be present.

The goal of serologic testing in ITP is to

detect autoantibody bound to the patient’s

own platelets with or without demonstra-

tion of similar reactivity in the patient’s

plasma. Most of the newer assays offered

for evaluation of patients suspected of hav-

ing ITP are Phase III assays, designed to de-

tect immunoglobulin binding to platelet-

specific epitopes found on platelet glyco-

protein complexes GPIIb/IIIa, GPIa/IIa,

and/or GPIb/IX.

These solid-phase GP-specific assays ap-

pear to have improved specificity in distin-

guishing ITP from nonimmune thrombo-

cytopenia when compared to Phase II assays,

but this is often balanced by a decrease in

sensitivity.64 Moreover, all of these methods

have limited usefulness in patients who

have very low platelet counts that prevent

adequate numbers of platelets to be collected

for use in the tests.

One commercially available Phase III

test, the GTI PAKAUTO,65 uses eluates pre-

pared from washed patient platelets. The

eluates are tested against a panel of MoAb-

immobilized platelet GP complexes, and

antibody binding is detected using an en-

zyme-linked antihuman immunoglobulin

probe. In the indirect phase of the assay,

patient plasma is tested against the same

glycoprotein panel. In general, plasma anti-

bodies are detected less often than anti-

bodies in the eluates. ITP patients may

have antibodies that are reactive with one

or several GP targets. To date, there is no

correlation between the specificity of auto-

antibodies in ITP and disease severity.

Drug-Induced Immune Platelet Disorders

Thrombocytopenia associated with specific

drugs is not uncommon. Drugs often im-

plicated include quinidine/quinine, sulfa

drugs, heparin, and colloidal gold. Both

drug-dependent and drug-independent

antibodies may be produced. Drug-inde-

pendent antibodies, although stimulated

by drugs, do not require the continued

presence of the drug to react with plate-

lets and are serologically indistinguish-

able from other platelet autoantibodies.

(Unlike typical autoimmune thrombocyto-

penia, these antibodies are transient ex-

cept when caused by therapy with gold,

which is excreted very slowly.) Drug-de-

pendent antibodies result when a drug

combines with platelets in such a way as to

create neoantigens to which antibodies are

formed. The drug must be present for the

antibody to react. These antibodies can

cause a thrombocytopenia of sudden and

rapid onset, usually resolving when the

drug is discontinued.

374 AABB Technical Manual

Testing for Drug-Dependent Platelet-

Reactive Antibodies

Serology. Virtually any platelet serology

test that is used to detect platelet-bound

immunoglobulin can be modified for use

in the detection of drug-dependent pla-

telet-reactive antibodies. In performing

drug-dependent antibody testing, it is es-

sential to establish the proper positive

and negative controls for the assay. Each

serum or plasma sample suspected of con-

taining drug-dependent antibody must be

tested against normal target platelets in

thepresenceandabsenceofdrug.More

over, at least one normal serum should be

tested with and without drug to control

for any possible drug-related platelet ef-

fect that does not require specific anti-

body. Finally, a positive control serum

known to be reactive with the drug being

assayedshouldbetestedwithandwith-

out drug to complete the evaluation. A

positive result must show that the serum

is positive against normal target platelets

inthepresenceofdrugandnotwithout

drug, and that the drug did not non-

specifically cause a positive result in the

target platelet. Likewise, the positive con-

trol must be positive with the drug and

negative without it.

Flow Cytometry. The flow cytometry test

can be readily adapted to detect both IgG

and IgM drug-dependent platelet antibod-

ies.53,66 In this modification, fluorescence of

normal platelets sensitized with the pa-

tient’s serum in the presence of drug can be

compared with that of the patient’s sample

without drug or to a normal serum with

drug to determine relative intensity of la-

beling. Flow cytometry has proven to have

superior sensitivity to other assays for de-

tection of quinine-quinidine- and sulfona-

mide-dependent platelet-reactive antibod-

ies.53 Table 16-6 shows other agents for

which drug-dependent platelet-reactive an-

tibodies have been detected and confirmed

by flow cytometry in a large platelet immu-

nology reference laboratory.

Flow cytometry has its limitations, as do

other antibody detection methods, in de-

tecting drug-dependent antibodies. For

many drugs, the optimal concentration to

demonstrate in-vitro binding of antibody

has not been determined. Probably the

most extensively studied drugs in this re-

gard are quinine or quinidine.67 Another

cause of poor sensitivity is the weak bind-

ing of drug to platelets, leading to rapidly

declining numbers of drug molecules on

the platelet surface once drug is removed

from the environment of the platelet. It is

therefore important to maintain a critical

concentration of drug in all washing buffers

before addition of probe at the end of an

assay.66 Yet another potential reason for in-

sensitivity is that a patient may not be sen-

sitized to the native drug but, rather, to a

metabolite of the drug. Antibodies depend-

ent on metabolites of acetaminophen and

sulfamethoxazole have been reported.68,69

A number of other assays have been

adopted for the detection of drug-depend-

ent platelet antibodies. Among these are

the SPRCA.70,71 Phase I assays have also been

modified for detection of heparin-depend-

ent platelet antibodies (see below). In some

cases, determination of the specific glyco-

protein to which antibody is directed by

Phase III assays may provide useful clinical

information. For example, drug-dependent

antibody to GPIb/IX was associated with a

more acute, but reversible, quinine-in-

duced thrombocytopenia, whereas anti-

body to GPIIb/IIIa was associated with a more

prolonged course.72

Heparin-Induced Thrombocytopenia

Two types of heparin-induced thrombo-

cytopenia (HIT) have been recognized.

Type I, of nonimmune origin, presents with

Chapter 16: Platelet and Granulocyte Antigens and Antibodies 375

mild transient thrombocytopenia within

minutes to several days after heparin ex-

posure but generally resolves despite on-

going heparin therapy and is not clinically

important. In contrast, immune, or Type

II, HIT may lead to life- and limb-threat-

ening thrombotic complications and requires

careful evaluation and management of af-

flicted patients.

The exact incidence of immune HIT is

unknown,butitmaydevelopinupto3%of

patients treated with unfractionated hepa-

rin. Low-molecular-weight heparin is less

likely to be associated with either antibody

production or thrombocytopenia.73 Bovine

heparin appears somewhat more likely to

causeHITthanporcineheparin.

74 Areduc

tion in baseline platelet count by at least

50% occurs generally within 5 to 14 days af-

ter primary exposure and sooner after sec-

ondary exposure to the drug. The platelet

count is often less than 100,000/µLand

usually recovers within 5 to 7 days upon

discontinuation of heparin.

About 30% of patients with HIT, or ap-

proximately 0.9% of patients who receive

heparin, develop thrombosis, which can

occur in the arterial, venous, or both sys-

tems.75,76 Patients may develop cardiovascu-

lar problems, myocardial infarction, limb

ischemia, deep venous thrombosis, or

ischemia of other organs. The thrombotic

complications may force limb amputation

or may prove fatal.

Thrombosis or an unexplained decrease

in platelet count while on heparin therapy

should raise concern about HIT. Heparin,

including heparin flushes and heparin-

coated catheters, should be discontinued,

and the patient should be evaluated for

laboratory evidence of HIT and signs of

thrombosis. In mild-to-moderate thrombo-

cytopenia, monitoring of platelet counts

and observation may be sufficient, but be-

cause of the high risk of thrombosis, treat-

ment with alternative anticoagulants is

generally recommended. Warfarin should

be avoided in the early treatment of HIT

because it does not prevent thrombosis in

this setting and may provoke limb-threat-

ening venous gangrene by reducing levels

of naturally occurring anticoagulants faster

than it reduces activated coagulation fac-

tors. However, warfarin can and should be

used after the patient is anticoagulated

with alternative drugs. Suitable anticoagu-

lants approved by the Food and Drug Ad-

ministration (FDA) include hirudin (a natu-

ral thrombin inhibitor) and argatroban.

Selected patients may also benefit from

376 AABB Technical Manual

Table 16-6. Drugs Confirmed to Elicit Drug-Dependent Platelet-Reactive Antibodies

In Vitro Using Flow Cytometry Testing

abciximab

acetaminophen

carbamazepine

ceftazidime

ceftizoxime

ceftriaxone

ciprofloxacin

eptifibatide

esomeprazole

fentanyl

fexofenadine

heparin

ibuprofen

levofloxacin

loracarbef

naproxen

orbofiban

oxaliplatin

phenytoin

propoxyphene

quinidine

quinine

ranitidine

rifampin

sulfamethoxazole

sulfisoxazole

suramin

tirofiban

trimethoprim

vancomycin

xemilofiban

thrombolectomy or thrombolytic therapy.

Platelet transfusion should be avoided,

given that bleeding is a rare complication in

HIT, and administration of platelets may

precipitate thrombosis.77-79

Heparin forms a complex with platelet

factor 4 (PF4), a tetrameric protein released

from platelet alpha granules. Antibodies

(IgG, IgM, and some IgA) form against vari-

ous epitopes on this complex and attach to

platelet FcγIIa receptors, whereby platelets

become activated. The antibody may also

bind to the complexes at other sites, nota-

bly on endothelial cells. Thus, HIT might

involve activation and damage not only of

platelets but also of endothelium, causing

increased susceptibility to thrombosis. This

new understanding of the mechanism of

heparin antibodies is exploited by ELISA

tests in which microwells are coated with

the complexes rather than with the plate-

lets themselves.80

Testing for Heparin-Dependent Antibodies

The PF4 ELISA is an example of a Phase

III assay for HIT. Target complexes of PF4

and heparin or heparin-like molecules are

immobilized on a solid phase. To perform

the test, patient serum is added to premade

complexes of PF4 and heparin or hepa-

rin-like molecules (eg, polyvinyl sulfate,

PVS) alone and in the presence of high-

dose (100 U/mL) heparin. Heparin-de-

pendent antibody binds to the complexes

and is detected via enzyme-conjugated

antihuman immunoglobulin. An optical

density value above 0.4 in the PF4-PVS

well that is inhibited by high-dose hepa-

rin confirms the presence of a heparin-

dependent antibody in the patient’s sam-

ple. Although IgG antibodies are the most

clinically relevant antibodies causing this

syndrome,81 occasional patients with HIT

appear to have only non-IgG (IgM or IgA)

antibodies.82 The PF4 ELISA detects but

does not differentiate IgG, IgM, and IgA

antibodies that bind to the PF4-heparin

complex.

The 14C-serotonin release assay (SRA) is

an example of a Phase I assay for detection

of heparin-dependent antibodies.83 Normal,

fresh target platelets are incubated with

14C-serotonin that is taken up into the

dense granules of the platelets. Then, target

platelets are exposed to patient serum in

thepresenceoflowandhighconcentra

tions of heparin. Release of at least 20% of

the radioactive label at the low dose of hep-

arin and inhibition of this release at the

high dose confirms the presence of hepa-

rin-dependent antibodies. Other functional

tests used to detect heparin-dependent an-

tibodies include the heparin-induced

platelet aggregation test and the heparin-

induced platelet activation test.

ThePF4ELISAandtheSRAareboth

more sensitive and specific than the plate-

let aggregation test for the detection of hep-

arin-dependent platelet antibodies in pa-

tients for whom there is clinical suspicion

of HIT.83-85 However, in asymptomatic pa-

tients receiving heparin or in those who

have not yet received the drug, neither test

is sufficiently predictive of HIT to warrant

its use in screening.

Granulocyte Antigens

Analogous to platelet alloantigens, neu-

trophil alloantigens are implicated in

clinical syndromes including neonatal

alloimmune neutropenia (NAN), transfu-

sion-related acute lung injury (TRALI),

immune neutropenia after marrow trans-

plantation, refractoriness to granulocyte

transfusion, and chronic benign autoim-

mune neutropenia of infancy. A neutrophil

equivalent of PTP has not been described.

To date, seven neutrophil alloantigens have

been described, including localization to

Chapter 16: Platelet and Granulocyte Antigens and Antibodies 377

neutrophil surface glycoprotein structures

and, in some cases, determination of DNA

polymorphisms in genes encoding for them

(see Table 16-7).

The first granulocyte-specific antigen,

NA1 (HNA-1a), was described in 1966 by

Lalezari and Bernard.86 HNA-1a and its anti-

thetical antigen, HNA-1b, are present on

FcγRIIIb. Antibodies to HNA-1a and -1b

have been implicated in TRALI, NAN, and

autoimmune neutropenia of infancy. About

0.1% of individuals of European ethnicity

have neutrophils with no detectable

FcγRIIIb (NAnull). The FcγRIII protein also

carries the neutrophil alloantigen SH

(HNA-1c).87 NB1 (HNA-2a) is found on an-

other granulocyte surface glycoprotein,

CD177, the function of which is still unde-

termined. HNA-2a has been reported to

have an allele, NB2, but the product of this

gene cannot be reliably identified with

alloantisera, and no MoAb specific for NB2

has been identified; therefore, the existence

of a second allele to HNA-2a (NB1)isun

proven.88 HNA-2a has been associated with

TRALI and NAN. The DNA sequence of the

NB1(HNA-2a) gene has been determined as

have the molecular polymorphisms associ-

ated with HNA-1a and HNA-1b and HNA-1c

on the gene for FcγRIII. Therefore, genotyp-

ing for these specificities can be performed

on genomic DNA using PCR-SSP.89 Reduced

expression of granulocyte antigens occurs in

paroxysmal nocturnal hemoglobinuria,

chronic myelogenous leukemia, and in pre-

mature infants.

Additional antigens on granulocytes are

shared with other cells and are not granulo-

cyte-specific. These include 5b (HNA-3a),

MARTa(HNA-4a), and ONDa(HNA-5a). The

HNA-3a is located on a 70- to 95-Kd protein

that has not yet been cloned. HNA-3a is also

expressed on the surface of lymphocytes.

The antibodies directed at this antigen are

378 AABB Technical Manual

Table 16-7. Neutrophil Alloantigens

Antigen Frequency (%)

Antigen

System Allele

HNA

Designation White Black

Glycoprotein

Location

Neutrophil-specific

NA1

HNA-1a 46 46 FcγRIIIb (CD16)

NA2

HNA-1b 88 84 FcγRIIIb (CD16)

HNA-1c 5 22 FcγRIIIb (CD16)

NB1

HNA-2a 97 CD177

Neutrophil-nonspecific

HNA-3a 97 70-95 kD GP

MART

HNA-4a 99 CD11b

OND

HNA-5a 99 CD11a

usually agglutinins; they occasionally occur

in women after pregnancy and may be as-

sociated with febrile transfusion reactions.

Potent anti-HNA-3a agglutinins in trans-

fused plasma have been responsible for fa-

tal TRALI.90-92 MARTaand ONDa,bothhigh-

incidence antigens, are also present on

monocytes and lymphocytes. MARTahas

been localized to the alpha M chain

(CD11b) of the C3bi receptor (CR3) and re-

sults from a single nucleic acid substitu-

tion. MARTahas been recently reported to

cause NAN.93 ONDais expressed on the al-

pha L integrin unit, leukocyte function an-

tigen-1 (CD11a) and also results from a sin-

gle nucleotide substitution. This marker,

found in a chronically transfused aplastic

anemia patient, has not been reported to

be associated with clinical disease.

Clinical Syndromes in Which

Granulocyte-Specific Alloantigens Are

Implicated

Nonhemolytic, febrile transfusion reac-

tions are often associated with granulo-

cyte antibodies. Although such reactions

are more often caused by antibodies to

Class I HLA antibodies directed at Class I

epitopes present on granulocytes, granu-

locyte-specific antibodies have been asso-

ciated with clinical syndromes similar to

those seen with antibodies to red cell and

platelet antigens.

Neonatal Alloimmune Neutropenia

NAN is caused by maternal antibodies

against alloantigens of fetal neutrophils;

the most frequent specificities seen are

against NA1, NA2, and NB1 antigens (see

Table 16-7). NAN most often occurs in

women of the neutrophil alloantigen phe-

notypes NA1/NA1 and NA2/NA2; it may

also occur in women of the rare NAnull

phenotype who lack the FcγRIII protein.

The neutropenia in all these cases can oc-

casionally be life-threatening because of

increased susceptibility to infection. Man-

agement with antibiotics, IGIV, granulo-

cyte colony-stimulating growth factor,

and/or plasma exchange may be helpful.

TRALI

TRALI is an acute, often life-threatening

reaction characterized by respiratory dis-

tress, hypo- or hypertension, and non-

cardiogenic pulmonary edema that oc-

curs within 6 hours of a transfusion of a

plasma-containing blood component.

TRALI has been reported to be induced by

neutrophil antibodies, although more re-

cent reports are more likely to implicate

antibodies to both Class I and II HLA anti-

gens.94 In TRALI, the causative antibodies

are most often found in the plasma of the

blood donor (see Chapters 17 and 27). Re-

cent reports have postulated that another

etiology for TRALI is possible. This theory

suggests that two events must coincide

for TRALI to occur: 1) the patient must

have a predisposing clinical condition

that releases cytokines or other factors

that prime neutrophils, causing adher-

ence to endothelium, and 2) the patient

must receive a transfusion of biologically

active lipids (which also stimulate neutro-

phils) from stored blood components.95 In

one study, neutrophil antibodies were de-

tected in only three of 10 incidents of

TRALI, and none of the donors had HLA

antibodies.

Autoimmune neutropenia, usually oc-

curring in adults, may be idiopathic or may

occur secondary to such diseases as rheu-

matoid arthritis, systemic lupus erythe-

matosus, or bacterial infections. In autoim-

mune neutropenia of infancy, usually

occurring in children between the ages of 6

months to 2 years, the autoantibody has a

neutrophil antigen specificity (usually

HNA-1a and -1b) in about half the cases.

Chapter 16: Platelet and Granulocyte Antigens and Antibodies 379

The condition is usually self-limiting (with

recovery usually in 7-24 months) and the

condition is relatively benign and manage-

able with antibiotics.96 Drug-dependent an-

tibodies can also cause neutropenia.

Testing for Granulocyte Autoantibodies

Tests for granulocyte antibodies are not

widely performed, although the implica-

tion of neutrophil antibodies as a cause of

TRALI has increased the demand for this

laboratory resource. Agglutination tests

performed in tube, capillary, or micro-

plate formats use heat-inactivated serum

inthepresenceofEDTAandrequirefresh

granulocytes. Immunofluorescence tests,

read with either a fluorescence micro-

scope or a flow cytometer, are also used

and are capable of detecting granulocyte-

bound antiglobulin. A combination of

agglutination and immunofluorescence

testsisbeneficial.

97 Other methods include

chemiluminescence and a MoAb-specific

immobilization of granulocyte antigens

(MAIGA) assay, similar to the MAIPA as-

say. An advantage of the MAIGA assay is

its ability to differentiate readily between

HLA and granulocyte-specific antibodies.

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Chapter 16: Platelet and Granulocyte Antigens and Antibodies 383

Chapter 17: The HLA System

Chapter 17

The HLA System

THE HLA SYSTEM includes a com-

plex array of genes and their pro-

tein products. HLA antigens con-

tribute to the recognition of self and

nonself, to the immune responses to anti-

genic stimuli, and to the coordination of

cellular and humoral immunity. The HLA

genes, which are located in the major

histocompatibility complex (MHC) on the

short arm of chromosome 6, code for

glycoprotein molecules found on cell sur-

face membranes. Class I molecules are

found on the surface of platelets and of all

nucleated cells of the body. Mature red

cells usually lack HLA antigens demon-

strable by conventional methods, but nu-

cleated immature erythroid cells express

them. MHC Class II antigens are restricted

to a few cell types; the most important are

B lymphocytes, macrophages, and den-

dritic cells. Other terms that have been

applied to antigens of the HLA system are:

major histocompatibility locus antigens,

transplantation antigens, and tissue anti-

gens.

The HLA antigen molecules play a key

role in antigen presentation. Immunologic

recognition of differences in HLA antigens

is probably the first step in the rejection of

transplanted tissue. The HLA system is sec-

ond in importance only to the ABO anti-

gens in influencing the long-term survival

of transplanted solid organs and is of para-

mount significance in hematopoietic pro-

genitor cell (HPC) transplantation. HLA an-

tigens and antibodies are also important in

such complications of transfusion therapy

as platelet refractoriness, febrile non-

hemolytic transfusion reactions (FNHTRs),

transfusion-related acute lung injury (TRALI),

and posttransplant and posttransfusion

graft-vs-host disease (GVHD).

Studies correlating HLA polymorphisms

with susceptibility and disease resistance

began soon after serologic techniques for

HLA Class I typing were developed. Histori-

cally, HLA antigen typing has been of value

in parentage testing and in forensic investi-

gations. Molecular analysis of the HLA re-

gion permits selection of more closely

385

matched donors for HPC transplantation,

for investigation of disease associations,

and for anthropologic population studies.

Becauseofthepolymorphicnatureofthe

HLA genes, a complex nomenclature has

been developed to refer to the unique allele

sequences based on the relationship of the

allele to the serologic specificity of the cor-

responding antigen.1

Genetics of the Major

Histocompatibility Complex

Class I and II HLA antigens are cell sur-

face glycoproteins that are products of

closely linked genes mapped to the p21.3

band on the short arm of chromosome 6

(Fig 17-1). This genomic region is called

the MHC and is usually inherited en bloc

as a haplotype. Each of the several loci

has multiple alleles with codominant ex-

pression of the products from each chro-

mosome. The HLA system is the most

polymorphic system of genes described in

humans.

The HLA-A,HLA-B,andHLA-C genes

encode the corresponding Class I antigens

A, B, and C. The HLA-DR,HLA-DQ,and

HLA-DP gene cluster codes for the synthe-

sis of correspondingly named Class II anti-

gens. Located between the Class I and Class

II genes is a group of non-HLA genes that

code for molecules that include the com-

plement proteins C2, Bf, C4A, C4B; a ste-

roid enzyme (21-hydroxylase); and a

cytokine (tumor necrosis factor). This re-

gion is referred to as MHC Class III.

Organization of HLA Genetic Regions

The HLA Class I region contains, in addi-

tion to the classical genes HLA-A,HLA-B,

and HLA-C, other gene loci designated

HLA-E, HLA-F, HLA-G, HFE, HLA-J, HLA-K,

HLA-L, MICA,andMICAB. These latter

genes encode the nonclassical or Class Ib

HLA proteins, characterized by limited

polymorphism and low levels of expres-

386 AABB Technical Manual

Figure 17-1. (A) The major histocompatibility complex located on the short arm of chromosome 6. The

centromere is to the left. The key Class I, II, and III genetic loci are shown. The Class III region con-

tains complement system genes (C2, Bf, C4A, C4B), the 21-hydroxylase gene (21OH), and the gene for

tumor necrosis factor (TNF). (B) Greater detail of the Class II region.

sion.2Some Class I genes express non-

functional proteins or are not able to ex-

press a protein. Genes unable to express a

functional protein product are termed

pseudogenes and presumably represent

an evolutionary dead end. HLA-E regu-

lates natural killer cells. HLA-G is ex-

pressed by the trophoblast and may be in-

volved in the development of maternal

immune tolerance of the fetus. Hereditary

hemochromatosis (HH), an iron overload

disorder with a 10% carrier frequency in

Northern Europeans, is associated with

two missense mutations in a Class I-like

gene.3ThegeneconferringHHwasini

tially named HLA-H; however, the HLA-H

designation had already been assigned to

an HLA Class I pseudogene by the World

Health Organization (WHO) Nomencla-

ture Committee.4ThegeneconferringHH

is now called HFE. Class I molecules are

also located outside the MHC, such as

CD1, which can present nonprotein anti-

gens (such as lipids) to T cells.

The genomic organization of the MHC

Class II region (HLA-D region) is more

complex. An MHC Class II molecule con-

sists of a noncovalent complex of two struc-

turally similar chains, the α-chain and the

β-chain. Both of these chains are encoded

within the MHC. The polymorphism of

HLA Class II molecules results from differ-

ences in both the α-chain and the β-chain;

this depends on the Class II isoform. For

example, with HLA-DR, the α-chain is

monomorphic, but the β-chain is very poly-

morphic. Multiple loci code for either alpha

or beta chains of the Class II MHC proteins.

Different haplotypes have different num-

bers of Class II genes and pseudogenes.

The proteins coded by the DRA gene and

the DRB1 gene result in HLA-DR1 through

HLA-DR18. The products of the Agene and

the B3 gene (if present) express HLA-DR52;

those of the Agene and the B4 gene (if pres-

ent) express HLA-DR53; and those of the A

gene and B5 gene (if present) express

HLA-DR51. The HLA-DQ1 through DQ9

antigens are expressed on the glycoproteins

coded by the DQA1 and DQB1 genes in the

DQ gene cluster. Many of the other genes of

the DQ cluster are probably pseudogenes. A

similar organization is found in the HLA-

DP gene cluster.

The MHC Class III region contains four

complement genes, whose alleles are gen-

erally inherited together as a unit, termed a

complotype.Therearemorethan10differ

ent complotypes inherited in humans. Two

of the Class III genes, C4A and C4B,code

for variants of the C4 molecule. These vari-

ants have distinct protein structure and

function; the C4A molecule (if present) car-

ries the Rodgers antigen and the C4B mole-

cule (if present) carries the Chido antigen,

both of which are adsorbed onto the red

cells of individuals who possess the gene.

Patterns of Inheritance

Although the organization of the MHC is

complicated, its inheritance follows the

established principles of genetics. Every

person has two different copies of chro-

mosome 6 and, thus, possesses two HLA

haplotypes, one from each parent. An in-

dividual’s haplotype is typically determined

by typing multiple family members from

different generations and observing which

alleles are inherited together. The ex-

pressed gene products constitute the phe-

notype, which can be determined for an

individual by typing for the HLA antigens.

Because the HLA genes are autosomal

and codominant, the phenotype repre-

sents the combined expression of both

haplotypes. Figure 17-2 illustrates inheri-

tance of haplotypes.

Finding HLA-Identical Siblings

Each child inherits one copy of chromo-

some 6 from each parent; hence, one MHC

Chapter 17: The HLA System 387

haplotype is inherited from each parent.

Because each parent has two different

copies of chromosome 6, four different

combinations of haplotypes are possible

in the offspring (assuming no recombina-

tion). This inheritance pattern is impor-

tant in predicting whether family members

will be compatible donors for transplan-

tation. The chance that two siblings will

be HLA-identical is 25%. The chance that

any one patient with “n” siblings will have

at least one HLA-identical sibling is 1-(3/4)n.

Having two siblings provides a 44% chance

and three siblings a 58% chance that one

sibling will be HLA-identical. No matter

how many siblings are available for typing

(aside from identical twins), the probabil-

ity will never be 100% for finding an HLA-

identical sibling.

Absence of Antigens

Usually, both copies of the genes within

the MHC are expressed as antigens; how-

388 AABB Technical Manual

Figure 17-2. The linked genes on each chromosome constitute a haplotype. To identify which haplo-

types a person possesses, one must know the antigens present and also the inheritance pattern in the

specific kindred. The observed typing results of the father in this family are interpreted into the follow-

ing phenotype: A1,3;B7,8;Cw7,-;DR15,17. The observed results plus the family study reveal the

haplotypes of the father to be: a = A1,Cw7,B8,DR17 andb=A3,Cw7,B7,DR15.Offspringofasingle

mating pair must have one of only four possible combinations of haplotypes, assuming there has been

no crossing-over.

ever, in certain individuals, only one anti-

gen can be identified. This may occur if

the individual is homozygous for the al-

lele, or if appropriate antisera are not

available to type the individual’s antigen

(referred to as a blank allele). Very rarely,

the absence of an antigen can result from

a null allele. A null allele is characterized

by substitutions within the coding region

ofthegenethatpreventtheexpressionof

a functional protein at the cell surface. Such

inactivation of a gene may be caused by

nucleotide substitutions, deletions, or in-

sertions, which lead to a premature cessa-

tion in the antigen’s synthesis. When re-

ferring to phenotypes, a blank is often

written as “x” (for A locus), “y” (for B lo-

cus), or “–” (for any locus) (eg, A1,x;B7,40

or A1,–;B7,40). Family studies must be per-

formed to determine the correct genotype.

Crossing-Over

The genes of the HLA region occasionally

demonstrate chromosome crossover, in

which segments containing linked genetic

material are exchanged between the two

chromosomes during meiosis or gameto-

genesis (see Fig 10-6). These recombinants

are then transmitted as new haplotypes to

the offspring. Crossover frequency is in

part related to the physical distance be-

tween genes. For example, the HLA-A,

HLA-B,andHLA-DR loci are close to-

gether, with 0.8% crossover between the A

and Bloci and 0.5% between the Band

DR loci. In family studies and in parent-

age testing, the possibility of recombina-

tion must be considered.

Linkage Disequilibrium

The MHC system is so polymorphic that,

theoretically, the number of possible uni-

que HLA phenotypes is greater than the

global human population. Moreover, new

HLA alleles are constantly being discov-

ered and characterized. As of 2004, there

were 309 HLA-A, 563 HLA-B, and 368

DRB1 alleles. In reality, many HLA haplo-

types are overrepresented compared with

what would be expected if the distribution

of HLA genes were random. The phenom-

enon of linkage disequilibrium accounts

for the discrepancy between expected and

observed HLA haplotype frequencies.

Expected frequencies for HLA haplo-

types are derived by multiplication of the

frequencies of each allele. For example, in

individuals of European ancestry, the over-

all frequency of the gene coding for HLA-A1

is 0.15 and that for HLA-B8 is 0.10; there-

fore, 1.5% (0.15 ×0.10) of all HLA haplo-

types in this population would be expected

to contain genes coding for both HLA-A1

and HLA-B8 if they were randomly distrib-

uted. The actual frequency of the A1 and B8

combination, however, is 7% to 8% in this

population. Certain allelic combinations

occur with increased frequency in different

racial groups and constitute common haplo-

types in those populations. These are called

ancestral haplotypes because they appear

to be inherited from a single common an-

cestor. The most common ancestral haplo-

type in Northern Europeans, the A1, B8,

DR3, DQ2 haplotype, includes both Class I

and Class II regions. It is unclear whether

ancestral haplotypes represent relatively

young haplotypes that have not had suffi-

cient time to undergo recombination, or

whether they are old haplotypes that are re-

sistant to recombination because of selec-

tion. Linkage disequilibrium in the HLA

system is important in studies of parent-

age because haplotype frequencies in the

relevant population make the transmis-

sion of certain gene combinations more

likely than others. Linkage disequilibrium

also affects the likelihood of finding suit-

able unrelated donors for HLA-matched

platelet transfusions and for HPC trans-

plantation.

Chapter 17: The HLA System 389

Biochemistry, Tissue

Distribution, and Structure

The HLA antigens are cell surface glyco-

proteins. HLA Class I molecules contain

one copy of two polypeptides: a heavy

chain, which is attached to the mem-

brane, and a light chain, which is called

β2-microglobulin. The HLA Class II mole-

cules are composed of one copy each of

an α-chain and a β-chain, both of which

are attached to the cell surface. HLA anti-

gens are divided into Class I and Class II

based on their function, tissue distribu-

tion, and biochemistry.

Characteristics of Class I and Class II

Antigens

Class I antigens (HLA-A, -B, and -C) have

a molecular weight around 57,000 daltons

and consist of two chains: a glycoprotein

heavy chain (45,000 daltons) encoded on

the short arm of chromosome 6 and, as a

light chain, the β2-microglobulin molecule

(12,000 daltons) encoded by a gene on

chromosome 15. The heavy chain pene-

trates the cell membrane. β2-microglobu-

lin is not attached to the cell membrane;

it associates with the heavy chain via the

latter’s nonvariable (α3) domain but is not

covalently bound to it (see Fig 17-3). The

external portion of the heavy chain con-

sists of three amino acid domains (α1, α2,

and α3), of which the outermost α1and

α2 domains contain the polymorphic re-

gions conferring the HLA antigen speci-

ficity.

Class I molecules are found on platelets

and on most nucleated cells in the body,

with some exceptions such as neurons, cor-

390 AABB Technical Manual

Figure 17-3. Stylized diagram of Class I and Class II MHC molecules showing αand βpolypeptide

chains, their structural domains, and attached carbohydrate units.

neal epithelium, trophoblast, and germinal

cells. Only vestigial amounts remain on

mature red cells, with certain allotypes

better expressed than others. These Class I

polymorphisms were independently recog-

nized as red cell alloantigens by serologists

and were designated as Bennett-Good-

speed (Bg) antigens. The specificities called

Bga,Bg

b,andBg

care identified as HLA-B7,

HLA-B17, and HLA-A28, respectively. Plate-

lets express primarily HLA-A and HLA-B

antigens. HLA-C antigens are present at

very low levels and Class II antigens are

generally not expressed at all on platelets.

ClassIIantigens(HLA-DR,-DQ,and

-DP) have a molecular weight of approxi-

mately 63,000 daltons and consist of two

structurally similar glycoprotein chains (α

and β), both of which traverse the mem-

brane (see Fig 17-3). The extramembranous

portion of each chain has two amino acid

domains, of which the outermost domain

contains the variable regions of the Class II

alleles. The expression of Class II antigens

is more restricted than that of Class I. Class

II antigens are expressed constitutively on

B lymphocytes, monocytes, and cells de-

rived from monocytes such as macro-

phages and dendritic cells, intestinal epi-

thelium, and early hematopoietic cells.

There is also constitutive expression of

Class II antigens on some endothelial cells,

especially those lining the microvasculature.

However, in general, endothelium, particu-

larly that of larger blood vessels, is negative

for Class II antigen expression, although its

presence can be induced (for instance, by

interferon-gamma during immune activa-

tion). T lymphocytes are negative for Class

II antigen expression but become positive

when activated. Class II antigens are ex-

pressed abnormally in autoimmune dis-

ease and on some tumor cells.

Soluble HLA Class I and Class II antigens

shed from cells are found in blood and

body fluids and may play a role in modulat-

ing immune reactivity.5Levels of soluble

HLA increase with infection [including hu-

man immunodeficiency virus (HIV)], in-

flammatory disease, and transplant rejec-

tion, and decline with progression of

malignancy. Levels of soluble HLA in blood

components are proportionate to the num-

ber of residual donor leukocytes and to the

length of storage.6Soluble HLA in blood

components may be involved in the im-

munomodulatory effect of blood transfu-

sion.

Configuration

A representative three-dimensional struc-

ture of these molecules can be obtained

by X-ray crystallographic analysis of puri-

fied HLA antigens. The outer domains,

which contain the regions of amino acid

variability and the antigenic epitopes of

the molecules, form a structure known as

the “peptide-binding groove.” Alleles de-

fined by polymorphisms in the HLA gene

sequences have unique amino acid se-

quences and, therefore, form unique

binding grooves, each able to bind differ-

ent classes of peptides. The peptide-bind-

inggrooveiscriticalforthefunctionalas

pects of HLA molecules (see section on

Biologic Function).

Nomenclature

An international committee sponsored by

the World Health Organization estab-

lishes the nomenclature of the HLA sys-

tem. It is updated regularly to incorporate

new HLA alleles. HLA antigens are desig-

nated by a number following the letter

that denotes the HLA series (eg, HLA-A1

or HLA-B8). Previously, antigenic speci-

ficities that were not fully confirmed car-

ried the prefix “w” (eg, HLA-Aw33). When

identification of the antigen became de-

finitive,theWHONomenclatureCommit

Chapter 17: The HLA System 391

tee dropped the “w” from the designation.

The Committee meets regularly to update

nomenclature by recognizing new speci-

ficities or genetic loci. The “w” prefix is no

longer applied in this manner and is now

used only for the following: 1) Bw4 and

Bw6, to distinguish these “public” antigens

from other B locus alleles; 2) all C locus

specificities, to avoid confusion with

members of the complement system; and

3) Dw and DP specificities that were de-

fined by mixed lymphocyte reactions and

primed lymphocyte typing. The numeric

designations for the HLA-A and HLA-B

specificities were assigned based on the

order of their discovery.

Splits

Refinement of serologic methods permit-

ted antigens previously believed to repre-

sent a single specificity to be “split” into

specificities that were serologically (and,

later, genetically) distinct. The designa-

tion for an individual antigen that is a

split of an earlier recognized antigen of-

ten includes the number of the parent an-

tigen in parentheses, eg, HLA-B44 (12).

Shared Determinants

As the specificity of HLA typing sera im-

proved, it was found that some epitopes,

the part of the antigen that binds anti-

body, were common to several antigens.

Antibodies to these epitopes identified

antigens that constituted a group, of which

the individual members could be identi-

fied by antisera with more restricted ac-

tivity.

Cross-Reactive Groups

In addition to “splits,” HLA antigens and

antigen groups may have other epitopes

in common. Antibodies that react with

these shared determinants often cause

cross-reactions in serologic testing. The

collective term for a group of HLA anti-

gens that exhibit such cross-reactivity is

cross-reactive group (CREG).

“Public” Antigens

In addition to splits and CREGs, HLA pro-

teins have reactivity that is common to

many different HLA specificities. Called

“public” antigens, these common amino

acid sequences appear to represent the

less variable portion of the HLA molecule.

Two well-characterized “public” antigens,

HLA-Bw4 and HLA-Bw6, are found in the

HLA-B series. The Bw4 antigen is also

found on some A locus molecules. “Pub-

lic” antigens are clinically important be-

cause patients exposed to foreign HLA

antigens via pregnancy, transfusion, or

transplantation can make antibodies to

them. A single antibody, when directed

against a “public” antigen, can resemble

the sum of multiple discrete alloanti-

bodies.

Nomenclature for HLA Alleles

As nucleotide sequencing is used to in-

vestigate the HLA system, increasing

numbers of HLA alleles are being identi-

fied, many of which share a common se-

rologic phenotype. The minimum re-

quirement for designation of a new allele

is the sequence of exons two and three for

HLA Class I and exon two for HLA Class II

(DRB1). These exons encode the variable

amino acids that confer HLA antigen spe-

cificity. A uniform nomenclature has been

adopted that takes into account the locus,

the major serologic specificity, and the al-

lele determined by molecular typing tech-

niques. For example, isoelectric focusing,

amino acid sequencing, and nucleotide

sequencing have identified several unique

variants of HLA-DR4. The first HLA-DR4

variant is designated DRB1*0401,indicat

392 AABB Technical Manual

ing the locus (DR), the protein (β1chain),

an asterisk to represent that an allele

name follows, the major serologic speci-

ficity (04 for HLA-DR4), and the allele

number (variant 01). For Class I alleles, the

name of the locus, for example HLA-B, is

followed by an asterisk and then a num-

ber of digits. The first two digits corre-

spond to the serologic specificity of the

antigen. The third and fourth digits are

used to list the subtypes, numbers being

assigned in the order in which the DNA

sequences have been determined. There-

fore, B*2704 represents the HLA-B locus,

with a serologic specificity of B27, and

was the fourth allele described in this

family (see Table 17-1). Finally, the no-

menclature can accommodate alleles

with silent mutations, ie, those that have

different DNA sequences but identical

amino acid sequences and null alleles.

Biologic Function

The essential function of the HLA system

is self/nonself discrimination. Discrimina-

tion of self from nonself is accomplished

by the interaction of T lymphocytes with

peptide antigens. T lymphocytes interact

with peptide antigen only when the T-cell

receptor (TCR) for antigen engages both

an HLA molecule and the antigenic pep-

tide contained with its peptide-binding

groove. This limitation is referred to as

“MHC restriction.”7

In the thymus, T lymphocytes whose

TCRs bind to a self HLA molecule are se-

lected (positive selection), with the excep-

tion of those whose TCRs also bind to a

peptide derived from a self antigen, in

which case they are deleted (negative selec-

tion). Some self-reactive T cells escape neg-

ative selection, however. If not functionally

inactivated, for instance, by the mechanism

of anergy, these self-reactive T cells may be-

come involved in an autoimmune process.

(See Chapter 11.)

Role of Class I

ClassImoleculesaresynthesized,and

peptide antigens are inserted into the

peptide-binding groove, in the endo-

plasmic reticulum. Peptide antigens that

fit into the Class I peptide-binding groove

are typically eight or nine amino acids in

lengthandarederivedfromproteinsthat

Chapter 17: The HLA System 393

Table 17-1. HLA Nomenclature

Genetic

Locus

Antigenic

Specificity Allele

Number of

Identified

Alleles

Polypeptide

Location

HLA-A

HLA-B

HLA-C

DRA

DRB1

DQA1

DQB1

DPA1

DPB1

A1 to A80

B7 to B81

Cw1toCw10

DR1 to DR18

DQ1 to DQ9

DPw1 to DPw6

A*0101 to *8001

B*0702 to *8301

Bw*0102 to *1802

DRA*0101 to *0102

DRB1*0101 to *1608

DQA1*0101 to *0601

DQB1*0501 to *0402

DPA1*0103 to *0401

DPB1*0101 to *8901

207

412

100

271

β1

are made by the cell (endogenous pro-

teins). These endogenous proteins, which

may be normal self proteins, altered self

proteins such as those found in cancer

cells, or viral proteins such as those found

in virus-infected cells, are degraded in the

cytosol by a large multifunctional prote-

ase (LMP) and transported to the endo-

plasmic reticulum by a transporter associ-

ated with antigen processing (TAP). The

LMP and TAP genes are both localized to

the MHC.

ClassImoleculesaretransportedtothe

cell surface where they are available to in-

teract with CD8-positive T lymphocytes. If

the TCR of a T lymphocyte can bind the an-

tigenic peptide in the context of the specific

Class I molecule displaying it, then this

binding activates the cytotoxic properties of

the T cell, which will then attack the cell,

characteristically eliciting an inflammatory

response. The presentation of antigen by

ClassImoleculesisespeciallyimportantin

a host’s defense against viral pathogens and

against malignant transformation. Tumor

cells that do not express Class I escape this

immune surveillance.

Role of Class II

Class II molecules, like Class I molecules,

are synthesized in the endoplasmic retic-

ulum, but peptide antigens are not in-

serted into the peptide-binding groove

here. Instead, an invariant chain (Ii) is in-

serted. The Class II-invariant chain mole-

cule is transported to an endosome where

the invariant chain is removed by a spe-

cialized Class II molecule called DM

(whose locus is also localized to the MHC).

A Class II antigenic peptide is then in-

serted into the peptide-binding groove.

Peptide antigens that fit into the Class II

peptide-binding groove are typically 12 to

25 amino acids in length and are derived

from proteins that are taken up by the cell

by endocytosis (exogenous proteins). Ex-

ogenous proteins, which may be normal

self proteins or proteins derived from

pathogens such as bacteria, are degraded

to peptides by enzymes in the endosomal

pathway. Class II molecules are then

transported to the cell surface where they

are available to interact with CD4-positive

T lymphocytes, which secrete immuno-

stimulatory cytokines in response. This

mechanism is especially important for the

production of antibodies.

Detection of HLA Antigens

and Alleles

Methods for the detection of HLA antigens

and alleles fall into three groups: molecu-

lar (DNA-based), serologic, and cellular

assays. Detailed procedures of commonly

used assays are provided in the current

edition of the American Society for Histo-

compatibility and Immunogenetics Labo-

ratory Manual. Depending on the clinical

situation, a particular HLA antigen detec-

tion or typing method may be preferable

(Table 17-2).

DNA-Based Assays

DNA-based typing has several advantages

over serologic and cellular assays: high

sensitivity and specificity; small sample

volumes; decreased turnaround time, for

some methods, as short as a few hours;

and absence of the need for cell surface

antigen expression or cell viability. Al-

though serologic methods can readily dis-

tinguish only about 21 serologic specifi-

cities, high-resolution DNA-based methods

can detect up to 368 alleles.

Polymerase Chain Reaction Testing

Polymerase chain reaction (PCR) technol-

ogy allows amplification of large quanti-

394 AABB Technical Manual

ties of a particular target segment of geno-

mic DNA. Low- to intermediate-resolution

typing detects the HLA serologic equiva-

lents with great accuracy; eg, it distin-

guishes DR15 from DR16, whereas high-

resolution typing distinguishes individual

alleles, eg, DRB1*0101 from DRB1*0102.

Several PCR-based methods have been

developed, of which two general approaches

are described below.

Oligonucleotide Probes. The first tech-

nique uses sequence-specific oligonucleo-

tide probes (SSOPs) and is known as PCR-

SSO, PCR-SSOP, or allele-specific oligo-

nucleotide (ASO) hybridization.8APCR

product amplified from genomic DNA is

applied to a membrane or filter to which

the labeled SSOPs are hybridized. These

short DNA probes will hybridize with the

complementary sequences and identify

groups of alleles or individual alleles. Ad-

vantages are that all Class II loci can be

typed and highly specific information ob-

tained. Disadvantages include potential

difficulty in interpretation of results and the

need to use multiple filters and perform

multiple amplifications and subsequent

hybridizations. A variation of this tech-

nique, the reverse line or dot blot, elimi-

nates the need for multiple filters and hy-

bridizations by incorporation of a label

(such as biotin) into the PCR product dur-

ing its amplification from genomic DNA.

The PCR product is then hybridized to a

membrane containing all the relevant

SSOPs and its pattern of hybridization with

the SSOPs revealed by detection of the in-

corporated label. In the past few years, a

new method for establishing HLA geno-

types that features arrays of oligonucleotide

probes on a solid phase has begun to ap-

pear in HLA typing laboratories. The

microbead array assay is an SSOP method

for HLA-A, -B, and -DR antigen level typing.

Chapter 17: The HLA System 395

Table 17-2. HLA Typing Methods and Appropriate Applications

Method Clinical Application Resolution

SSP (PCR) Solid organ, related and unrelated HPC

transplantation

Serologic to allele level,

higher resolution with

large number of primers

DNA sequencing Unrelated HPC transplantation, resolu-

tion of typing problems with other

methods, characterization of new

alleles

Allele level

Forward SSOP

hybridization

Solid organ and HPC transplantation

(can accommodate high-volume

testing)

Serologic to allele level

Reverse SSOP

hybridization

Solid organ, related and unrelated HPC

transplantation

Serologic, higher resolution

with larger number of

probes

Microlympho-

cytotoxicity

Solid organ transplantation, evaluation

of platelet refractoriness, HLA typing

(Class I only) of platelet recipients

and platelet donors

Serologic specificity

This technique also has the ability for low-

to-intermediate resolution DNA-based tis-

sue typing, with a reduction in sample pro-

cessing time.

Sequence-Specific Primers. A second

major technique uses sequence-specific

primer pairs (SSPs) that target and amplify

a particular DNA sequence.9This method

requires the performance of multiple PCR

reactions in which each reaction is specific

for a particular allele or group of alleles. Di-

rect visualization of the amplified alleles is

seen after agarose gel electrophoresis. Be-

cause SSPs have such specific targets, pres-

ence of the amplified material indicates

presence of the corresponding allele(s). The

pattern of positive and negative PCR ampli-

fications is examined to determine the HLA

alleles present. Primer pair sets are avail-

able that can determine the full HLA-A, -B,

-C,-DR,-DQ,and-DPtype.

Sequence-Based Typing

High-resolution nucleic acid sequencing

of HLA alleles generates allele-level se-

quences that are used to characterize new

allele(s). With the ever-increasing avail-

ability and ease of use of automated se-

quencers, sequence-based typing has be-

come a routine HLA typing method in

some HLA laboratories.

Serologic Assays

Lymphocytotoxicity

The microlymphocytotoxicity test can be

used to detect HLA-A, -B, -C, -DR, and

-DQ antigens. Lymphocytes are used for

testing because they are readily obtained

from anticoagulated peripheral blood

and, unlike granulocytes, give reproduc-

ible results. Lymphocytes obtained from

lymph nodes or spleen may also be used.

HLA typing sera are obtained primarily

from multiparous women. Some mouse

monoclonal antisera are also available.

HLA sera of known specificities are

placed in wells of a microdroplet test plate.

A suspension of lymphocytes is added to

each well. Rabbit complement is then

added and, if sufficient antibody has bound

to the lymphocyte membranes, the com-

plement cascade will be activated through

the membrane attack complex, leading to

lymphocytotoxicity. Damage to the cell

membrane can be detected by the addition

of dye: cells that have no attached antibody,

no activated complement, and no damage

to the membrane keep the vital dyes from

penetrating; cells with damaged mem-

branes allow the dye to enter. The cells are

examined for dye exclusion or uptake un-

der phase contrast microscopy. If a fluores-

cent microscope is available, fluorescent vi-

tal dyes can also be used.

Because HLA-DR and HLA-DQ antigens

are expressed on B cells and not on resting

T cells, typing for these antigens usually re-

quires that the initial lymphocyte prepara-

tion be manipulated before testing to yield

an enriched B-cell population. This is typi-

cally accomplished by the use of magnetic

beads, to which monoclonal antibodies to

B cells have been bound.

The interpretation of serologic reactions

requires skill and experience. Control wells

of known reactivity and careful quality con-

trol of reagents are required, especially for

the activity of the complement used to in-

duce lymphocytotoxicity. In addition, anti-

gen assignments can be made only on the

basis of results obtained with multiple anti-

sera because few reagent antisera have suf-

ficient monospecific reliability to be used

alone. The extreme polymorphism of the

HLA system, the variation in antigen fre-

quencies among different racial groups, the

reliance on biologic antisera and living tar-

get cells, and the complexities introduced

by splits, CREGs, and “public” antigens all

contribute to difficulties in accurate sero-

logic HLA typing.

396 AABB Technical Manual

Antibodies in Patients

Microlymphocytotoxicity testing can be

used to test serum specimens against se-

lected target cells. This is routinely done

in HLA crossmatching, which consists of

testing serum from a potential recipient

against unfractionated lymphocytes (or

fractionated T and B lymphocytes) from a

potential donor. A variation of the micro-

lymphocytotoxicity test, which uses an anti-

globulin reagent, is one of the methods

used to increase sensitivity. Flow cyto-

metryisalsousedasanindependent

method to increase the sensitivity of the

crossmatch.

Testing the patient’s serum against a

panelof30to60ormoredifferenttarget

cells can assess the extent of HLA allo-

immunization. The percent of the panel

cells to which the recipient has formed

cytotoxicantibodiesisreferredtoasthe

panel reactive antibody (PRA) level. Deter-

mination of PRA can be useful in the inves-

tigation of FNHTRs, in the workup of

platelet refractoriness, and in following pa-

tients who are awaiting cadaver solid organ

transplants. This “HLA antibody screen” not

only detects the presence of HLA antibod-

ies but also may allow their specificity to be

determined. The presence of HLA antibod-

ies can also be demonstrated by using an

enzyme-linked immunosorbent assay with

solid-phase HLA antigens or by flow cyto-

metric analysis using antigen-coated beads.

Cellular Assays

Historically, the mixed lymphocyte cul-

ture (MLC) (also called mixed leukocyte

culture, mixed lymphocyte reaction, or

MLR) was used to detect genetic differ-

ences in the Class II region. In the MLR,

lymphocytes from different individuals

are cultured together and have the oppor-

tunity to recognize foreign HLA-D region

antigens and to respond by proliferating.

The HLA System and

Transfusion

HLA system antigens and antibodies play

important roles in a number of transfu-

sion-related events. They include allo-

immunization and platelet refractoriness,

FNHTR, TRALI, and posttransfusion GVHD.

HLA antigens are highly immunogenic. In

response to pregnancy, transfusion, or

transplantation, immunologically normal

individuals are more likely to form anti-

bodies to HLA antigens than to any other

antigen system.

Platelet Refractoriness

The incidence of HLA alloimmunization

and platelet refractoriness among pa-

tients receiving repeated transfusions of

cellular components is 20% to 71%.10 The

refractory state exists when transfusion of

suitably preserved platelets fails to in-

crease the recipient’s platelet count. Pla-

telet refractoriness may be due to clinical

factors such as sepsis, high fever, dissemi-

nated intravascular coagulopathy, medi-

cations, hypersplenism, complement-me-

diated destruction, or a combination of

these, or it may have an immune basis.

(See Chapter 16 for more information

about platelets.)

Antibody Development

Antibodies against HLA antigens usually

cause immune-mediated platelet refrac-

toriness, but antibodies to platelet-spe-

cific or ABH antigens may also be in-

volved. HLA alloimmunization can follow

pregnancy, transfusion, or organ trans-

plantation because the foreign antigens

are the donor MHC antigens themselves.

A common example of this is the develop-

ment of HLA antibodies directed against

Class I antigens that occurs with transfu-

sion of platelets, which express only Class

Chapter 17: The HLA System 397

I antigens. The presence, in the trans-

fused component, of leukocytes bearing

Class I and II antigens elicits alloimmuni-

zation. The likelihood of immunization

can be lessened with leukocyte-reduced

blood components, or by treatment with

ultraviolet light, which alters the co-

stimulatory molecules or impairs anti-

gen-presenting cell activity. The threshold

level of leukocytes required to provoke a

primary HLA alloimmune response is un-

clear and probably varies among different

recipients. Some studies have suggested

that 5 ×106leukocytes per transfusion

may represent an immunizing dose. In

patients who have been previously sensi-

tized by pregnancy or transfusion, expo-

sure to even lower numbers of allogeneic

cells is likely to provoke an anamnestic

antibody response.

Finding Compatible Donors

The HLA antibody response of transfused

individuals may be directed against indi-

vidual specificities present on donor cells

or against “public” alloantigens. Precise

characterization may be difficult. An over-

all assessment of the degree of HLA allo-

immunization can be obtained by mea-

suring the PRA of the recipient’s serum.

Platelet-refractory patients with a high

PRA are broadly alloimmunized and may

be difficult to support with platelet trans-

fusions. HLA-matched platelets, obtained

by plateletpheresis, benefit some, but not

all, of these refractory patients. Because

donors with a four-antigen match for an

immunized recipient are hard to find, strat-

egies for obtaining HLA-matched platelets

vary. Selection of partially mismatched

donors, based on serologic cross-reactive

groups, has been emphasized, but such

donors may fail to provide an adequate

transfusionresponseinvivo.Analterna

tive approach to the selection of donors is

based on matching for “public” speci-

ficities rather than cross-reactive private

antigens. Obtaining an adequate number

of readily available HLA-typed donors can

prove difficult; it has been estimated that

a pool of 1000 to 3000 or more donors

would be needed to provide the transfu-

sion requirements of most HLA-allo-

immunized patients.11 Useofsingleanti

gen beads to identify HLA antibody

specificities precisely can allow a better

selection of donors who have acceptable

mismatched antigens.12

In the past, it was recommended that

patients who were at risk of becoming

alloimmunized and refractory be serologi-

cally Class I HLA-typed early in the course

of their illness, when enough lymphocytes

were present in the peripheral blood to ob-

tain a reliable HLA type. Intensive chemo-

therapy makes it very difficult to obtain

enough cells for such typing. More recently,

with the advent of molecular typing tech-

niques, a patient’s HLA alleles can be deter-

mined using genomic DNA isolated from

very small numbers of white cells or even

nonblood tissue (eg, buccal swabs).

HLA-alloimmunized patients often re-

spond to crossmatch-compatible platelets

selected using patient serum and samples

of apheresis platelets in a platelet antibody

assay. Crossmatching techniques may as-

sess compatibility for both HLA and plate-

let-specific antibodies.13 These histocom-

patible platelet components are further

discussed in Chapter 21.

Febrile Nonhemolytic Transfusion

Reactions

HLA antibodies, as well as granulocyte

and platelet-specific antibodies, have

been implicated in the pathogenesis of

FNHTRs. The recipient’s antibodies, re-

acting with transfused antigens, elicit the

release of cytokines (eg, interleukin-1) ca-

398 AABB Technical Manual

pableofcausingfever.Serologicinvesti

gation, if undertaken, may require multi-

ple techniques and target cells from a

number of different donors (see Chapter

27).

Transfusion-Related Acute Lung Injury

In TRALI, a transfusion reaction that is

being recognized with increasing fre-

quency, acute noncardiogenic pulmonary

edema develops in response to transfusion.

Pathogenesis appears to reflect the pres-

ence of HLA antibodies in donor blood,

which react with and fix complement to

granulocytes of the recipient, leading to

severe capillary leakage and pulmonary

edema. Rarely, HLA antibodies of the re-

cipient react with transfused leukocytes

from the donor (see Chapter 27). Cases of

TRALI have been reported that appear to

be caused by donor antibodies against

Class II antigens in recipients. Because

ClassIIantigensarenotexpressedon

neutrophils, an alternate explanation for

activation of neutrophils in these instances

is required. One hypothesis is that Class II

antigens on the recipient’s pulmonary

macrophages are targeted by these com-

plement-activating antibodies. Subse-

quent release of cytokines and chemokines

results in the recruitment and activation

of neutrophils in the lungs.14

Chimerism and Posttransfusion

Graft-vs-Host Disease

Chimerism refers to the presence of do-

nor cells in the recipient. Persistent chi-

merism after blood transfusion may lead

to the development of GVHD in the recip-

ient. The development of posttransfusion

GVHD depends on several factors: the de-

gree to which the recipient is immuno-

compromised; the number and viability

of lymphocytes in the transfused compo-

nent; and the degree of HLA similarity be-

tween donor and recipient. The observa-

tion of posttransfusion GVHD with the use

of fresh blood components from blood

relatives has highlighted the role of the

HLA system in GVHD.

Figure 17-4 illustrates the conditions for

increased risk of GVHD. The parents have

one HLA haplotype in common. Each child,

therefore, has a one in four chance of inher-

iting the same haplotype from each parent,

and Child #1 is homozygous for the shared

parental HLA haplotype. Transfusion of

blood from this person to an unrelated re-

cipient who did not have this haplotype

would have no untoward consequences. If,

however, Child #1 were a directed donor for

the relatives heterozygous for that haplo-

type (both parents and Child #3), the recipi-

ent would not recognize any foreign anti-

gens on the transfused lymphocytes and

would not eliminate them. The donor cells,

however, would recognize the recipient’s

foreign HLA antigens, would become acti-

vated, proliferate, and attack the host. To

avoid this situation, it is recommended that

all cellular components known to be from

blood relatives be irradiated before transfu-

sion. Other specially chosen donor units,

such as HLA-matched platelets, may also

present an increased risk of posttransfusion

GVHD. Rarely, transfusion-associated GVHD

has occurred after the transfusion of blood

from an unrelated donor.15

Chimerism is also proposed to be re-

sponsible for the maintenance of tolerance

in some organ transplant recipients16 and

for the maintenance of HLA sensitization.17

It has been postulated that scleroderma is a

form of GVHD resulting from chimeric cells

derived from fetal cells transferred across

the placenta during pregnancy.18

Hemolytic Transfusion Reactions

HLA incompatibility has rarely been im-

plicated as a cause of shortened red cell

Chapter 17: The HLA System 399

survival in patients with antibodies to HLA

antigens such as Bga(B7), Bgb(B17), and

Bgc(A28) that are expressed, although

weakly, on red cells (see Chapter 15). Such

an incompatibility may not be detected

with conventional pretransfusion testing.

HLA Testing and

Transplantation

HLA testing is an integral part of organ

transplantation. The extent of testing dif-

fers for different types of transplants. Or-

gan transplantation is discussed in great-

er detail in Chapter 26.

Hematopoietic Progenitor Cell Transplants

It has long been recognized that disparity

within the HLA system represents an im-

portant barrier to successful HPC trans-

plantation.19 HLA similarity and compati-

bility between the donor and the recipient

are required for engraftment and to pre-

vent GVHD, but some degree of rejection

or GVHD remain common problems for

recipients of allogeneic HPCs, despite

immunosuppressive conditioning.

Candidate donors and recipients are

typed for their HLA-A, -B, -C, -DR and -DQ

alleles. The goal is to match, as closely as

possible, the alleles of the prospective do-

nor and recipient at the HLA-A,-B,and

-DRB1 loci, with the optimal match being

an allele-level match.20 Some transplant

400 AABB Technical Manual

Figure 17-4. HLA haplotypes in a family at risk for transfusion-associated GVHD. In contrast to the fam-

ily shown in Fig 17-2, each parent shares a common HLA haplotype, HLA-A1,B8,DR17. Child 1 is ho-

mozygous for the haplotype shared by the parents and by child 3. The lymphocytes of child 1 are capa-

ble of producing posttransfusion GVHD if transfused to either parent or to child 3.

programs additionally match for HLA-C

and -DQ alleles. Molecular HLA typing is

performed on samples from both the donor

and recipient for optimal assessment of

Class I and II region compatibility. Al-

though HLA-identical sibling donors re-

main the best choice for HPC transplanta-

tion, there is increasing use of unrelated

donors identified by searching the file of 5

million HPC donors listed in the National

Marrow Donor Program’s registry of volun-

teer donors. The use of umbilical cord

blood stem cells and hematopoietic stem

cell grafts that have undergone T-cell deple-

tion may allow greater donor-recipient mis-

matches.21,22

Kidney and Pancreas Transplants

ABO compatibility is the most important

factor determining the immediate survival

of kidney transplants. Because ABH anti-

gens are expressed in varying amounts on

all cells of the body, transplanted ABO-in-

compatible tissue comes into continuous

contact with the recipient’s ABO antibod-

ies. Of particular importance is the ex-

pression of ABH antigens on vascular en-

dothelial cells because the vascular supply

in the transplant is a common site for re-

jection.

Both the recipient and the donor are or-

dinarilytestedforABO,HLA-A,-B,and-DR

antigens. HLA-C and -DQ testing is also

usually performed. Before surgery, a major

crossmatch of recipient serum against do-

nor lymphocytes is required. ASHI Stan-

dards for Histocompatibility Testing23 re-

quire that the crossmatch be performed

using a method more sensitive than routine

microlymphocytotoxicity testing, such as

prolonged incubation, washing, augmenta-

tion with antihuman globulin reagents, or

flow cytometry. Flow cytometry is the most

sensitive method and is especially useful

because it can best predict early acute re-

jection and delayed graft function, both of

which are strong predictors of chronic re-

jection and long-term allograft survival.24 In

patients undergoing cadaveric kidney re-

transplantation, the 7-year graft survival

rate using the T-cell flow crossmatch to se-

lect the donor kidney was comparable to

that of patients undergoing primary cada-

veric transplantation (68% vs 72%) and was

significantly better than that of regraft pa-

tients for whom only the antiglobulin

lymphocytotoxicity crossmatch was used

(45%).25 Because HLA antibody responses

are dynamic, the serum used for the cross-

match is often obtained within 48 hours of

surgery and is retained in the frozen state

for any required subsequent testing. An in-

compatible crossmatch with unfractionated

or T lymphocytes is a contraindication to

kidney transplantation. The significance of

a positive B-cell crossmatch is unclear.

Serumfromapatientawaitingcadaver-

donor kidney transplant surgery is tested at

regular intervals for the degree of alloim-

munization by determining the PRA. In ad-

dition, many laboratories identify the

specificities of HLA alloantibodies formed.

If an antibody with a defined HLA specific-

ity is identified in a recipient, it is a com-

mon practice to avoid the corresponding

antigen when allocating a deceased donor

allograft. The serum samples used for peri-

odic PRA testing are usually frozen. The

samples with the highest PRA are often

used, in addition to the preoperative sam-

ple, for pretransplant crossmatching. The

necessity of a prospective crossmatch for

recipients with no evidence of HLA sensiti-

zation has been questioned. Prompt trans-

plantation with reduced cold ischemia time

for the renal allograft may provide greater

benefit to the patient than prospective

crossmatching, provided 1) a very sensitive

method for antibody detection, such as

flow cytometry, has been used26,27 and2)itis

absolutely certain that the patient has had

Chapter 17: The HLA System 401

no additional sensitizing event (ie, immuni-

zation or transfusions within 2 weeks be-

fore or any time since that serum was

screened).

The approach to kidney transplants us-

ing living donors is different. In the past,

when several prospective living donors

were being considered, MLC testing be-

tween the recipient and the donors was

sometimes performed, but it is rarely per-

formed today. HLA matching of recipients

with kidney donors (both living and cada-

veric donor) contributes to long-term allo-

graft survival by decreasing the likelihood

of chronic rejection. In 1996, the projected

20-year allograft survival rates were 57% for

two-haplotype-matched sibling donors,

30% for one-haplotype-matched parental

donors, and 18% for cadaver donors.28 For

cadaver donors with no mismatches with

the recipient for HLA-A, -B, and -DR, the

projected 20-year allograft survival was

40%. Surprisingly, projected survival for

allografts from living, unrelated donors is

similar to those from parental donors.29 Re-

cently, the 1-year survival rates for grafts

from living and cadaver renal donors were

93.9% and 87.7% respectively, and the half-

lives of living-donor and cadaver-donor re-

nal allografts were 21.6 and 13.8 years, re-

spectively.30

Other Solid Organ Transplants

For liver, heart, lung, and heart/lung trans-

plants, ABO compatibility remains the

primary immunologic system for donor

selection, and determining pretransplant

ABO compatibility between donor and re-

cipient is mandatory. HLA-A, -B, and -DR

testing of potential recipients is required,

and the transplant crossmatch must be

available before transplantation when the

recipient has demonstrated presensitiza-

tion, except for emergency situations.

Levels of HLA compatibility do correlate

with graft survival after heart transplanta-

tion, but prospective HLA matching has

been difficult to implement.31

Parentage and Other

Forensic Testing

HLA typing (particularly DNA-based HLA

typing) has proven useful in forensic test-

ing. In parentage testing, HLA typing

alone can exclude about 90% of falsely ac-

cused males. With the addition of red cell

antigen typing, the exclusion rate rises to

95% and exceeds 99% when typing for red

cell enzymes and serum proteins is in-

cluded. Haplotype frequencies, rather

than gene frequencies, are used in these

calculations because linkage disequilib-

rium is so common in the HLA system. It

is important, however, to keep in mind

the racial differences that exist in HLA

haplotype frequencies; recombination

events must also be considered.

Other useful DNA-based assays for fo-

rensic testing are detection of alleles with

variable numbers of tandem repeats and al-

leles with variation in the number of short

tandem repeats, which assess other poly-

morphic, non-HLA genetic regions. DNA-

based assays allow identification of individ-

uals on the basis of extremely small sam-

ples of fluid or tissue, such as hairs, epithe-

lial cells, or semen.

HLA and Disease

For some conditions, especially those be-

lieved to have an autoimmune etiology,

an association exists between HLA phe-

notype and occurrence of clinical disease

(see Table 17-3).32-34 HLA-associated dis-

eases have several features in common.

They are known or suspected to be inher-

ited, display a clinical course with acute

402 AABB Technical Manual

exacerbations and remissions, usually have

characteristics of autoimmune disorders,

and the exact cause is unknown. Evidence

has been accumulating that implicates

the HLA molecules themselves in disease

susceptibility. For instance, resistance to

cerebral malaria results from a strong

cytotoxic T-cell response to particular ma-

larial peptides that are restricted by (fit

into the peptide-binding grooves of) two

specific HLA molecules.35 Another mecha-

nism that could lead to the association of

HLA phenotype and disease is the pres-

ence of a Class I or II heterodimer en-

coded by a specific allele that preferen-

tially presents autoantigens to the T-cell

receptor.

The ancestral haplotype A1, B8, DR3,

DQ2 discussed previously (under Linkage

Disequilibrium) is associated with suscepti-

bility to Type 1 diabetes, lupus, celiac dis-

ease, common variable immunodeficiency

and IgA deficiency, myasthenia gravis, and

also with an accelerated course of HIV in-

fection, likely due to the presence of multi-

ple genes.36 However, HLA typing has only

limited value in assessing risk for most dis-

eases because the association is incom-

plete, often giving false-negative and

false-positive results. The association of

HLA-B27 and ankylosing spondylitis in

those of European ancestry is instructive.

The test is highly sensitive; more than 90%

of such patients with ankylosing spondylitis

possess the HLA-B27 antigen. On the other

hand, specificity is low; only 20% of individ-

uals with the B27 antigen will develop an-

kylosing spondylitis. A second condition,

narcolepsy, is strongly associated with the

HLA allele DQB1*0602.37 As with the case of

HLA-B27 and ankylosing spondylitis, over

90% of individuals with narcolepsy are pos-

itive for HLA-DQB1*0602, but only a minor-

ity of individuals with this marker develop

the disease.

The degree of association between a

given HLA type and a disease is often de-

scribed in terms of relative risk (RR), which

is a measure of how much more frequently

a disease occurs in individuals with a spe-

cific HLA type when compared to individu-

als not having that HLA type. Calculation of

RR is usually based on the cross-product

ratio of a 2 ×2 contingency table. However,

because the HLA system is so highly poly-

morphic, there is an increased possibility of

Chapter 17: The HLA System 403

Table 17-3. HLA-Associated Diseases

Disease HLA RR 32-37

Celiac disease DQ2 >250

Ankylosing spondylitis B27 >150

Narcolepsy DQ6 >38

Subacute thyroiditis B35 14

Type I diabetes DQ8 14

Multiple sclerosis DR15, DQ6 12

Rheumatoid arthritis DR4 9

Juvenile rheumatoid arthritis DR8 8

Grave’s disease DR17 4

RR = relative risk.

finding an association between an HLA an-

tigen and a disease by chance alone. There-

fore, calculation of RR for HLA disease as-

sociations is more complex and is typically

done by Haldane’s modification of Woolf’s

formula.38-39 The RR values for some dis-

eases associated with HLA are shown in Ta-

ble 17-3.

References

1. Schreuder GMTh, Hurley CK, Marsh SGE, et

al. The HLA dictionary 2001: A summary of

HLA-A, -B, -C, -DRB1/3/4/5, -DQB1 alleles and

their association with serologically defined

HLA-A, -B, -C, -DR, and -DQ antigens. Hum

Immunol 2001;62:826-49.

2. Braud VM, Allan DSJ, McMichael AJ. Func-

tions of nonclassical MHC and non-MHC-en-

coded class I molecules. Curr Opin Immunol

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3. Feder JN, Gnirke A, Thomas W, et al. A novel

MHC class I-like gene is mutated in patients

with hereditary haemochromatosis. Nat Genet

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4. BodmerJG,ParhamP,AlbertED,MarshSG.

Putting a hold on “HLA-H.” Nat Genet 1997;

15:234-5.

5. McDonald JC, Adamashvili I. Soluble HLA: A

review of the literature. Hum Immunol 1998;

59:387-403.

6. Ghio M, Contini P, Mazzei C, et al. Soluble

HLA class 1, HLA class II, and Fas ligand in

blood components: A possible key to explain

the immunomodulatory effects of allogeneic

blood transfusion. Blood 1999;93:1770-7.

7. Zinkernagel RM, Doherty PC. The discovery

of MHC restriction. Immunol Today 1997;18:

14-7.

8. CaoK,ChopekM,Fernandez-VinaMA.High

and intermediate resolution DNA typing sys-

tems for class I HLA-A,-B,-C genes by hy-

bridization with sequence-specific oligo-

nucleotide probes (SSOP). Rev Immunogenet

1999;1:177-208.

9. Welsh K, Bunce M. Molecular typing for the

MHC with PCR-SSP. Rev Immunogenet 1999;

1:157-76.

10. Dzik WH. Leukoreduced blood components:

Laboratory and clinical aspects. In: Simon

TL, Dzik WH, Snyder EL, et al, eds. Rossi’s

principles of transfusion medicine. 3rd ed.

Baltimore, MD: Lippincott Williams and

Wilkins, 2002:270-87.

11. Bolgiano DC, Larson EB, Slichter SJ. A model

to determine required pool size for HLA-

typed community donor apheresis programs.

Transfusion 1989;29:306-10.

12. PeiR,LeeJH,ShihNJ,etal.Singlehuman

leukocyte antigen flow cytometry beads for

accurate identification of human leukocyte

antigen antibody specificities. Transplanta-

tion 2003;75:43-9.

13. Friedberg RC. Independent roles for platelet

crossmatching and HLA in the selection of

platelets for alloimmunized patients. Trans-

fusion 1994;34:215-20.

14. Kopko PM, Popovsky MA, MacKenzie MR, et

al. HLA class II antibodies in transfusion-re-

lated acute lung injury. Transfusion 2001;41:

1244-8.

15. Gorman TE, Julius CJ, Barth RF, et al. Transfu-

sion-associated graft-vs-host disease. A fatal

case caused by blood from an unrelated HLA

homozygous donor. Am J Clin Pathol 2000;

113:732-7.

16. Starzl TE, Demetris AJ, Murase N, et al.

Chimerism after organ transplantation. Curr

Opin Nephrol Hypertens 1997;6:292-8.

17. SivasaiKSR,JendrisakM,DuffyBF,etal.

Chimerism in peripheral blood of sensitized

patients waiting for renal transplantation.

Transplantation 2000;69:538-44.

18. Artlett CM, Smith JB, Jimenez SA. Identifica-

tion of fetal DNA and cells in skin lesions

from women with system sclerosis. N Engl J

Med 1998;338:1186-91.

19. Thomas ED. Bone marrow transplantation: A

review. Semin Hematol 1999;36:95-103.

20. Mickelson EM, Petersdorf E, Anasetti PM, et

al. HLA matching in hematopoietic cell trans-

plantation. Hum Immunol 2000;61:92-100.

21. KurtzbergJ,LaughlinM,GrahamML,etal.

Placental blood as a source of hematopoietic

stem cells for transplantation into unrelated

recipients. N Engl J Med 1996;335:157.

22. Aversa F, Tabilio A, Velardi A. Treatment of

high-risk acute leukemia with T cell depleted

stem cells from related donors with one fully

mismatched HLA haplotype. N Engl J Med

1998;339:1186-93.

23. Standards for histocompatibility testing. Mt.

Laurel, NJ: American Society for Histo-

compatibility and Immunogenetics, 1998.

24. Utzig MJ, Blumke M, Wolff-Vorbeck G, et al.

Flow cytometry cross-match: A method for

predicting graft rejection. Transplantation

1997;63:551-4.

25. Bryan CF, Baier KA, Nelson PW, et al. Long-

term graft survival is improved in cadaveric

renal retransplantation by flow cytometric

crossmatching. Transplantation 2000;66:

1827-32.

404 AABB Technical Manual

26. Taylor CJ, Smith SI, Morgan CH, et al. Selec-

tive omission of the donor crossmatch before

renal transplantation: Efficacy, safety, and ef-

fects of cold storage time. Transplantation

2000;69:719-23.

27. Gebel HM, Bray RA. Sensitization and sensi-

tivity: Defining the unsensitized patient.

Transplantation 2000;69:1370-4.

28. Tera sa ki PI, Cho Y, Takemot o S, et al. Twent y-

year follow-up on the effect of HLA matching

on kidney transplant survival and prediction

of future twenty-year survival. Transplant

Proc 1996;28:1144-5.

29. Terasaki PI, Cecka JM, Gjertson DW, Takemoto

S. High survival rates of kidney transplants

from spousal and living unrelated donors. N

Engl J Med 1995;333:333-6.

30. HariharanS,JohnsonCP,BresnahanBA,etal.

Improved graft survival after renal transplan-

tation in the United States, 1988 to 1996. N

Engl J Med 2000;342:605-12.

31. Ketheesan N, Tay GK, Witt CS, et al. The sig-

nificance of HLA matching in cardiac trans-

plantation. J Heart Lung Transplant 1999;18:

226-30.

32. Thorsby E. Invited anniversary review: HLA

associated diseases. Hum Immunol 1997;53:

1-11.

33. Pile KS. HLA and disease associations. Pa-

thology 1999;31:202-12.

34. Howell WM, Jones DB. The role of human

leukocyte antigen genes in the development

of malignant disease. J Clin Pathol Mol Pathol

1995;48:M302-6.

35. Hill AV. The immunogenetics of resistance to

malaria. Proc Assoc Am Physicians 1999;111:

272-7.

36. PriceP,WittC,AllcockR,etal.Thegenetic

basis for the association of the 8.1 ancestral

haplotype (A1, B8, DR3) with multiple im-

munopathological diseases. Immunol Rev

1999;167:257-74.

37. PelinZ,GuilleminaultC,RischN,etal.HLA-

DQB1*0602 homozygosity increases relative

risk for narcolepsy but not for disease sever-

ity in two ethnic groups. US Modafinil in

Narcolepsy Multicenter Study Group. Tissue

Antigens 1998;51:96-100.

38. Haldane JBS. The estimation and significance

of the logarithm of a ratio of frequencies. Ann

Hum Genet 1955;20:309-11.

39. Woolf B. On estimating the relation between

blood groups and disease. Ann Hum Genet

1955;19:251-3.

Suggested Reading

ASHI Clinical Affairs Committee. Guidelines for

clinical histocompatibility practice. Mt. Laurel,

NJ: American Society for Histocompatibility and

Immunogenetics, 1999.

PhelanDL,MickelsonEM,NoreenHS,etal.ASHI

laboratory manual. 4th ed. Mt. Laurel, NJ: Ameri-

can Society for Histocompatibility and Immuno-

genetics, 2001.

Standards for histocompatibility testing. Mt. Lau-

rel, NJ: American Society for Histocompatibility

and Immunogenetics, 1998.

Chapter 17: The HLA System 405

Chapter 18: Pretransfusion Testing

Chapter 18

Pretransfusion Testing

THE PURPOSE OF pretransfusion

testing is to select blood compo-

nents that will not cause harm to

the recipient and will have acceptable

survival when transfused. If performed

properly, pretransfusion tests will confirm

ABO compatibility between the compo-

nent and the recipient and detect most

clinically significant unexpected antibod-

ies.

The AABB Standards for Blood Banks

and Transfusion Services1requires that the

following procedures be performed before

blood components are issued for transfu-

sion:

■Positive identification of the recipi-

ent and the recipient’s blood sample.

■ABO group and Rh typing of the re-

cipient’s blood.

■Red cell antibody detection tests for

clinically significant antibodies us-

ing the recipient’s serum or plasma.

■Comparison of current findings on

the recipient’s sample with records

of previous results.

■Confirmation of the ABO group of

red cell components.

■Confirmation of the Rh type of Rh-

negative red cell components.

■Selection of components of ABO

group and Rh type appropriate for

the recipient.

■Performanceofaserologicorcom

puter crossmatch.

■Labeling of products with the recipi-

ent’s identifying information.

Transfusion Requests

Requests for transfusion may be submit-

ted electronically or on paper and must

contain sufficient information for positive

recipient identification. Standards1(p36) re-

quires two independent identifiers to

identify the patient. These identifiers

could be the patient’s first and last names,

an identification number unique to that

individual, a birth date, or other identify-

ing system. Other information necessary

407

to process the request includes identifica-

tion of the component needed, the quan-

tity, any special requests such as irradia-

tion, gender and age of the recipient (42

CFR 493.1241), and the name of the re-

sponsible physician. The diagnosis and

the recipient’s history of transfusion and

pregnancy may be helpful in problem-

solving. Each facility should have a writ-

ten policy defining request acceptance

criteria. Blood requests that lack the re-

quired information, are inaccurate, or are

illegible should not be accepted.1(p36) Tele-

phoned requests are acceptable in urgent

situations but should be documented, for

example, in a telephone log; a subsequent

request as a written authorization is to be

made within 30 days.2

Patient Identification

Collection of a properly labeled blood

sample from the intended recipient is

critical to safe blood transfusion. Most

hemolytic transfusion reactions result

from errors in sample or patient identifi-

cation.3,4 Thepersondrawingtheblood

sample must identify the intended recipi-

ent in a positive manner. Each facility

must develop and implement policies and

procedures for patient identification and

specimen collection.

Most hospitals identify patients with an

identification wristband. Ideally, this wrist-

band is placed on the patient before speci-

mencollectionandremainsonthepatient

until discharge. The same identifying infor-

mation on the specimen tube submitted for

testing will be used to label blood compo-

nents and this information will be com-

pared against the patient’s wristband at the

time of transfusion. Some hospitals use an

internally generated or commercially avail-

able identification band with a substitute or

additional “blood bank number” as the

unique patient identifier. Commercial sys-

tems vary in design: color-coded numbers

on wristbands, tubes, and units; a wristband

with an embosser for label printing; and a sys-

tem for barcoding that provides positive sam-

ple and patient identification.

Hospital policies generally require phle-

botomists to collect blood specimens only

from patients who have an attached patient

identification wristband. However, in some

circumstances, it may not be possible for

the patient to wear an identification wrist-

band, and an alternative means of positive

patient identification may be needed. The

use of wristbands is difficult when the pa-

tient has total body burns, when the patient

is an extremely premature infant, or when

the wristband is inaccessible during sur-

gery. Some facilities allow identifying infor-

mation to be placed on a patient’s ankle or

forehead. Intraoperative patient identifica-

tion procedures may allow the use of an al-

ternative identification process in lieu of an

inaccessible wristband. It is important to

remind clinical personnel that transfusions

should not be administered to a patient

who lacks positive identification.

When the patient’s identity is unknown,

an emergency identification method may

be used. This patient identification must be

attached to the patient and affixed or repro-

duced on blood samples. This identifica-

tion must be cross-referenced with the pa-

tient’s name and hospital identification

number or code when they become known.

When hospitals allow the use of confiden-

tial or alias names, the facility must have

policies and procedures that govern their

use.

Outpatients may be identified with the

use of a patient wristband for the purpose

of blood sample collection. Alternative

methods of positive patient identification

include a driver’s license or other photo-

graphic identification. Whenever possible,

the patient should be asked to state his or

her name and to provide confirmation of

408 AABB Technical Manual

birth date and address. If a discrepancy is

noted, the sample must not be collected

until the patient’s identity has been clari-

fied.

The identification of specimens used for

preadmission testing must meet the same

requirements as those used for inpatient

transfusion—there must be no doubt about

the identity of the specimen and the pa-

tient. With the advent of patient admission

on the same day as surgery, hospitals have

devised several mechanisms to identify pa-

tients when specimens have been collected

several days or weeks before surgery. One

option is to require that the patient wear an

identification wristband. Other facilities use

a unique number on specimens and on a

patient identification form that the patient

must provide on the day of surgery in order

for the preadmission specimen to be valid

for transfusion.5An alternative procedure is

to place on the patient’s medical record the

wristbandusedduringspecimencollec-

tion. This wristband would be attached to

the patient upon arrival on the day of sur-

gery after proper identification of the pa-

tient.6

Regardless of the system used, it must be

well known to all those who collect blood

specimens and be followed routinely.

Ideally, patient identification procedures

should be used as a matter of course to

identify patients for all treatment methods,

not solely transfusions.

Sample Labeling

Before leaving the patient, the phleboto-

mist must label the blood sample tubes

with two independent patient identifiers

and the date of collection. Either hand-

written or imprinted labels may be used

as long as the information on the label is

identical to that on the wristband and

request. There must be a mechanism to

identify the phlebotomist1(p37); this certifi-

cation may be placed on the label of the

tube, placed on the requisition, or docu-

mented in a computer system.

Confirming Sample Identity in the

Laboratory

When a sample is received in the labora-

tory, a trained member of the staff must

confirm that the information on the label

and on the transfusion request is identi-

cal. If there is any doubt about the iden-

tity of the patient, a new sample must be

obtained.1(p37) It is unacceptable for any-

one to correct identifying information on

an incorrectly labeled sample. Each labo-

ratory should establish policies and pro-

cedures that define identifying information

and describe how to document receipt of

mislabeled specimens.

Blood Sample

Pretransfusion testing may be performed

on either serum or plasma. Plasma may be

the preferred specimen for some methods

such as tests using gel technology. Incom-

pletely clotted blood samples may cause

small fibrin clots that trap red cells into

aggregates that could resemble aggluti-

nates. Plasma collected from patients with

high levels of fibrinogen or patients with

dysproteinemia may demonstrate rouleaux.

Rouleaux formation can be mistaken for

agglutination. Clotting may be incomplete

in specimens not intended to be anti-

coagulated, such as patients who have been

treated with heparin. Adding thrombin or

protamine sulphate to the sample usually

corrects the problem.

It is permissible to collect blood from an

infusion line. To avoid interference from re-

sidual intravenous fluid, the tubing should

be flushed with saline, and 5 mL or a vol-

ume of blood approximately twice the fluid

Chapter 18: Pretransfusion Testing 409

volume in the line should be withdrawn

and discarded before sample collection.7

Appearance of Sample

The appearance of the serum or plasma

may create difficulties in detecting antibody-

induced hemolysis. Whenever possible, a

hemolyzed sample should be replaced with

a new specimen. Test results observed with

lipemic serum can be difficult to evaluate.

On occasion, it may be necessary to use

hemoglobin-tinged or lipemic serum or

plasma. If hemolyzed samples are used, it

should be noted in the patient testing re-

cords to differentiate hemolysis as a result

of an antigen-antibody reaction. Each in-

stitution should have a procedure describ-

ing the indications for using hemolyzed

and lipemic specimens.

Age of Sample

Blood samples intended for use in cross-

matching should be collected no more

than 3 days before the intended transfu-

sion unless the patient has not been preg-

nant or transfused within the preceding 3

months. If the patient’s transfusion or

pregnancy history is uncertain or unavail-

able, compatibility tests must be per-

formed on blood samples collected within

3 days of RBC transfusions.1(p38) This is to

ensure that the sample used for testing

reflects the recipient’s current immuno-

logic status because recent transfusion or

pregnancy may stimulate production of

unexpected antibodies. Because it is not

possible to predict whether or when such

antibodies will appear, a 3-day limit has

been selected as an arbitrary interval ex-

pected to be both practical and safe. It is

short enough to reflect acute changes in

immunologic status but long enough to

allow the results of preadmission testing

completed on Friday (day 0) to be used

for surgical cases performed on Monday

(day 3). The 3-day requirement applies

only to patients who have been transfused

or pregnant within the last 3 months, but

many laboratories prefer to standardize

their operations by setting a 3-day limit

on all specimens used for pretransfusion

testing.

Each institution should have a policy that

defines the length of time samples may be

used. Testing of stored specimens should

be based on the specimen storage limita-

tions in the reagent manufacturer’s infor-

mation circulars. Lack of appropriate stor-

age space may also limit the length of time

specimens are stored.

Retaining and Storing Blood Samples

The recipient’s blood specimen and a

sample of the donor’s red cells must be

stored at refrigerator temperature for at

least 7 days after each transfusion.1(p37) Do-

nor red cells may be from the remainder

of the segment used in the crossmatch or

a segment removed before issuing the

blood. If the opened crossmatch segment

is saved, it should be placed in a tube la-

beled with the unit number and sealed or

stoppered. Keeping the patient’s and do-

nor’s samples allows repeat or additional

testing if the patient experiences adverse

effects.

Serologic Testing

The patient’s ABO group and Rh type must

be determined in order to transfuse ABO-

and Rh-compatible components. Stan-

dards1(pp37,38) requires that the red cells of

the intended recipient be typed for ABO

and Rh and the serum/plasma be tested

for expected and unexpected antibodies

before components containing red cells

are issued for transfusion. There should

be written procedures for exceptions dur-

ing emergencies. When only plasma and

410 AABB Technical Manual

platelets or Cryoprecipitated AHF are be-

ing infused, historical testing information

in the patient’s record may be used. Refer

to Table 18-1 for selection of the ABO types

for red cell and plasma transfusion when

ABO-identical products are not available.

ABO Grouping and Rh Typing of the

Recipient

To determine the ABO group of the recipi-

ent, red cells must be tested with anti-A

and anti-B, and the serum or plasma with

A1and B red cells. The techniques used

and interpretation of the results are de-

scribed in Chapter 13. Any discrepant re-

sults should be resolved before blood is

given. If transfusion is necessary before

resolution, the patient should receive group

O red cells.

The patient’s red cells must be tested

with anti-D, with suitable observations or

controls to avoid a false-positive interpreta-

tion. Chapter 14 contains a more extensive

discussion of Rh typing reagents, appropri-

ate control techniques, and weak D types. If

problems in D typing arise, the patient

should be given Rh-negative blood until the

problem has been resolved. Testing a recipi-

ent’s red cells for weak D is not necessary

because giving Rh-negative cells causes no

harm to recipients with the weak D pheno-

type. Omitting the test for weak D prevents

misinterpretations arising from the presence

of a positive direct antiglobulin test (DAT).

However, some transfusion services test pa-

tient pretransfusion specimens for weak D

in order to identify patients who could be

given Rh-positive blood components, thus

reserving the Rh-negative components for

patients who are D–. Routine testing for

other Rh antigens is not required.

Detecting Unexpected Antibodies to Red

Cell Antigens

Before deciding upon routine procedures

for antibody detection, the blood bank di-

rector must approve which antibodies are

considered potentially clinically signifi-

cant. In general, an antibody is consid-

ered potentially clinically significant if an-

tibodies of that specificity have been

associated with hemolytic disease of the

fetus and newborn, a hemolytic transfusion

reaction, or notably decreased survival of

transfused red cells. Antibodies reactive at

37 C and/or in the antiglobulin test are

more likely to be clinically significant

than cold-reactive antibodies.8

Numerous serologic techniques have

been developed that are suitable for detec-

Chapter 18: Pretransfusion Testing 411

Table 18-1. Selection of Components When ABO-Identical Donors Are Not Available

ABO Requirements

Whole Blood Must be identical to that of the recipient.

Red Blood Cells Must be compatible with the recipient’s plasma.

Granulocytes Pheresis Must be compatible with the recipient’s plasma.

Fresh Frozen Plasma* Must be compatible with the recipient’s red cells.

Platelets Pheresis All ABO groups are acceptable; components compatible

with the recipient’s red cells are preferred.

Cryoprecipitated AHF All ABO groups are acceptable.

*Also see Table 21-5.

tion of blood group antibodies (see Chapter

12 and Chapter 19). Goals in providing

compatible blood for a recipient are to:

■Detect as many clinically significant

antibodies as possible.

■Detect as few clinically insignificant

antibodies as possible.

■Complete the procedure in a timely

manner.

Standards1(p38) requires that tests for unex-

pected antibodies use unpooled reagent

red cells in a method that detects clinically

significant antibodies and includes an anti-

globulin test preceded by incubation at 37 C.

Each negative antiglobulin test must be

followed by a control system of IgG-sensi-

tized cells (check cells). If alternative proce-

dures are used, there must be documenta-

tion of equivalent sensitivity, and the

manufacturer’s specified controls must be

used.

The method chosen should have suffi-

cient sensitivity to detect very low levels of

antibody in a recipient’s serum. Transfusion

of antigen-incompatible red cells to a recip-

ient with a weakly reactive antibody may

result in rapid anamnestic production of

antibody, with subsequent red cell destruc-

tion. The same antibody detection proce-

dure may be used for all categories of spec-

imens, including pretransfusion and prenatal

testsonpatientsandscreeningofdonor

blood. Once a procedure has been adopted,

the method must be described in the facil-

ity’s standard operating procedures manual.

Reading and Interpreting Reactions

In serologic testing, the hemolysis or ag-

glutination that constitutes the visible end-

point of a red cell antigen-antibody inter-

action must be observed accurately and

consistently. The strength of agglutination

or degree of hemolysis observed with each

cell sample should be recorded immedi-

ately after reading. All personnel in a lab-

oratory should use the same interpreta-

tions and notations and be consistent in

grading (eg, 0-4+) reactions. Some labora-

tories prefer to use a numeric scoring (eg,

0-12) system to indicate reaction strength.

Refer to Method 1.8 for grading and scor-

ing. An optical aid such as a concave mir-

ror enhances visualization in reading tube

tests. Microscopic observation is not rou-

tinely recommended in manufacturer’s in-

serts for enhancement media. A micro-

scope can be useful in distinguishing

rouleaux from true agglutination. Micro-

scopic reading may also allow for the

detection of specific patterns of aggluti-

nation that are characteristic of some an-

tibodies. For example, anti-Sdatypically

produces small refractile agglutinates in a

sea of free red cells, giving the appearance

of a mixed-field appearance. If gel or solid

phase is used, the manufacturer’s direc-

tions must be followed for reading and in-

terpreting positive and negative reactions.

Autologous Control

An autologous control or DAT is not re-

quired as a part of pretransfusion testing.

It is of limited value, even for patients

who have recently been transfused, and

should be used only when antibody iden-

tification is required.

Practical Considerations

Antibody detection tests may be performed

in advance of, or together with, a cross-

match between the patient’s serum/plasma

and donor red cells. Performing antibody

detection tests before crossmatching per-

mits early recognition and identification

of clinically significant antibodies. This

allows for the selection of the appropriate

crossmatch procedure and acquisition of

units with special antigen blood types (eg,

e– units).

412 AABB Technical Manual

Comparison with Previous Records

Results of ABO and Rh tests on a current

specimen must be compared with previ-

ous transfusion service records if there has

been prior testing during the past 12

months, and the comparison must be doc-

umented.1(p39) Errors in identification and/

or testing may be detected when discrep-

ancies are found between previous and

current ABO and Rh results.

Records are also to be reviewed for the

presence of clinically significant red cell an-

tibodies, for difficulties in testing, for the

occurrence of significant adverse reactions,

and for special transfusion requirements.1(p39)

Clinically significant red cell alloantibodies

may become undetectable in a recipient’s

serum over time. Between 30% and 35% of

antibodies become undetectable within 1

year and nearly 50% become undetectable

after10ormoreyears.

Crossmatching Tests

Unless there is an urgent need for blood, a

crossmatch must be performed for red

cell transfusion. The crossmatch shall use

procedures to demonstrate ABO incom-

patibility and clinically significant anti-

bodies to red cell antigens. Blood lacking

the relevant antigens is to be selected for

transfusion when a patient has clinically

significant antibody identified currently

or historically, even though the antibody

is presently nonreactive.1(pp39,40) The cross-

match shall include the antiglobulin test.

When no clinically significant antibodies

are detected in current antibody screen-

ing tests and there is no record of previ-

ous detection of such antibodies,1(p40) then

only a method to detect ABO incompati-

bility, such as an immediate-spin or com-

puter crossmatch, is required. It is very

rare for the antiglobulin phase of the

crossmatch to detect a clinically significant

unexpected antibody if the patient’s anti-

body detection test is negative.10,11

The potential benefits of omitting a rou-

tine antiglobulin crossmatch include de-

creased turnaround time, decreased work-

load, reduced reagent costs, and more effective

use of blood inventory. Omitting the anti-

globulin phase of the crossmatch for patients

who meet the criteria must be in approved

written standard operating procedures. The

methods used for serologic crossmatching

may be the same as those used for red cell

antibody detection or identification or they

may be different. For example, gel methods

may be used for both antibody detection

and identification, but the crossmatch may

be performed using a tube test.

Repeat Testing of Donor Blood

A serologic test to confirm the ABO group

of all RBC units and the Rh type of RBC

units labeled as Rh-negative must be per-

formed before transfusion. Confirmatory

testing for weak D is not required. This

confirmatory testing is to be performed

on a sample obtained from an attached

segment. Any discrepancies are to be re-

solved before the unit is issued for trans-

fusion.1(p37)

Suggested Procedures for Routine

Crossmatching

Red cells used for crossmatching must be

obtained from a segment of tubing origi-

nally attached to the blood container. For

routine tube testing, the cells may be

washed and resuspended to 2% to 5% in

saline. Washing the donor’s red cells re-

moves small fibrin clots and some cold

agglutinins that may interfere with inter-

pretation of results. Because the ratio of

serum to cells markedly affects the sensi-

tivity of agglutination tests, it is important

to stay within the 2% to 5% cell suspen-

sion range or as specified by the manufac-

Chapter 18: Pretransfusion Testing 413

turer’s instructions. For example, if too many

red cells are present, weak antibodies may

be missed because too few antibody mol-

ecules bind to each cell. Many laborato-

rians find that a 2% to 3% concentration

yields the best results. For tests using col-

umn (gel) or solid-phase microplate sys-

tems, follow the manufacturer’s directions.

The simplest serologic crossmatch method

is the immediate-spin saline technique, in

which serum is mixed with saline-sus-

pended red cells at room temperature and

the tube is centrifuged immediately. The

immediate-spin crossmatch method is de-

signed to detect ABO incompatibilities be-

tween donor red cells and recipient serum.

It can be used as the sole crossmatch

method only if the patient has no present

or previous clinically significant antibodies.

Because the testing is performed at room

temperature, antibodies such as anti-M, -N,

and -P1may be detected that were not ob-

served if antibody detection tests omitted

room-temperature testing. A sample imme-

diate-spin crossmatch technique is de-

scribed in Method 3.1. An antiglobulin

crossmatch procedure that meets the re-

quirements of Standards for all routine situ-

ations is described in Method 3.2.

Type and Screen (Antibody Detection Test)

Type and screen is a policy in which the

patient’s blood sample is tested for ABO,

Rh, and unexpected antibodies, then stored

in the transfusion service for future cross-

match if a unit is needed for transfusion.

Crossmatched blood is not labeled and

reserved for patients undergoing surgical

procedures that rarely require transfu-

sion. The blood bank must have enough

donor blood available to meet unexpected

needs of patients undergoing operations

under a type and screen policy. If transfu-

sion becomes necessary, ABO- and Rh-

compatible blood can be safely released

after an immediate-spin or computer

crossmatch, if the antibody screen is neg-

ative and there is no history of clinically

significant antibodies. However, if the anti-

body screen is positive, the antibody(ies)

must be identified and antigen-negative

units for the clinically significant antibod-

ies identified must be available for use if

needed.

Routine Surgical Blood Orders

Blood ordering levels for common elective

procedures can be developed from previ-

ous records of blood use. Because surgical

requirements vary among institutions,

routine blood orders should be based on

local transfusion utilization patterns. The

surgeons, anesthesiologists, and the med-

ical director of the blood bank should

agree on the number of units required for

each procedure. See Chapter 3 for a more

detailed discussion of blood ordering pro-

tocols. Routine blood order schedules are

successful only when there is cooperation

and confidence among the professionals

involved in setting and using the guidelines.

Once a surgical blood ordering schedule

has been established, the transfusion ser-

vice routinely crossmatches the predeter-

mined number of units for each patient un-

dergoing the designated procedures. Routine

orders may need to be modified for pa-

tients with anemia, bleeding disorders, or

other conditions in which increased blood

use is anticipated. As with other circum-

stances that require rapid availability of

blood, the transfusion service staff must be

prepared to provide additional blood if the

need arises.

Computer Crossmatch

When no clinically significant antibodies

have been detected by antibody screening

414 AABB Technical Manual

and history review, it is permissible to omit

the antiglobulin phase of the crossmatch

and perform only a procedure to detect

ABO incompatibility. Computerized match-

ing of blood can be used to fulfill the re-

quirement, provided that the following

conditions have been met1(pp40,41):

■The computer system has been vali-

dated, on site, to ensure that only

ABO-compatible whole blood or red

cells have been selected for transfu-

sion.

■Two determinations of the recipi-

ent’s ABO group as specified in Stan-

dards are made, one on a current

specimen and a second one by one

of the following methods: by retest-

ing the same sample, by testing a

second current sample, or by com-

parisonwithpreviousrecords.

■The computer system contains the

unit number, component name,

ABOgroup,andRhtypeofthecom-

ponent; the confirmed donor unit

ABO group; two unique recipient

identifiers; and the recipient’s ABO

group, Rh type, and antibody screen

results.

■A method exists to verify correct en-

try of data before the release of

blood components.

■The system contains logic to alert

the user to discrepancies between

the donor ABO group and Rh type

on the unit label and the interpreta-

tion of the blood group confirmatory

test, and to ABO incompatibility be-

tween recipient and donor unit.

Butch et al12,13 and Safwenberg et al14 have

described in detail a model computer cross-

match system. Potential advantages of a

computer crossmatch include decreased

workload, reduced sample volume required

for testing, reduced exposure of personnel

to blood specimens, and better use of blood

inventory.

Compatibility Testing for Neonates Less

than 4 Months of Age

Requirements for compatibility testing for

neonates less than 4 months of age are

discussed in Chapter 24. An initial pre-

transfusionspecimenmustbeobtained

from the infant to determine ABO and Rh

type. For ABO typing, testing the cells

with anti-A and anti-B is the only test re-

quired. Serum or plasma from either the

infant or the mother may be used to de-

tect unexpected red cell antibodies and

for crossmatching. The infant’s serum

need not be tested for ABO antibodies un-

less a non-group-O infant is to receive

non-group-O cells that are incompatible

with passively acquired anti-A or anti-B.

This antibody most often comes from the

mother but can be present from group O

RBCs or plasma from incompatible plate-

lets. Test methods in this case are to in-

clude an antiglobulin phase using either

donororreagentA

1or B red cells. If no

clinically significant unexpected antibod-

ies are present, it is unnecessary to cross-

match donor red cells for the initial or

subsequent transfusions. Repeat testing

may be omitted for infants less than 4

months of age during any one hospital

admission as long as they are receiving

only group O cells.1(p42)

Interpretation of Antibody

Screening and Crossmatch

Results

Most samples tested have a negative anti-

body screen and are crossmatch-compati-

ble with units selected. A negative anti-

body screen, however, does not guarantee

that the serum does not have clinically

significant red cell antibodies, only that it

contains no antibodies that react with the

screening cells by the techniques employed.

Chapter 18: Pretransfusion Testing 415

Furthermore, a compatible crossmatch

does not guarantee normal red cell sur-

vival.

Table18-2reviewsthepossiblecausesof

positive pretransfusion tests. Most of what

is known about red cell antigen and anti-

body reactions comes from work per-

formed in tube testing. This information is

not necessarily applicable to antibody de-

tection tests using other technologies such

as solid-phase microtiter plates or column

technologies. Spurious or unexpected reac-

tions in these other technologies may have

the same or other causes than those seen in

the tube tests. Depending on the antigen/

antibody reaction strength and the testing

conditions, not all of the scenarios will

result in positive tests.

The cause of the serologic problems

should be identified before transfusion.

Chapter 19 reviews techniques for problem

resolution. If the patient is found to have

clinically significant antibodies, units is-

sued for transfusion should be nonreactive

for such antigens when tested with licensed

reagents (see Selection of Units, Other

Blood Groups). When the antibody identi-

fied is considered clinically insignificant

(eg, anti-A1,-M,-N,-P

1,-Le

a,and/or-Le

and is not reactive at 37 C,15 random units

may be selected for crossmatch.

Labeling and Release of

Crossmatched Blood at the

Time of Issue

Standards1(p44) requires that the following

activities take place at the time of issue:

■A tag or label indicating the recipi-

ent’s two independent identifiers,

donor unit number, and compatibil-

ity test interpretation, if performed,

must be attached securely to the

blood container.

■A final check of records maintained

in the blood bank for each unit of

blood or component must include:

1. Two independent identifiers,

one of which is usually the pa-

tient’s name.

2. The recipient’s ABO group and

Rh type.

3. The donor unit or pool identifi-

cation number.

4. Donor’s ABO and, if required,

Rh type.

5. The interpretation of the cross-

match tests (if performed).

6. Thedateandtimeofissue.

7. Special transfusion requirements

(eg, cytomegalovirus-reduced-

risk, irradiated, or antigen-neg-

ative).

■There must be a process to confirm

that the identifying information, the

request, the records, and the blood

or component are in agreement and

that discrepancies have been re-

solved before issue.

Additional records that may be of use in-

clude those that identify the person issuing

the blood and the person to whom the

blood was issued or the destination of the

unit.

After the transfusion, a record of the

transfusion shall be made a part of the pa-

tient’s medical record. This information

may be part of a computer record or a pa-

per form. Records must contain the identi-

fication of the person(s) performing the test

and, if blood is issued before the resolution

of compatibility problems, the final status

of the serologic findings.

There should be a system to ensure that

the proper blood component is issued for

theintendedpatient.Beforeissuingaunit

of blood or component, personnel must

ensure that the product is acceptable for

use, the unit is checked to make sure it

does not have an abnormal color or ap-

416 AABB Technical Manual

Chapter 18: Pretransfusion Testing 417

Table 18-2. Causes of Positive Pretransfusion Tests*

Negative Antibody Screen, Incompatible Immediate-Spin Crossmatch

■Donor red cells are ABO-incompatible, caused by error in selecting donor unit, patient

specimen, or labeling the donor unit.

■Donor red cells are ABO-incompatible, caused by failure to detect weak expressions of antigens.

■Donor red cells are polyagglutinable.

■Anti-A1in the serum of an A2or A2B individual.

■Other alloantibodies are reactive at room temperature (eg, anti-M).

■Rouleaux formation.

■Cold autoantibodies (anti-I), especially if immediate spin is not tested on antibody detection

screen.

Negative Antibody Screen, Incompatible Antiglobulin Crossmatch

■Donor red cells have a positive direct antiglobulin test.

■Antibody reacts only with red cells having strong expression of a particular antigen either

because of dosage effect (eg, Rh, Kidd, Duffy, and MN antigens) or because of intrinsic variation

in antigen strength (eg, P1).

■Antibody reacts with a low-incidence antigen present on the donor red cells.

■Passively transferred antibody is present—significant levels of circulating anti-A or -B may

be present after infusion of ABO-incompatible platelets to a recipient.

Positive Antibody Screen, Compatible Crossmatches

■Auto-anti-IH (-H).

■Anti-LebH.

■Antibodies are dependent on reagent cell diluent.

■Antibodies demonstrating dosage effect and red cells of the unit are from heterozygotes

(ie, express a single dose of antigen).

Positive Antibody Screen, Incompatible Crossmatches, Negative Auto Control

■Alloantibody(ies).

■Unexpected interactions with reagent red cells.

Positive Antibody Screen, Incompatible Crossmatches, Positive Auto Control, Negative

Direct Antiglobulin Test

■Antibody to ingredient in enhancement media.

Positive Antibody Screen, Incompatible Crossmatches, Positive Auto Control

■Alloantibody is present and patient is experiencing either a delayed serologic or hemolytic

transfusion reaction.

■Passively transferred alloantibody from a derivative reactive with the recipient’s cells (eg,

intravenous immune globulin).

■Cold-reactive autoantibody.

■Warm-reactive autoantibody.

■Rouleaux formation.

■Reagent-related problems.

*Causes are dependent upon serologic methods used.

pearance, the container is not leaking, and

the product is not outdated.

Final identification of the recipient and

the blood container rests with the trans-

fusionist, who must identify the patient and

donor unit and certify that identifying in-

formation on forms, tags, and labels is in

agreement (see Chapter 22).

Selection of Units

ABO Compatibility

Whenever possible, patients should re-

ceive ABO-identical blood; however, it

may be necessary to make alternative se-

lections. If the component to be trans-

fused contains 2 mL or more of red cells,

the donor’s red cells must be ABO-com-

patible with the recipient’s plasma.1(p40) Be-

cause plasma-containing components

can affect the recipient’s red cells, the

ABO antibodies in transfused plasma

should be compatible with the recipient’s

red cells when feasible. Requirements for

components and acceptable alternative

choices are summarized in Table 18-1.

Rh Type

Rh-positive blood components should

routinely be selected for D+ recipients.

Rh-negative units will be compatible but

should be reserved for D– recipients. D–

patients should receive red-cell-contain-

ing components that are Rh-negative to

avoid immunization to the D antigen. Oc-

casionally, ABO-compatible Rh-negative

components may not be available for D–

recipients. In this situation, the blood

bank physician and the patient’s physi-

cian should weigh alternative courses of

action. Depending on the childbearing

potential of the patient and the volume of

red cells transfused, it may be desirable to

administer Rh Immune Globulin to a D–

patient given Rh-positive blood.16

Other Blood Groups

Antigens other than ABO and D are not

routinely considered in the selection of

units of blood. However, if the recipient

has a clinically significant unexpected an-

tibody, antigen-negative blood should be

selected for crossmatching. If the anti-

body is weakly reactive or no longer de-

monstrable, a licensed reagent should be

used to confirm that the donor units are

antigen negative. If there is an adequate

quantity of the patient’s serum, or if an-

other patient’s serum with the same anti-

body specificity is available, and that anti-

body reacts well with antigen-positive red

cells, that serum may be used to screen

for antigen-negative units. Those units

found to be antigen negative must be

confirmed with a licensed reagent, when

available. When licensed reagents are not

available (eg, anti-Lan or anti-Yta), expired

reagents or stored serum specimens from

patients or donors can be used, provided

that controls tested on the day of use are

acceptable (see the Food and Drug Ad-

ministration Compliance Program Guid-

ance Manual, Chapter 42, Blood and Blood

Products). When crossmatch-compatible

units cannot be found, the medical direc-

torshouldbeinvolvedinthedecisionto

transfuse the patient. (See Chapter 19 and

Chapter 20 for additional information on

issuing crossmatch-incompatible units.)

Antigen-negative units are not usually

provided for the patient who has antibod-

ies that are not clinically significant.

Sometimes, problems associated with

crossmatching units for patients with

these antibodies may be avoided by alter-

ing the serologic technique used for the

crossmatch.

418 AABB Technical Manual

Blood Administered in Urgent Situations

When blood is urgently needed, the pa-

tient’s physician must weigh the risk of

transfusing uncrossmatched or partially

crossmatched blood against the risk of

delaying transfusion until compatibility

testing is complete. Ideally, a transfusion

service physician should provide consul-

tation. The risk that the transfused unit

might be incompatible may be judged to

be less than the risk of depriving the pa-

tient of oxygen-carrying capacity of that

transfusion. See Chapter 21.

Required Procedures

When blood is released before pretrans-

fusion testing is complete, the records

must contain a signed statement of the

requesting physician indicating that the

clinical situation was sufficiently urgent

to require release of blood.1(p46) Such a

statement does not absolve blood bank

personnel from their responsibility to is-

sue properly labeled donor blood that is

ABO-compatible with the patient. When

urgent release is requested, blood bank

personnel should:

1. Issue uncrossmatched blood, which

should be:

a. Group O Red Blood Cells if the

patient’s ABO group is unknown.

It is preferable to give Rh-nega-

tive blood if the recipient’s Rh

type is unknown, especially if the

patient is female with the po-

tential to bear children.

b. ABOandRhcompatible,ifthere

has been time to test a current

specimen. Previous records must

not be used, nor should infor-

mation be taken from other re-

cordssuchascards,identifica

tion tags, or driver’s license.

2. Indicate in a conspicuous fashion on

the attached tag or label that com-

patibility testing was not complete

atthetimeofissue.

3. Begin compatibility tests and com-

plete them promptly (for massive

transfusion, see below). If incompat-

ibility is detected at any stage of test-

ing, the patient’s physician and the

transfusion service physician should

be notified immediately.

Massive Transfusion

Massive transfusion is defined as infusion,

within a 24-hour period, of a volume of

blood approximating the recipient’s total

blood volume. Exchange transfusion of an

infant is considered a massive transfusion.

Following massive transfusion, the pre-

transfusion sample no longer represents

the blood currently in the patient’s circula-

tion and its use for crossmatching has lim-

ited benefit. It is only important to confirm

ABO compatibility of units administered

subsequently. The blood bank director may

implement a more limited pretransfusion

testing protocol to be used in these situa-

tions. This protocol should be in writing to

ensure consistent application by all labora-

tory personnel.

Blood Administered After

Non-Group-Specific Transfusion

Transfusion services sometimes release

units for transfusion during emergencies

before they receive a sample for blood

typing. When it arrives, the sample is a

pretransfusion specimen. In most cases,

thesampleistestedandunitsofthatABO

group are issued for transfusion without

concern for anti-A and/or anti-B remain-

ing from the initial emergency-release

units. Because most donor units are RBCs

with comparatively little supernatant

plasma, or RBCs (Additive Solution Added)

with even less residual plasma, the risks

involved in following this practice are

Chapter 18: Pretransfusion Testing 419

minimal. For example, the patient may

exhibit a transient positive DAT.

In some cases, when large volumes of

red cells are transfused, or when young

children or infants receive transfusions,

passively acquired ABO antibodies may be

detected,17 and it may be appropriate to

demonstrate compatibility of red cells of

the patient’s original ABO group with a

freshly drawn serum specimen. If the cross-

match is incompatible because of ABO an-

tibodies, transfusion with red cells of the al-

ternative group should be continued.

If the change in blood type involves only

the Rh system, return to type-specific blood

is simple because antibodies are unlikely to

be present in the plasma of either the recip-

ient or the donor. If a patient has received

blood of an Rh type other than his or her

own before a specimen has been collected

for testing, it may be difficult to determine

the correct Rh type. If there is any question

about the recipient’s D type, Rh-negative

blood should be transfused if possible. The

use of Rh Immune Globulin prophylaxis

should be considered when Rh-positive

components are transfused to Rh-negative

patients. See Chapter 21.

References

1. Silva MA, ed. Standards for blood banks and

transfusion services. 23rd ed. Bethesda, MD:

AABB, 2005.

2. Code of federal regulations. Title 42 CFR

493.1241. Washington, DC: US Government

Printing Office, 2004 (revised annually).

3. Sazama K. Reports of 355 transfusion associ-

ated deaths: 1976 through 1985. Transfusion

1990;30:583-90.

4. LindenJV,WagnerK,VoytovichAE,Sheehan

J.TransfusionerrorsinNewYorkState:An

analysis of 10 years’ experience. Transfusion

2000;40:1207-13.

5. Butch SH, Stoe M, Judd WJ. Solving the same-

day admission identification problem (ab-

stract). Transfusion 1994;34(Suppl):93S.

6. AuBuchon JP. Blood transfusion options: Im-

proving outcomes and reducing costs. Arch

Pathol Lab Med 1997;121:40-7.

7. Procedures for the collection of diagnostic

blood specimens by venipuncture. 3rd ed.

NCCLS document H3-A2, approved standard.

Villanova, PA: National Committee for Clini-

cal Laboratory Standards, 1991.

8. Issitt PD, Anstee DJ. Applied blood group se-

rology. Durham, NC: Montgomery Scientific

Publications, 1998:873-905.

9. Ramsey G, Smietana SJ. Long term follow-up

testing of red cell alloantibodies. Transfusion

1994;34:122-4.

10. Oberman HA. The present and future cross-

match. Transfusion 1992;32:794-5.

11. Meyer EA, Shulman IA. The sensitivity and

specificity of the immediate-spin crossmatch.

Transfusion 1989;29:99-102.

12. Butch SH, Judd WJ, Steiner EA, et al. Elec-

tronic verification of donor-recipient com-

patibility: The computer crossmatch. Trans-

fusion 1994;34:105-9.

13. Butch SH, Judd WJ. Requirements for the

computer crossmatch (letter). Transfusion 1994;

34:187.

14. SafwenbergJ,HögmanCF,CassemarB.Com-

puterized delivery control a useful and safe

complement to the type and screen compati-

bility testing. Vox Sang 1997;72:162-8.

15. Shulman IA, Petz LD. Red cell compatibility

testing: Clinical significance and laboratory

methods. In: Petz LD, Swisher SN, Kleinman

S, eds. Clinical practice of transfusion medi-

cine.3rded.NewYork:ChurchillLivingstone,

1996:199-244.

16. Pollack W, Ascari WQ, Crispen JF, et al. Stud-

ies on Rh prophylaxis II: Rh immune prophy-

laxis after transfusion with Rh-positive blood.

Transfusion 1971;11:340-4.

17. Garratty G. Problems associated with pas-

sively transfused blood group alloantibodies.

Am J Clin Pathol 1998;109:169-77.

Suggested Reading

Beck ML, Tilzer LL. Red cell compatibility testing:

A perspective for the future. Transfus Med Rev 1996;

10:118-30.

Brecher ME. Collected questions and answers. 7th

ed. Bethesda, MD: AABB, 2001:49-56.

Butch SH for the Scientific Section Coordinating

Committee. Guidelines for implementing an elec-

tronic crossmatch. Bethesda, MD: AABB, 2003.

420 AABB Technical Manual

Butch SH, Oberman HA. The computer or elec-

tronic crossmatch. Transfus Med Rev 1997;11:256-

64.

Frohn C, Dumbgen L, Brand JM, et al. Probability

of anti-D development in D– patients receiving D+

RBCs. Transfusion 2003;43:893-8.

Garratty G. How concerned should we be about

missing antibodies to low-incidence antigens?

(editorial) Transfusion 2003;43:844-7.

Issitt PD. From kill to overkill: 100 years of (per-

haps too much) progress. Immunohematology 2000;

16:18-25.

Lumadue JA, Biyd JS, Ness PM. Adherence to a strict

specimen-labeling policy decreases the incidence

of erroneous blood grouping of blood bank speci-

mens. Transfusion 1997;37:1169-72.

Padget BJ, Hannon JL. Variations in pretransfusion

practices. Immunohematology 2003;19:1-6.

Rossmann SN for the Scientific Section Coordinat-

ing Committee. Guidelines for the labeling of spec-

imens for compatibility testing. Bethesda, MD:

AABB, 2002.

Schonewille H, van Zijl AM, Wijermans PW. Im-

portance of antibodies against low-incidence RBC

antigens in complete and abbreviated cross-

matching. Transfusion 2003;43:939-44.

Chapter 18: Pretransfusion Testing 421

Chapter 19: Initial Detection and Identification of Alloantibodies to Red Cell Antigens

Chapter 19

Initial Detection and

Identification of

Alloantibodies to Red Cell

Antigens

RED CELL ALLOANTIBODIES other

than naturally occurring anti-A or

-B are called unexpected red cell

alloantibodies. Depending upon the group

of patients or donors studied and the sen-

sitivity of the test methods used, alloanti-

bodies can be found in 0.3% to 38% of the

population.1,2 Alloantibodies react only with

allogeneic red cells, whereas red cell auto-

antibodies react with the red cells of the

antibody producer. Immunization to red

cell antigens may result from pregnancy,

transfusion, transplantation or from in-

jections with immunogenic material. In

some instances, no specific immunizing

event can be identified. These naturally

occurring antibodies are a result of expo-

sure to environmental, bacterial, or viral

antigens that are similar to blood group

antigens. Also, antibodies detected in se-

rologic tests can be passively acquired.

These antibodies may be acquired from

injected immunoglobulins, donor plasma,

or passenger lymphocytes in transplanted

organs or hematopoietic progenitor cells.

Significance of

Alloantibodies

Alloantibodies to red cell antigens may be

initially detected in any test that uses se-

rum or plasma (eg, ABO test, antibody de-

tection test, crossmatch) or in an eluate

prepared from red cells coated with allo-

antibody. Once an antibody is detected, its

specificity should be determined and its

clinical significance assessed.

A clinically significant red cell antibody,

although difficult to define, could be char-

acterized as an antibody that shortens the

survival of transfused red cells or has been

associated with hemolytic disease of the fe-

tus and newborn (HDFN). The degree of

clinical significance varies with antibodies

ofthesamespecificity.Someantibodies

423

cause destruction of incompatible red cells

within hours or even minutes, others de-

crease the survival by only a few days, and

some cause no discernible red cell destruc-

tion. Antibodies of some specificities are

known to cause HDFN, whereas others may

cause a positive direct antiglobulin test

(DAT) in the fetus without clinical evidence

of HDFN.

Reported experience with other exam-

ples of antibody with the same specificity

can be used in assessing clinical signifi-

cance. Table 15-1 summarizes the expected

reactivity and clinical significance of com-

monly encountered alloantibodies. Daniels

et al3have published a review of these and

other specificities. For some antibodies, few

or no data exist, and decisions must be

based on the premise that clinically signifi-

cant antibodies are usually those active at

37 C and/or by an indirect antiglobulin test

(IAT). It is not true, however, that all anti-

bodies active in vitro at 37 C and/or by an

IAT are clinically significant.

Antibodies encountered in pretransfu-

sion testing should be identified to assess

the need to select antigen-negative red cell

components for transfusion. Patients with

clinically significant antibodies should,

whenever practical, receive red cells that

have been tested and found to lack the cor-

responding antigen. In prenatal testing, the

specificity and immunoglobulin class of an

antibody influence the likelihood of HDFN.

The results of antibody identification tests

on donor blood may be used to character-

ize the units for labeling before transfusion

and to procure blood typing reagents or

teaching samples.

General Procedures

The techniques employed for antibody de-

tection and antibody identification are

similar. Antibody identification methods

can be more focused and based on the re-

activity patterns seen in the antibody de-

tection test.

Each facility should establish which

techniques for antibody detection and

identification will be employed routinely.

Sometimes, it is valuable to develop flow-

charts to clearly guide the technologist

through the process of selecting additional

techniques to identify antibody specifici-

ties. This approach is helpful in expediting

the identification process and minimizing

unnecessary testing.

Specimen Requirements

Either serum or plasma may be used for

antibody detection and identification.

Plasma is not suitable for detecting com-

plement-activating antibodies. A 5- to

10-mL aliquot of whole blood usually

contains enough serum or plasma for

identifying simple antibody specificities;

more may be required for complex studies.

When autologous red cells are studied,

the use of a sample anticoagulated with

EDTA avoids problems associated with

the in-vitro uptake of complement com-

ponents by red cells, which may occur in

clotted samples.

Medical History

It is useful to know a patient’s clinical di-

agnosis, history of transfusions or preg-

nancies, and recent drug therapy when

performing an antibody identification.

For example, in patients who have had re-

cent red cell transfusions, the circulating

blood may contain sufficient donor cells

to make red cell phenotyping studies dif-

ficult to interpret. Special procedures to

separate the autologous red cells for typ-

ing may be required (see Method 2.15).

Other special procedures may be required

for patients with autoantibodies.

424 AABB Technical Manual

Reagents

Antibody Detection Red Cells

Group O red cells suitable for antibody

screening are commercially available and

are offered as sets of either two or three

vials of single-donor red cells. Pooled an-

tibody detection cells (usually from two

donors) can only be used in testing serum

samples from donors.

Thedecisiontousetwoorthreecellsin

an antibody detection test should be based

on circumstances in each individual labo-

ratory. The reagent red cells are selected to

express the antigens associated with most

commonly encountered antibodies. Re-

agent cells licensed by the Food and Drug

Administration (FDA) for this purpose must

express the following antigens: D, C, E, c, e,

M, N, S, s, P1,Le

a,Le

b,K,k,Fy

a,Fy

b,Jk

a,and

Jkb.4Some weakly reactive antibodies react

only with red cells from donors who are ho-

mozygous for the genes controlling the ex-

pression of these antigens, a serologic phe-

nomenon called dosage. Antibodies in the

Rh,Duffy,MNS,andKiddsystemsmost

commonly demonstrate dosage. Reagent

red cells should be refrigerated when not in

use and should not be used for antibody

detection beyond their expiration date.

Antibody Identification Panels

Identification of an antibody to red cell an-

tigen(s) requires testing the serum against

a panel of selected red cell samples with

known antigen composition for the major

blood groups. Usually, they are obtained

from commercial suppliers, but institutions

may assemble their own by using red cells

from local sources. Panel cells are (except

in special circumstances) group O, allow-

ing serum of any ABO group to be tested.

Each cell of the panel is from a different

individual. The cells are selected so that,

taking all the cells into account, a distinc-

tive pattern of positive and negative reac-

tions exists for each of many antigens. To

be functional, a reagent red cell panel must

make it possible to identify with confidence

those clinically significant alloantibodies

that are most frequently encountered, such

as anti-D, -E, -K, and -Fya. The phenotypes

of the reagent red cells should be distrib-

uted such that single specificities of the

common alloantibodies can be clearly

identified and most others excluded. Ide-

ally, the pattern of reactivity for most exam-

ples of single alloantibodies will not overlap

with any other; eg, all of the K+ samples

should not be the only ones that are also

E+. It may also be valuable to include red

cell samples with a double dose of the anti-

gen in question for antibodies that fre-

quently show dosage. To lessen the possi-

bility that chance alone has caused an

apparently definitive pattern, there must be

a sufficient number of red cell samples that

lack, and sufficient red cell samples that ex-

press, most of the antigens listed in Table

19-1.

Commercially prepared panels are gen-

erally issued every 2 to 4 weeks. Each panel

contains different red cell samples with dif-

ferent antigen patterns, so it is essential to

use the phenotype listing sheet that comes

with the panel in use. Commercial cells

usually come as a 2% to 5% suspension in a

preservative medium that can be used di-

rectly from the vial. Washing is generally

unnecessary unless the media in which the

reagent cells are suspended are suspected

of interfering with alloantibody identifica-

tion.

Panel cells should not be used beyond

the expiration date; however, this is not always

practical. Most serologists use in-date reagent

cells for initial antibody identification pan-

els and, if necessary, use expired reagent

cells for exclusion or confirmation of speci-

ficity. Each laboratory must establish and

validate a policy for the use of expired re-

agent cells.5

Chapter 19: Initial Detection and Identification of Alloantibodies to Red Cell Antigens 425

426 AABB Technical Manual

Table 19-1. A Reagent Red Cell Panel for Alloantibody Identification

Sample

Phenotype

Rh Kell Duffy Kidd P Lewis MNS

wcDEe K Fy

aFybJkaJkbP1LeaLebMNS s

1r′r +0+00+ 0 +0 ++ + 0+ ++0+

1w++0+0+ + ++ 0+ + +0 ++++

1+00+0+ 0 ++ ++ 0 0+ +0+0

200+++0 0 0+ 0+ + +0 0+0+

5r″r 00+0++ 0 ++ 0+ 0 0+ +++0

6 r 00+00+ 0 0+ +0 + 00 ++0+

7 r 00+00+ + 0+ +0 + 0+ +0+0

8 r 00+00+ 0 +0 0+ + +0 0+0+

9 r 00+00+ 0 0+ +0 0 0+ 0++0

10 R000++0+ 0 00 ++ + 00 ++++

+ Denotes presence of antigen; 0 denotes absence of antigen.

Antiglobulin Reagents

To detect clinically significant antibodies,

most antibody identification tests include

an antiglobulin phase. Either polyspecific

or IgG-specific antiglobulin reagents may

be used. Polyspecific reagents may detect,

or detect more readily, antibodies that bind

complement. This may be of value in the

detection of certain Kidd antibodies.6Al-

though this may be advantageous in some

instances, many serologists prefer to use

IgG-specific reagents to avoid unwanted

reactivity resulting from in-vitro comple-

ment binding by cold-reactive antibodies.

Enhancement Media

Although the test system may consist solely

of serum and red cells (reagent red cells as

provided by the manufacturer or saline-

suspended red cells), most serologists use

some type of enhancement medium. Many

different media are available, including

low-ionic-strength saline (LISS), polyeth-

ylene glycol (PEG), and 22% to 30% albu-

min. For the initial antibody identifica-

tion panel, most laboratories use the

same enhancement method used in their

routine antibody detection tests. Addi-

tional enhancement techniques may be

employed for more complex studies. En-

hancement techniques are discussed later

in this chapter.

Autologous Control (Autocontrol)

It may be helpful to know how a serum

under investigation reacts with autologous

red cells. This helps determine whether

alloantibody, autoantibody, or both are

present. Serum that reacts only with the

reagent red cells usually contains only

alloantibody, whereas reactivity with both

reagent and autologous red cells suggests

the presence of autoantibody or autoanti-

body plus alloantibody. However, a pa-

tient with alloantibodies to antigens ex-

pressed on recently transfused red cells may

have circulating donor red cells coated

with alloantibodies, resulting in a positive

autocontrol. Because this result may be

misinterpreted as being due to autoanti-

body, a detailed history of recent transfu-

sions should be obtained for all patients

with a positive DAT or positive auto-

control.

The autologous control, in which serum

and autologous cells undergo the same test

conditions as serum and reagent cells, is

not the same as a DAT. Incubation and the

presence of enhancement reagents may

cause reactivity in the autologous control

that is only an in-vitro phenomenon. If the

autocontrol is positive in the antiglobulin

phase, a DAT should be performed. If the

DAT is positive, elution studies should be

considered if the patient has been recently

transfused, if there is evidence of immune

hemolysis, or if the results of serum studies

prove inconclusive. A reactive DAT may

also indicate the presence of autoantibody.

If autoantibody is detected in the serum,

adsorption studies may be necessary to

detect coexisting alloantibodies.

Basic Antibody Identification

Techniques

For initial panels, it is common to use the

same methods and test phases used in the

antibody detection test or crossmatch.

Some serologists may choose to include

an immediate centrifugation reading and/

or a room temperature incubation and

reading without adding an enhancement

medium. This may enhance the detection

of certain antibodies (anti-M, -N, -P1,-I,

-Lea,or-Le

b) and may help to explain re-

actions detected at other phases. Many in-

stitutions omit these steps to avoid finding

antibodies that react only at lower tem-

peratures and have little or no clinical sig-

Chapter 19: Initial Detection and Identification of Alloantibodies to Red Cell Antigens 427

nificance. Test observation after 37 C in-

cubation may detect some antibodies (eg,

potent anti-D, -K, or -E) that can cause di-

rect agglutination of red cells. Other anti-

bodies (eg, anti-Lea,-Jk

a) may be detected

by their lysis of antigen-positive red cells

during the 37 C incubation. Some serolo-

gists believe that because clinically signif-

icant antibodies will be detected with the

IAT, the reading after 37 C can be safely

omitted. This omission will lessen the de-

tection of unwanted positive reactions re-

sulting from clinically insignificant cold-

reactive auto- and alloantibodies.7,8

Thephenotypeofthereactiveantibody

detection cells will provide clues to the

specificity or help exclude specificities. This

information is useful for selecting cells that

would be most informative in additional

testing.

If the patient has previously identified

antibodies, this may affect panel selection.

For example, if the patient is known to have

anti-e,itwillnotbehelpfultotestthese-

rum against a panel of 10 red cell samples,

nine of which are e+. Testing a panel of se-

lected e– red cell samples will better reveal

any newly formed antibodies.

Sometimes, the patient’s phenotype in-

fluences the selection of reagent cells. For

example, if the patient is D– and the serum

is reactive with D+ cells in the screening

test, an abbreviated panel or select cell panel

of D– red cell samples may be tested. This

can both confirm the presence of anti-D

and demonstrate the presence or absence

of additional antibodies, while minimizing

the amount of testing required.9

Interpreting Results

Antibody screening results are interpreted

as positive or negative based on the pres-

ence or absence of reactivity (eg, aggluti-

nation). Interpretation of panel results can

be a more complex process combining

technical knowledge and intuitive skills.

Panel results generally will include both

positive and negative results at different

phases of testing, each of which should be

explained by the final conclusion. Deter-

mination of the patient’s red cell pheno-

type and the probability of antibody spec-

ificity can also play roles in the final

interpretation.

Positives and Negatives

Both positive and negative reactions are

important in antibody identification. Pos-

itive reactions indicate the phase and

strength of reactivity (see Method 1.8 for

grading agglutination), which can suggest

certain specificities. Positive reactions also

can be compared to the antigen patterns

expressed by the panel cells to help assign

specificity. Single alloantibodies usually

yield definite positive and negative reac-

tions that create a clear-cut pattern with

antigen-positive and -negative reagent

red cell samples. For example, if a serum

reacts only with cells 4 and 5 of the re-

agent red cell panel shown in Table 19-1,

anti-E is very likely present. Both reactive

samples express E and all nonreactive

samples lack E.

Negative reactions are important in anti-

body identification because they allow ten-

tative exclusion of antibodies to antigens

expressed on the nonreactive cells. Exclu-

sion of antibodies is an important step in

the interpretation process and must be per-

formed to ensure proper identification of

all the antibodies present.

Exclusion or “Crossing Out”

A widely used first approach to the inter-

pretation of panel results is to exclude

specificities based on nonreactivity with

the serum tested. Such a system is some-

times referred to as a “cross-out” or “rule-

428 AABB Technical Manual

out” method. Once results have been re-

corded on the worksheet, the antigen pro-

file of the first nonreactive cell is exam-

ined. If an antigen is present on the cell

and the serum did not react with the cell,

the presence of the corresponding anti-

body may be, at least tentatively, excluded.

Many technologists will cross out that an-

tigen from the listing on the panel sheet

to facilitate the process. After all antigens

present on that cell have been crossed off,

interpretation proceeds with the other non-

reactive cells and additional specificities

are excluded. In most cases, this process

will leave a group of antibodies that still

have not been excluded.

Next, the cells reactive with the serum

are evaluated. The pattern of reactivity for

each nonexcluded specificity is compared

to the pattern of reactivity obtained with

the test serum. If there is a pattern that

matches exactly, that is most likely the

specificity of the antibody in the serum.

However, if there are remaining speci-

ficities that have not been excluded, addi-

tional testing may be needed to eliminate

remaining possibilities and to confirm the

specificity identified. This requires testing

the serum against cells selected for specific

antigenic characteristics. For example, this

approach could be employed if the pattern

of positive reactions exactly fits anti-Jka,but

anti-K and anti-S have not been excluded.

Then serum should be tested against se-

lected cells, ideally with the following phe-

notypes: Jk(a–), K–, S+; Jk(a–), K+, S–; and

Jk(a+), K–, S–. The reaction pattern with

these cells should both confirm the pres-

ence of anti-Jkaand include or exclude

anti-K and anti-S.

Although the exclusion (cross-out) ap-

proach often identifies simple antibody

specificities, it should be considered only a

provisional step, particularly if the cross-out

was completed based on the nonreactivity

of cells with weaker (eg, heterozygous) ex-

pression of an antigen (see Variations in

Antigen Expression).

Probability

To ensure that an observed pattern is not

theresultofchancealone,conclusivean

tibody identification requires serum to be

tested against sufficient reagent red cell

samples that lack, and that express, the

antigen that corresponds to the apparent

specificity of the antibody.

A standard approach (based on Fisher’s

exact method10) has been to require, for

each specificity identified, three antigen-

positive cells that react and three anti-

gen-negative cells that fail to react. This

standard is not always possible, but it works

well in practice, especially if cells with

strong antigen expression are available. A

somewhat more liberal approach is derived

from calculations by Harris and Hochman,11

whereby minimum requirements for a

probability (p) value of 0.05 are met by hav-

ing two positive and three negative cells, or

one positive and seven negative cells (or

the reciprocal of either combination). Com-

parative p values are shown in Table 19-2.

The use of two positive and two negative

cells is also an acceptable approach for an-

tibody confirmation.12 Additional details on

calculating probability may be found in the

Suggested Reading

Boorman KE, Dodd BE, Lincoln PJ. Blood group

serology.6thed.Edinburgh,Scotland:Churchill

Livingstone, 1988.

Crookston MC. Soluble antigens and leukocyte re-

lated antibodies. Part A. Blood group antigens in

plasma: An aid in the identification of antibodies.

In: Dawson RD, ed. Transfusion with “crossmatch

incompatible” blood. Washington, DC: AABB, 1975:

20-5.

Daniels G. Human blood groups. 2nd ed. Oxford,

England: Blackwell Scientific Publications, 2002.

Engelfriet CP, Overbeeke MAM, Dooren MC, et al.

Bioassays to determine the clinical significance of

red cell antibodies based on Fc receptor-induced

destruction of red cells sensitized by IgG. Transfu-

sion 1994;14:617-26.

Garratty G. In-vitro reactions with red blood cells

that are not due to blood group antibodies: A re-

view. Immunohematology 1998;14(1):1-11.

Issitt PD, Anstee DJ. Applied blood group serology.

4th ed. Durham, NC: Montgomery Scientific Pub-

lications, 1998.

JohnsonST,RudmannSV,WilsonSM,eds.Sero-

logic problem-solving strategies: A systematic ap-

proach. Bethesda, MD: AABB, 1996.

Judd WJ. Elution of antibody from red cells. In:

Bell CA, ed. A seminar on antigen-antibody reac-

tions revisited. Washington, DC: AABB, 1982:175-

221.

Judd WJ. Methods in immunohematology. 2nd ed.

Durham, NC: Montgomery Scientific Publications,

1994.

Kanter MH. Statistical analysis. In: Busch MP, Brecher

ME, eds. Research design and analysis. Bethesda,

MD: AABB, 1998:63-104.

Mallory D, ed. Immunohematology methods and

procedures. Rockville, MD: American Red Cross,

1993.

Marsh WL, Reid ME, Kuriyan M, et al. A handbook

of clinical and laboratory practices in the transfu-

sion of red blood cells. Moneta, VA: Moneta Medi-

cal Press, 1993.

Menitove JE. The Hardy-Weinberg principle: Se-

lection of compatible blood based on mathematic

principles. In: Fridey JL, Kasprisin CA, Chambers

LA,RudmannSV,eds.Numbersforbloodbankers.

Bethesda, MD: AABB, 1995:1-11.

Chapter 19: Initial Detection and Identification of Alloantibodies to Red Cell Antigens 451

Mollison PL, Engelfriet CP, Contreras M. Blood

transfusion in clinical medicine. 10th ed. London:

Blackwell Scientific Publications, 1997.

Race RR, Sanger R. Blood groups in man. 6th ed.

Oxford, England: Blackwell Scientific Publications,

1975.

Reid ME, Lomas-Francis C. The blood group anti-

genfactsbook.2nded.NewYork:AcademicPress,

2004.

Rolih S. A review: Antibodies with high-titer, low-

avidity characteristics. Immunohematology 1990;

6:59-67.

Telen MJ. New and evolving techniques for anti-

body and antigen identification. In: Nance ST, ed.

Alloimmunity: 1993 and beyond. Bethesda, MD:

AABB, 1993:117-39.

452 AABB Technical Manual

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction

Chapter 20

The Positive Direct

Antiglobulin Test and

Immune-Mediated Red

Cell Destruction

THE DIRECT ANTIGLOBULIN test

(DAT) is generally used to determine

if red cells have been coated in vivo

with immunoglobulin, complement, or both.

A positive DAT, with or without shortened

red cell survival, may result from:

1. Autoantibodies to intrinsic red cell

antigens.

2. Alloantibodies in a recipient’s circu-

lation, reacting with antigens on re-

cently transfused donor red cells.

3. Alloantibodies in donor plasma,

plasma derivatives, or blood frac-

tions that react with antigens on the

red cells of a transfusion recipient.

4. Alloantibodies in maternal circula-

tion that cross the placenta and coat

fetal red cells.

5. Antibodies directed against certain

drugs that bind to red cell mem-

branes (eg, penicillin).

6. Nonspecifically adsorbed proteins,

including immunoglobulins, associ-

ated with hypergammaglobulinemia

or recipients of high-dose intravenous

gammaglobulin,1,2 or modification of

the red cell membrane by certain

drugs, eg, some cephalosporins.

7. Red-cell-bound complement. This may

be due to complement activation by

alloantibodies, autoantibodies, drugs,

or bacterial infection.

8. Antibodies produced by passenger

lymphocytes in transplanted organs

or hematopoietic components.3

A positive DAT does not necessarily

mean that a person’s red cells have short-

ened survival. Small amounts of both IgG

and complement appear to be present on

all red cells. A range of 5 to 90 IgG mole-

cules/red cell4and5to40C3dmolecules/

red cell5appears to be normal on the red

cells of healthy individuals.

The DAT can detect a level of 100 to 500

molecules of IgG/red cell and 400 to 1100

molecules of C3d/red cell, depending on

the reagent and technique used. Positive

DATs without clinical manifestations of im-

453

mune-mediated red cell destruction are re-

ported in 1 in 1000 up to 1 in 14,000 blood

donors and 1% to 15% of hospital patients.4

Most blood donors with positive DATs

appear to be perfectly healthy, and most

patients with positive DATs have no obvi-

ous signs of hemolytic anemia, although

some may show slight evidence of in-

creased red cell destruction.4,6(p222) Elevated

levels of IgG or complement have been

noted on the red cells of patients with sickle

cell disease, β-thalassemia, renal disease,

multiple myeloma, autoimmune disorders,

AIDS, and other diseases with elevated se-

rum globulin or blood urea nitrogen (BUN)

levels with no clear correlation between a

positive DAT and anemia.1,2,7 Interpretation

of positive DATs should include the pa-

tient’s history, clinical data, and the results

of other laboratory tests.

The Direct Antiglobulin Test

The principles of the DAT are discussed in

Chapter 12. Although any red cells may be

tested, EDTA-anticoagulated blood sam-

ples are preferred to prevent in-vitro fixa-

tion of complement. If red cells from a

clotted blood sample have a positive DAT

due to complement, the results should be

confirmed on cells from a freshly col-

lected or EDTA-anticoagulated specimen

if those results are to be used for diagnos-

tic purposes.

Most DATs are initially performed with

a polyspecific antihuman globulin (AHG)

reagent capable of detecting both IgG and

C3d (see Method 3.6). If positive, tests with

specific anti-IgG and anticomplement re-

agents may be appropriate. Occasionally,

polyspecific AHG reagents react with cell-

bound proteins other than IgG or C3d (eg,

IgM, IgA, or other complement compo-

nents); specific reagents to distinguish

these proteins are not readily available in

most laboratories. If cord blood samples

aretobetested,itisappropriatetouse

anti-IgG only because hemolytic disease of

the fetus and newborn (HDFN) results from

the fetal red cells becoming sensitized with

maternally derived IgG antibody and com-

plement activation rarely occurs.3

The Pretransfusion DAT and the

Autologous Control

Neither the AABB, in the Standards for

Blood Banks and Transfusion Services,8nor

any other accrediting agency requires a DAT

or an autologous control (autocontrol) as

part of pretransfusion testing. Studies

have shown that eliminating the DAT/

autocontrol portion of routine pretransfu-

sion testing carries minimal risk.9

Evaluation of a Positive DAT

Extent of Testing

Clinical considerations should dictate the

extent to which a positive DAT is evalu-

ated. Dialogue with the attending physi-

cian is important. Interpretation of the

significance of serologic findings requires

knowledge of the patient’s diagnosis; re-

cent drug, pregnancy, and transfusion

history; and information on the presence

of acquired or unexplained hemolytic

anemia. The results of serologic tests alone

are not diagnostic; their significance must

be assessed in conjunction with clinical

information and such laboratory data as

hematocrit, bilirubin, haptoglobin, and

reticulocyte count. When investigating a

transfusion reaction, performance of the

DAT on postreaction specimens is part of

the initial transfusion reaction investiga-

tion. The DAT may be positive if sensi-

tized red cells have not been destroyed or

negative if hemolysis and rapid clearance

have occurred. Positive DAT results should

be further evaluated. The patient’s history

454 AABB Technical Manual

is important in interpreting a posttransfu-

sion reaction positive DAT (see Chapter 27).

Answers to the following questions may

help decide what investigations are appro-

priate:

1. Is there any evidence of in-vivo red

cell destruction? Reticulocytosis,

spherocytes observed on the periph-

eral blood film, hemoglobinemia,

hemoglobinuria, decreased serum

haptoglobin, and elevated levels of

serum unconjugated bilirubin or

lactate dehydrogenase (LDH), espe-

cially LDH1, may be associated with

increased red cell destruction. If an

anemic patient with a positive DAT

does show evidence of hemolysis,

testingtoevaluateapossibleim-

mune etiology is appropriate. IF

THEREISNOEVIDENCEOFIN-

CREASED RED CELL DESTRUC-

TION,NOFURTHERSTUDIESARE

NECESSARY, unless the patient needs

transfusion and the serum contains

incompletely identified unexpected

antibodies to red cell antigens.

2. Has the patient been recently trans-

fused? Many workers routinely at-

tempt to determine the cause of a

positive DAT when the patient has

received transfusions within the pre-

vious 3 months because the first in-

dication of a developing immune re-

sponse may be the attachment of

antibody to recently transfused red

cells. Antibody may appear as early

as7to10days(buttypically2weeks

to several months) after transfusion

in primary immunization and as

earlyas2to7days(butusuallywith

in 20 days) in a secondary response;

these alloantibodies could shorten the

survival of red cells already trans-

fused or given subsequently.

Studies have shown that the posi-

tive DAT and reactive eluates can

persist for more than 300 days fol-

lowing a transfusion reaction, which

is far longer than the transfused cells

would be expected to survive, sug-

gesting that autologous as well as

transfused red cells are sensitized

following a transfusion reaction.10,11 A

mixed-field appearance in the post-

transfusion DAT may or may not be

observed.

3. Is the patient receiving any drugs, such

as cephalosporins, procainamide, intra-

venous penicillin, or -methyldopa?

Cephalosporins are associated with

positive DATs; the second- and third-

generation cephalosporins can be as-

sociated with immune red cell destruc-

tion.12 In one study, 21% of patients

receiving procainamide developed a

positive DAT (three of whom had evi-

dence of hemolytic anemia).13 Ahighin-

cidence (39%) of positive DATs has been

reported in patients taking Unasyn.14

Although not commonly seen in recent

years, approximately 3% of patients re-

ceiving intravenous penicillin, at very

high doses, and 15% to 20% of patients

receiving α-methyldopa will develop a

positive DAT. However, fewer than 1%

of those patients who develop a posi-

tive DAT have hemolytic anemia. Posi-

tive DATs associated with other drugs

are rare. If a positive DAT is found in a

patient receiving such drugs, the at-

tending physician should be alerted so

that appropriate surveillance for red

cell destruction can be maintained. If

redcellsurvivalisnotshortened,no

further studies are necessary.

4. Has the patient received marrow, pe-

ripheral blood stem cells, or an organ

transplant? Passenger lymphocytes of

donor origin produce antibodies di-

rected against ABO or other antigens

on the recipient’s cells, causing a pos-

itive DAT.3

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction 455

5. Is the patient receiving IGIV or intra-

venous RhIG? Immune Globulin, In-

travenous (IGIV) may contain ABO

antibodies, anti-D, or, sometimes, other

antibodies. Intravenous Rh Immune

Globulin (RhIG) causes Rh-positive

patients to develop a positive DAT.15

6. Is the patient septic? Complement

activation can occur in septic patients,

leading to intravascular hemolysis.

This is most often seen in cases of

polyagglutination resulting from or-

ganisms that produce neuraminidase.

Serologic Studies

Three investigative approaches are help-

ful in the evaluation of a positive DAT.

1. Test the DAT-positive red cells with

anti-IgG and anti-C3d reagents to

characterize the types of proteins

coating the red cells.

2. Test the serum/plasma to detect and

identify clinically significant anti-

bodies to red cell antigens.

3. Test an eluate (see Methods 4.1 through

4.5) prepared from the coated red

cells with a panel of reagent red cells

to define whether the coating protein

has red cell antibody activity. When

the only coating protein is comple-

ment, eluates are frequently nonreac-

tive. However, an eluate from the pa-

tient’s red cells coated only with

complement should be tested if there

is clinical evidence of antibody-me-

diated hemolysis. The eluate prepa-

ration can concentrate small amounts

of IgG that may not be detectable on

direct testing using routine methods.

Results of these tests combined with

the patient’s history and clinical data

should assist in classification of the

problems involved.

See Appendix 20-1 for an example of an

algorithm for investigating a positive DAT

(excluding investigation of HDFN).

Elution

Elution frees antibody from sensitized red

cells and recovers antibody in a usable form.

Details of eluate preparation are given in

Chapter 19 and in Methods 4.1 through

4.5. Commercial elution kits are also

available. Table 20-1 lists the advantages

and disadvantages of several common

elution methods; no single elution method

is ideal in all situations. Although many

elution methods damage or destroy the

red cells, certain techniques (see Methods

2.11, 2.12, 2.13, and 2.14) remove anti-

body but leave the cells sufficiently intact

to allow testing for various antigens or for

use in adsorption procedures. Some anti-

gens may be altered by elution, however,

and appropriate controls are essential.

In cases of HDFN or hemolytic transfu-

sion reactions, specific antibody (or anti-

bodies) is usually detected in the eluate,

which may or may not be detectable in the

serum.Inthecaseoftransfusionreactions,

newly developed antibodies initially detect-

able only in the eluate are usually detect-

able in the serum after about 14 to 21 days.

Eluate preparation from the patient’s red

cells often concentrates antibody activity

and may facilitate identification of weakly

reactive serum antibodies.

When the eluate reacts with all the cells

tested, autoantibody is the most likely ex-

planation, especially if the patient has not

been recently transfused. WHEN NO UN-

EXPECTED ANTIBODIES ARE PRESENT IN

THE SERUM, AND IF THE PATIENT HAS

NOT BEEN RECENTLY TRANSFUSED, NO

FURTHER SEROLOGIC TESTING OF AN

AUTOANTIBODY IS NECESSARY.

A nonreactive eluate prepared from IgG-

coated red cells may have several causes.

456 AABB Technical Manual

Onecausemaybethattheeluatewasnot

tested against cells positive for the corre-

sponding antigen, notably group A or

group B cells, or antigens of low incidence,

which are absent from most reagent cell

panels. If a non-group-O patient has re-

ceived plasma containing anti-A or anti-B

(as in transfusion of group O platelets), and

the recipient appears to have immune

hemolysis, the eluate can be tested against

A and/or B cells. If the expected ABO anti-

bodies are not detected, other causes of the

positive DAT should be sought. It may be

appropriate to test the eluate against red

cells from recently transfused donor units,

which could have caused immunization to

a rare antigen, or, in HDFN, against cells

from the father, from whom the infant may

have inherited a rare gene. Pursuing the

cause of a nonreactive eluate for patients

with no evidence of hemolysis is usually

not indicated. Toy et al1showed that 79% of

hospital patients with a positive DAT have a

nonreactive eluate. It is suggested that at

least one contributing factor to these posi-

tive DAT results is nonspecific uptake of

proteins on the red cells, which occurs in

patients with elevated gamma globulin lev-

els.1,2

Reactivity of eluates can be enhanced by

testing them against enzyme-treated cells

or by the use of enhancement techniques

such as polyethylene glycol (PEG). Anti-

body reactivity can be increased by the use

of a concentrated eluate, either by alter-

ation of the fluid-to-cell ratio or by use of

commercial concentration devices. Wash-

ing the red cells with low-ionic-strength sa-

line (LISS) or cold wash solutions may pre-

vent the loss of antibody while the cells are

being prepared for elution.

Certain elution methods give poor re-

sults with certain antibodies. When eluates

are nonreactive yet clinical signs of red cell

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction 457

Table 20-1. Antibody Elution Techniques

Method Advantages Disadvantages

Heat (56 C) Good for ABO-HDFN; quick and

easy

Poor recovery of other blood

group allo- and

autoantibodies

Freeze-thaw Good for ABO-HDFN; quick

method; requires small vol-

ume of red cells

Poor recovery of other blood

group allo- and

autoantibodies

Cold acid Quick and easy; adequate for

most warm auto- and

alloantibodies; commercial

kits available

Possible false-positive elution

(see Leger et al16)

Digitonin acid Nonhazardous; good recovery of

most antibodies

Time-consuming washing of

stroma

Dichloromethane/

Methylene chloride

Noncarcinogenic, nonflamma-

ble; good for IgG auto- and

alloantibodies

Vapors harmful

Compiled from Judd17 andSouthetal.

destruction are present, elution by a differ-

ent method may be helpful. If both serum

and eluate are nonreactive at all test phases

and if the patient has received high-dose

intravenous penicillin or other drug ther-

apy, testing to demonstrate drug-related

antibodies should be considered.

Immune-Mediated

Hemolysis

Immune-mediated hemolysis (immune

hemolysis) is the shortening of red cell

survival by the product(s) of an immune

response. If marrow compensation is ade-

quate, the reduced red cell survival may

not result in anemia. Immune hemolysis

is only one cause of hemolytic anemia,

and many causes of hemolysis are unre-

lated to immune reactions. The serologic

investigations carried out in the blood bank

do not determine whether a patient has a

“hemolytic” anemia. The diagnosis of

hemolytic anemia rests on clinical find-

ings and such laboratory data as hemo-

globin or hematocrit values; reticulocyte

count; red cell morphology; bilirubin,

haptoglobin, and LDH levels; and, some-

times, red cell survival studies. The sero-

logic findings help determine whether the

hemolysis has an immune basis and, if so,

what type of immune hemolytic anemia is

present. This is important because the

treatment for each type is different.

The terms hemolysis and hemolytic are

frequently used to indicate both intravas-

cular and extravascular red cell destruction;

however, this may be misleading. In-vivo

lysis of cells and release of free hemoglobin

within the intravascular compartment (ie,

resulting in hemoglobinemia and hemoglo-

binuria) is uncommon and, often, dramatic.

Extravascular hemolysis, which is more

common, is characterized by an increase in

serum bilirubin, but not by hemoglobinemia

and hemoglobinuria. As a description of

in-vitro antibody reactivity, hemolysis or

lysis of the red cells with release of free he-

moglobin to the surrounding media is both

obvious and rare.

Immune hemolytic anemias can be clas-

sified in various ways. One classification

system is shown in Table 20-2. Autoim-

mune hemolytic anemias (AIHAs) are sub-

divided into five major types: warm anti-

body AIHA (WAIHA), cold agglutinin

syndrome (CAS), mixed-type AIHA, parox-

ysmal cold hemoglobinuria (PCH), and

DAT-negative AIHA. Not all cases fit neatly

into these categories. Drugs (discussed in a

later section of this chapter) may also in-

duce immune hemolysis. The prevalence of

each type can vary depending on the pa-

tient population studied. Table 20-3 shows

the serologic characteristics of the autoim-

muneanddrug-inducedhemolyticane-

mias.

DATs performed with IgG- and C3-spe-

cific AHG reagents as well as the serum and

eluate studies described earlier can be used

to help classify AIHAs. Three additional pro-

cedures may be useful: a cold agglutinin ti-

ter and thermal amplitude studies (Meth-

ods 4.7 and 4.8) and the Donath-Landsteiner

test for PCH (Method 4.13).

The binding of antibody to red cells does

not, in itself, damage the cells. It is the phe-

nomena that the bound antibody-antigen

complex promotes that may eventually

damage cells. These include complement

binding, adherence to Fc receptors on

macrophages leading to phagocytosis, and

cytotoxic lysis. The IgG subclass of bound

antibody may be significant. IgG1 is the

subclass most commonly found, some-

times alone but often in combination with

other subclasses. The other IgG subclasses

occur more often in combination with

other subclasses than alone. In general,

IgG3 antibodies have the most destructive

458 AABB Technical Manual

effects, followed by IgG1. IgG2 antibodies

are associated with less destruction and

IgG4 with little to no destruction.

The number of antibody molecules per

red cell also plays a role. The number of an-

tibody molecules on the red cells of appar-

ently healthy blood donors with positive

DATs (<200 molecules/red cell) is far less

than that usually seen in patients with

AIHA.4,5 Some patients with apparent im-

mune hemolysis may have negative DATs.

Warm Antibody Autoimmune Hemolytic

Anemia

The most common type of AIHA is associ-

ated with warm-reactive (37 C) antibod-

ies. Typical serologic findings are described

below.

DAT

When IgG-specific and complement-spe-

cificAHGreagentsareused,threepat

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction 459

Table 20-2. Classification of Immune Hemolytic Anemias

Autoimmune Hemolytic Anemia (AIHA)

1. Warm autoimmune hemolytic anemia

a. primary (idiopathic)

b. secondary [to such conditions as lymphoma, systemic lupus erythematosus (SLE),

carcinoma, or to drug therapy]

2. Cold agglutinin syndrome

a. primary (idiopathic)

b. secondary (to such conditions as lymphoma, mycoplasma pneumonia, infectious

mononucleosis)

3. Mixed-type AIHA

a. primary (idiopathic)

b. secondary (to such conditions as SLE, lymphoma)

4. Paroxysmal cold hemoglobinuria

a. primary (idiopathic)

b. secondary (to such conditions as syphilis, viral infections)

5. DAT-negative AIHA

a. primary (idiopathic)

b. secondary (to such conditions as lymphoma, SLE)

Drug-Induced Hemolytic Anemia

Alloimmune Hemolytic Anemia

1. Hemolytic disease of the fetus and newborn

2. Hemolytic transfusion reaction

460 AABB Technical Manual

Table 20-3. Serologic Findings in Immune Hemolytic Anemias

WAIHA CAS Mixed-Type AIHA PCH Drug-Induced

Percent of cases 48%19 to 70%316%3to 32%19 7%19 to 8%20 Rare in adults; 32% in

children21

12%3to 18%19

DAT IgG:

20%3to 66%19

IgG + C3:

24%19 to 63%3

C3:

7%19 to 14%3

C3 only:

91%19 to 98%3

IgG + C3:

71%19 to 100%3

C3:

13%3

C3 only:

94%19 to 100%3

IgG:

94%19

IgG + C3:

6%19

Immuno-

globulin type

IgG (sometimes IgA or

IgM, rarely alone)

IgM IgG, IgM IgG IgG

Eluate IgG antibody Nonreactive IgG antibody Nonreactive IgG antibody

Serum 57% react by saline-IAT;

13% hemolyze

enzyme-treated RBCs

at 37 C; 90%

agglutinate enzyme-

treated RBCs at 37 C;

30% agglutinate

untreated RBCs at

20 C; rarely

agglutinate untreated

RBCs at 37 C3

IgM agglutinating

antibody; titer usually

>1000 at 4 C; usually

react at 30 C in

albumin; monoclonal

antibody in chronic

disease3

IgG IAT-reactive antibody

plus IgM

agglutinating

antibody, usually

react at 30-37 C in

saline; high titer at

4 C (classic CAS) or

low titer (<64) at

3,19,20,22

IgG biphasic hemolysin

(Donath-Landsteiner

antibody)3

IgG antibody similar to

WAIHA3

Specificity Rh specificity; other

specificities have

been reported*

Usually anti-I but can be

anti-i; rarely anti-Pr23

Usually specificity is

unclear19,20,22

Can be anti-I, -i, or other

cold agglutinin

specificities

Anti-P (nonreactive with

pandP

kRBCs)

Specificity often Rh

related23

*See text.

terns of reactivity may be found: coating

with IgG alone, with complement alone,

or with both. In approximately 1% of cases,

the DAT will be positive with a poly-

specific AHG reagent but negative with

IgG- and complement-specific AHG re-

agents. Some of these may be due to at-

tachment of IgM or IgA alone if reactivity

with these immunoglobulins has not been

excluded by the manufacturer.3,19

Serum

Autoantibody in the serum typically is IgG

and reacts by indirect antiglobulin testing

against all cells tested.3If the autoanti-

body has been adsorbed by the patient’s

red cells in vivo, the serum may contain

verylittlefreeantibody.Theserumwill

contain antibody after all the specific an-

tigen sites on the red cells have been oc-

cupied and no more antibody can be

bound in vivo. In such cases, the DAT is

usually strongly positive. Approximately

50% of patients with WAIHA have serum

antibodies that react with untreated sa-

line-suspended red cells. When testing

with PEG, enzyme-treated red cells, or

solid-phase methods, over 90% of these

sera can be shown to contain autoantibody.

Approximately one-third of patients with

WAIHA have cold-reactive autoagglutinins

demonstrable in tests at 20 C, but cold ag-

glutinin titers at 4 C are normal. The pres-

ence of this cold agglutinin does not

mean the patient has CAS in addition to

WAIHA.3

Eluate

The presence of the IgG autoantibody on

the red cells may be confirmed by elution

at least upon initial diagnosis and/or at

pretransfusion testing. (See Methods 4.1,

4.2, and 4.5.) Typically, the eluate reacts

with virtually all cells tested, with reactiv-

ity enhanced in tests against enzyme-

treated cells or when PEG is used. The

eluate will usually have no serologic activ-

ity if the only protein coating the red cells

is complement components. Occasion-

ally, antibody not detected by the DAT will

be detected in the eluate, possibly due to

the concentrating effect of eluate prepa-

ration.

Specificity of Autoantibody

The specificity of autoantibodies associ-

ated with WAIHA is complex. In routine

tests, all cells tested are usually reactive.

Some autoantibodies that have weaker or

negative reactivity with cells of rare Rh

phenotypes, such as D– – or Rhnull, appear

to have broad specificity in the Rh system.

Apparent specificity for simple Rh antigens

(D,C,E,c,e)isoccasionallyseen,either

as the sole autoantibody or as a predomi-

nant portion, based on stronger reactivity

with cells of certain phenotypes. Such re-

activity is often termed a “relative” speci-

ficity. Such relative specificity in a serum

may be mistaken for alloantibody, but cells

negative for the apparent target antigen

can adsorb and remove the “mimicking”

specificity.23,24

Unusual Specificities. Apart from Rh

specificity, warm autoantibodies with many

other specificities have been reported, eg,

specificities in the LW, Kell, Kidd, Duffy, and

Diego systems.24 Dilution and selective ad-

sorption of eluates may uncover specificity

or relative specificity of autoantibodies. Pa-

tients with autoantibodies of Kell, Rh, LW,

Ge, Sc, Lu, and Lan specificities may have

depressed expression of the respective anti-

gen and the DAT may be negative or very

weakly positive.24

Practical Significance. Tests against red

cells of rare phenotype and by special tech-

niques have limited clinical application. In

rare instances of WAIHA involving IgM ag-

glutinins, determining autoantibody speci-

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction 461

ficity may help differentiate such cases

from typical CAS.25 It is rarely, if ever, neces-

sary to ascertain autoantibody specificity in

order to select antigen-negative blood for

transfusion. If apparent specificity is di-

rected to a high-incidence antigen (eg,

anti-U), or when the autoantibody reacts

with all red cells except those of a rare Rh

phenotype (eg, D– –, Rhnull), compatible do-

nor blood is unlikely to be available and

there is little point in determining specific-

ity. Such blood, if available, should be

reserved for alloimmunized patients of that

uncommon phenotype.

Transfusion-Stimulated Autoantibodies.

Transfusion itself may lead to the produc-

tion of autoantibodies that may persist and

cause positive DATs for some time after

transfusion yet not cause obvious red cell

destruction. Such cell-bound autoantibodies

sometimes display blood group specificity

(eg, E, K, Jka). The positive DAT may persist

long after transfused red cells should have

disappeared from the circulation, appar-

ently adsorbed to the patient’s own anti-

gen-negative red cells.11

Transfusion of Patients with Warm-Reactive

Autoantibodies

Inherent Risks. Patients with warm-reac-

tive autoantibodies range from those with

no apparent decreased red cell survival to

those with life-threatening anemia. Pa-

tients with little or no evidence of signifi-

cant in-vivo red cell destruction tolerate

transfusion quite well.

When autoantibody is active in serum, it

may be difficult to exclude the presence of

alloantibodies, which increases the risk of

an adverse reaction. Transfusion may stim-

ulate alloantibody production, complicat-

ing subsequent transfusions. Transfusion

may intensify the autoantibody, inducing

or increasing hemolysis and making sero-

logic testing more difficult. Transfusion

may depress compensatory erythropoiesis.

Destruction of transfused cells may increase

hemoglobinemia and hemoglobinuria. In pa-

tients with active hemolysis, transfused red

cells may be destroyed more rapidly than the

patient’s own red cells. In rare cases, this may

promote hypercoagulability and dissemi-

nated intravascular coagulation (DIC). Trans-

fusion reactions, if they occur, may be diffi-

cult to investigate.

TransfusioninWAIHA.Transfusion is

especially problematic for patients with

rapid in-vivo hemolysis, who may present

with a very low hemoglobin level and hypo-

tension. Reticulocytopenia may accompany

a rapidly falling hematocrit, and the patient

may exhibit coronary insufficiency, conges-

tive heart failure, cardiac decompensation,

or neurologic impairment. Under these cir-

cumstances, transfusion is usually required

as a lifesaving measure. The transfused

cells may support oxygen-carrying capacity

until the acute hemolysis diminishes or

other therapies can effect a more lasting

benefit. These patients represent a signifi-

cant challenge because serologic testing

may be complex while clinical needs are

acute.

Transfusion should not be withheld

solely because of serologic incompatibility.

The volume transfused should usually be

the smallest amount required to maintain

adequate oxygen delivery, not necessarily

toreachanarbitraryhemoglobinlevel.Vol

umes of about 100 mL may be appropriate.3

The patient should be carefully monitored

throughout the transfusion.

TransfusioninChronicWAIHA.Most

patients with WAIHA have a chronic stable

anemia, often at relatively low hemoglobin

levels. Those with hemoglobin levels above

8 g/dL rarely require transfusion, and many

patients with levels of 5 g/dL (or even

lower) can be managed with bed rest and

no transfusions. Transfusion will be re-

quired if the anemia progresses or is ac-

462 AABB Technical Manual

companied by such symptoms as severe

angina, cardiac decompensation, respira-

tory distress, and cerebral ischemia.

Most patients with chronic anemia due

to WAIHA tolerate transfusion without

overt reactions, even though the transfused

cells may not survive any better than their

own. Because transfusion may lead to cir-

culatory overload or to increased red cell de-

struction, the decision to transfuse should

be carefully considered. A few patients

without acute hemolysis have had severe

hemolytic reactions after transfusion. This

may be due to the sudden availability of

large volumes of donor cells and the expo-

nential curve of decay, by which the num-

ber of cells hemolyzed is proportional to

the number of cells present.3,6(pp230,369)

Selecting Blood. If the decision is made

to transfuse, selection of appropriate donor

blood is essential. It is important to deter-

mine the patient’s ABO and Rh type and, if

time permits, to detect potentially clinically

significant alloantibodies. Adsorption and

other special techniques described later in

this chapter can greatly reduce the risk of

undetected alloantibodies but may be

time-consuming. If clinically significant

alloantibodies are present, the transfused

cells should lack the corresponding anti-

gen(s).

If the autoantibody has apparent and

relatively clear-cut specificity for a single

antigen (eg, anti-e) and there is active on-

going hemolysis, blood lacking that antigen

may be selected. There is evidence that, in

some patients, such red cells survive better

than the patient’s own red cells.6(p230),20 In the

absence of hemolysis, autoantibody speci-

ficity is not important, although donor units

negative for the antigen may be chosen be-

cause this is a simple way to circumvent the

autoantibody and detect potential alloanti-

bodies. If the autoantibody shows broader

reactivity, reacting with all cells but show-

ing some relative specificity (eg, it reacts

preferentially with e+ red cells), the use of

blood lacking the corresponding antigen is

debatable. It may be undesirable to expose

the patient to Rh antigens absent from

autologous cells, especially D and espe-

cially in females who may bear children

later, merely to improve serologic compati-

bility testing with the autoantibody (eg,

when a D– patient has autoanti-e).

In many cases of WAIHA, no autoanti-

body specificity is apparent. The patient’s

serum reacts with all red cell samples to the

same degree or reacts with red cells from

different donors to varying degrees for rea-

sons seemingly unrelated to Rh phenotypes.

Even if specificity is identified, the exotic

cells used for such identification are not

available for transfusion. The most impor-

tant consideration in such cases is to ex-

clude the presence of clinically important

alloantibodies before selecting either pheno-

typically similar or dissimilar, crossmatch-

incompatible red cells for transfusion. In

extremely rare cases in which there is se-

vere and progressive anemia, it may be es-

sential to transfuse blood that does not re-

act with the patient’s autoantibody.

Frequency of Testing. Although AABB

Standards8(p38) requires that a sample be

tested every 3 days, some serologists con-

tend that, in these difficult cases, the con-

tinued collection and testing (to include

antibody investigation) of patient samples

are unnecessary.26 Others disagree with that

opinion. In studies of patients with WAIHA,

there was 12% to 40% alloimmunization,

with many alloantibodies developing after

recent transfusions.27,28 These two papers

offer methods to assist in the detection of

alloantibodies in the presence of autoanti-

bodies. For patients with previously identi-

fied clinically significant antibodies, Stan-

dards8(p38) requires that methods of testing

shall be those that identify additional clini-

cally significant antibodies. It is the exclu-

sion of newly formed alloantibodies that is

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction 463

of concern. Autoantibodies that react with

all reagent red cells, even weakly, are capa-

ble of masking alloantibody reactivity; the

serologic reactivity is not necessarily addi-

tive.29 Due to the presence of autoantibodies,

all crossmatches will be incompatible. This

is unlike the case of clinically significant

alloantibodies, where a compatible cross-

match with antigen-negative red cells can

be obtained. Monitoring for evidence of red

cell destruction due to alloantibodies is dif-

ficult in patients who already have AIHA;

the patient’s own red cells and transfused

red cells will have shortened survival. In pa-

tients who have autoantibodies without

hemolytic anemia, transfused red cells

should have normal survival.

An alternative transfusion management

protocol proposed by one group uses pro-

phylactic antigen-matched units for pa-

tients with warm autoantibodies where fea-

sible, in combination with streamlined

adsorption procedures.30 Such a protocol

depends on the ability to maintain an

adequate inventory of antigen-negative

units.31

IgM Warm AIHA

AIHA associated with IgM agglutinins that

react at 37 C is unusual but is character-

ized by severe hemolysis.25,32-34 The prog-

nosisforthesepatientsispoor.

DAT

The patient’s red cells are typically spon-

taneously agglutinated, requiring disrup-

tion of the IgM agglutinin by dithiothrei-

tol in order to obtain accurate DAT (and

ABO/Rh) results. Complement is usually

detected on the red cells. In one series,

IgG was detected in 17% of cases and IgM

in 28%. By a more sensitive flow cyto-

metric method, red cell-bound IgM was

detected on 82% of patients’ red cells not

reacting with anti-IgM by tube DAT.33

Serum

Warm IgM autoagglutinins are typically

weak and sometimes are enhanced in the

presence of albumin or when the serum is

acidified.33 Occasionally, optimal reactiv-

ity is between 20 C and 30 C, rather than

37 C. These antibodies have low or negli-

gible antibody titers; a 4 C titer of <64 eas-

ily differentiates this IgM warm antibody

from those seen in CAS.

Eluate

IgM agglutinins are often detected in an

eluate when inspected at the agglutinin

phase before proceeding to the antiglo-

bulin test.

Cold Agglutinin Syndrome

Cold agglutinin syndrome (also called cold

hemagglutinin disease, CHD) is the hemoly-

tic anemia most commonly associated with

cold-reactive autoantibodies and accounts

for approximately 16% to 32% of all cases

of immune hemolysis.3,19 (See Table 20-3.)

It occurs as an acute or chronic condition.

The acute form is often secondary to

lymphoproliferative disorders (eg, lym-

phoma) or Mycoplasma pneumoniae in-

fection. The chronic form is often seen in

elderly patients, sometimes associated

with lymphoma, chronic lymphocytic

leukemia, or Waldenstrom’s macroglo-

bulinemia. Acrocyanosis and hemoglo-

binuria may occur in cold weather. CAS is

often characterized by rapid agglutina-

tion, at room temperature, of red cells in

an EDTA specimen. Clumping of red cells

may be obvious in such a sample, some-

times so strong that the cells appear to be

clotted. Problems with ABO and Rh typing

and other tests are not uncommon. Main-

taining the EDTA specimen at 37 C and

washing the red cells with 37 C saline is

usually necessary to disperse the cold

464 AABB Technical Manual

autoagglutinin before performing ABO and

Rh typing and the DAT.

DAT

Complement is the only protein detected

on the red cells in almost all cases. If other

proteins are detected, a negative control

for the DAT, eg, 6% to 10% albumin,

should be tested to ensure that the cold

autoagglutinin is not causing a false-posi-

tive test.

The cold-reactive autoagglutinin is usu-

ally IgM, which binds to red cells in the

comparatively low temperature of the pe-

ripheral circulation and causes comple-

ment components (C3 and C4 in particular)

to attach to the red cells. As the red cells cir-

culate to warmer areas, the IgM dissociates,

but the complement remains. Red-cell-

bound C3b can react with the CR1 or CR3

receptors of macrophages in the reticulo-

endothelial system. More of the red cell de-

struction occurs in the liver. Regulatory

proteins convert the bound C3 and C4 to

C3dg and C4d, and it is the anti-C3d com-

ponent of polyspecific AHG reagents that

accounts for the positive DAT. The presence

of C3dg alone does not shorten red cell sur-

vival because macrophages have no C3dg

or C3d receptors.

Serum

IgM cold-reactive autoagglutinins associ-

ated with immune hemolysis usually re-

act ≥30 C and have a titer ≥1000 when

testedat4C;theyrarelyreactwithsa

line-suspended red cells above 32 C. If

30% bovine albumin is included in the re-

action medium, 100% and 70% of clini-

cally significant examples will react at 30

C or 37 C, respectively.35 Occasionally,

pathologic cold agglutinins will have a

lower titer (ie, <1000), but they will have a

high thermal amplitude (ie, reactive at 30

C with or without the addition of albu-

min). Hemolytic activity against untreated

red cells can be demonstrated sometimes

at 20 to 25 C, and, except in rare cases

with Pr specificity, enzyme-treated red

cells are hemolyzed in the presence of ad-

equate complement.

Determination of the true thermal am-

plitude or titer of the cold autoagglutinin

requires that the specimen be collected and

maintained strictly at 37 C until the serum

and cells are separated, to avoid in-vitro

autoadsorption. Alternatively, plasma can

be used from an EDTA-anticoagulated

specimen that has been warmed for 10 to

15 minutes at 37 C (with repeated mixing)

and then separated from the cells, ideally at

37 C. This should release autoadsorbed an-

tibody back into the plasma.

In chronic CAS, the IgM autoagglutinin

is usually a monoclonal protein with kappa

light chains. In the acute form induced by

Mycoplasma or viral infections, the anti-

body is polyclonal IgM with normal kappa

and lambda light-chain distribution. Rare

examples of IgA and IgG cold-reactive

autoagglutinins have also been described.

Eluate

Elution is seldom necessary in obvious

cases of CAS. If the red cells have been

collected properly and washed at 37 C,

there will be no immunoglobulin on the

cells and no reactivity will be found in the

eluate.

Specificity of Autoantibody

The autoantibody specificity in CAS is

usually of academic interest only. CAS is

most often associated with antibodies with

I specificity.3,23 Less commonly, i specific-

ity is found, usually associated with infec-

tious mononucleosis.3On rare occasions,

cold-reactive autoagglutinins with Pr or

other specificities are seen3,23 (see Method

4.7). Dilution of the serum may be neces-

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction 465

sary to demonstrate specificity of very

high-titer antibodies.

Autoantibody specificity is not diagnos-

tic for CAS. Autoanti-I may be seen in

healthysubjectsaswellaspatientswith

CAS. The nonpathologic forms of auto-

anti-I, however, rarely react to titers above

64 at 4 C, and are usually nonreactive with

I– (i cord and i adult) red cells at room tem-

perature. In contrast, the autoanti-I of CAS

may react quite strongly with I– red cells in

tests at room temperature, and equal or

even stronger reactions are observed with

I+ red cells. Autoanti-i reacts in the oppo-

site manner, demonstrating stronger reac-

tions with I– red cells than with red cells

that are I+. Procedures to determine the

titers and specificities of cold-reactive auto-

antibodies are given in Method 4.6 and

Method 4.7.

Pretransfusion Testing. Antibody detec-

tion tests should be performed in ways that

minimize cold-reactive autoantibody activ-

ity yet still permit detection of clinically sig-

nificant alloantibodies. The use of albumin

and other potentiators may increase the re-

activity of the autoantibodies. To avoid the

detection of bound complement, most

serologists use an IgG-specific reagent,

rather than a polyspecific AHG serum. Ad-

ditionally, a prewarming technique may be

used (see Method 3.3).

Adsorption Procedures. When cold-re-

active autoantibody reactivity continues to

interfere with antibody detection tests (eg,

when performed strictly at 37 C), cold

autoadsorption studies (see Method 4.6)

can be helpful. One or two cold auto-

adsorptions should remove enough auto-

antibody to make it possible to detect

alloantibodies at 37 C that were otherwise

masked by the cold-reactive autoantibody;

many cold autoadsorptions would be re-

quired to remove enough of the cold-reac-

tive autoantibody for room temperature

testing. If the patient has been recently

transfused, rabbit red cells may be used to

remove autoanti-I and -IH from sera36;clini

cally significant alloantibodies, notably

anti-B,-D,-E,Vel,andothers,havebeenre

moved by this method.37,38 A preparation of

rabbit red cell stroma is commercially avail-

able. Alternatively, allogeneic adsorption

studiesat4Ccanbeperformedasfor

WAIHA (see below).

Mixed-Type AIHA

Although about one-third of patients with

WAIHA have nonpathologic IgM antibod-

ies that react to high titer at low tempera-

ture, another group of patients with

WAIHA have cold agglutinins that react at

or above 30 C. This latter group is referred

to as “mixed-type” AIHA and can be sub-

divided: patients with high titer, high

thermal amplitude IgM cold antibodies

(therareWAIHAplusclassicCAS)andpa-

tients with normal titer (<64 at 4 C), high

thermal amplitude cold antibodies.19,20,22,39

Patients with mixed-type AIHA often

present with hemolysis and complex se-

rum reactivity present in all phases of

testing. Typical serologic findings are de-

scribed below.

DAT

When the patient has WAIHA plus classic

CAS, both IgG and C3 are usually detect-

able on the patient’s red cells. When the

cold agglutinin has a normal titer, but

high thermal amplitude (greater than or

equalto30C),IgGand/orC3maybede

tectable on the red cells.3

Serum

Both warm-reactive IgG autoantibodies

and cold-reactive, agglutinating IgM auto-

antibodies are present in the serum. These

usually result in reactivity at all phases of

testing, with virtually all cells tested. The

IgM agglutinating autoantibody(ies) re-

466 AABB Technical Manual

acts at 30 C or above. If adsorption studies

are done to detect alloantibodies, it may

be necessary to perform adsorptions at both

warmandcoldtemperatures.

Eluate

A suitably prepared eluate will contain a

warm-reactive IgG autoantibody.

Specificity of Autoantibodies

The unusual cold-reactive IgM agglutinat-

ing autoantibody can have specificities

typical of CAS (ie, I or i) but often has no

apparent specificity.19,20,22 The warm-reac-

tive IgG autoantibody often appears sero-

logically indistinguishable from specifi-

cities encountered in typical WAIHA.

Transfusion in Mixed-Type AIHA

If blood transfusions are necessary, the

considerations in the selection of blood

for transfusion are identical to those de-

scribed for patients with acute hemolysis

due to WAIHA (see above).

Paroxysmal Cold Hemoglobinuria

The rarest form of DAT-positive AIHA is

PCH. In the past, it was characteristically

associated with syphilis, but this associa-

tion is now unusual. More commonly, PCH

presents as an acute transient condition

secondary to viral infections, particularly

in young children. In such cases, the

biphasic hemolysin (see below) may only

be transiently detectable. PCH can also

occur as an idiopathic chronic disease in

older people. One large study found that

none of 531 adults having well-defined

immune hemolytic anemias had Donath-

Landsteiner hemolysins, whereas 22 of 68

(32%) children were shown to have Donath-

Landsteiner hemolysins.21

DAT

PCH is caused by an IgG complement-fix-

ing antibody, but, as with IgM cold-reac-

tive autoagglutinins, it reacts with red

cells in colder areas of the body (usually

the extremities), causes C3 to bind irre-

versibly to red cells, and then the anti-

body dissociates from the red cells as the

blood circulates to warmer parts of the

body. Red cells washed in a routine man-

nerfortheDATareusuallycoatedonly

with complement components, but IgG

may be detectable on cells that have been

washed with cold saline and tested with

cold anti-IgG reagent.3Keeping the system

nearer its optimal binding temperature

allows the cold-reactive IgG autoantibody

to remain attached to its antigen.

Serum

The IgG autoantibody in PCH is classically

described as a biphasic hemolysin because

binding to red cells occurs at low temper-

atures but hemolysis does not occur until

the coated red cells are warmed to 37 C.

This is the basis of the diagnostic test for

the disease, the Donath-Landsteiner test

(see Method 4.13). The autoantibody may

agglutinate normal red cells at 4 C but

rarely to titers greater than 64. Because the

antibody rarely reacts above 4 C, the se-

rum is usually compatible with random

donor cells by routine crossmatch proce-

dures and pretransfusion antibody detec-

tion tests are usually nonreactive.

Eluate

Because complement components are

usually the only globulins present on cir-

culating red cells, eluates prepared from

red cells of patients with PCH are almost

always nonreactive.

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction 467

Specificity of Autoantibody

The autoantibody of PCH has most fre-

quently been shown to have P specificity,

reacting with all red cells by the Donath-

Landsteiner test (including the patient’s

own red cells) except those of the very

rare p or Pkphenotypes. Exceptional ex-

amples with other specificities have been

described.6(p221),23

Transfusion in PCH

Transfusion is rarely necessary for adult

patients with PCH, unless their hemolysis

is severe. In children, especially under age

6, the thermal amplitude of the antibody

tends to be much wider than in adults

and hemolysis more brisk, so transfusion

may be required as a lifesaving measure.

Althoughthereissomeevidencethatp

red cells survive better than P+ (P1+orP

1–)

red cells,6(p221) theprevalenceofpbloodis

approximately 1 in 200,000 and the urgent

need for transfusion usually precludes at-

tempts to obtain this rare blood. Transfu-

sion of random donor blood should not

be withheld from PCH patients whose

need is urgent. Red cells negative for the P

antigen should be considered only for

those patients who do not respond ade-

quately to random donor blood.3

DAT-Negative AIHA

Clinical evidence of hemolytic anemia is

present in some patients whose DAT is non-

reactive. Frequently, autoantibody cannot

be detected in either eluate or serum.

There may be several reasons the DAT is

negative. The autoantibody may be IgA or

IgM.3,40 Antibodies with low binding affin-

ity may dissociate from the red cells dur-

ing saline washing of the cells for the DAT.

Washing with ice cold (eg, 4 C) LISS or sa-

line may help retain antibody on the cells;

a control (eg, 6-10% albumin) is necessary

to confirm that cold autoagglutinins are

not causing the positive results.3,40 There

may be too few antibody molecules on

the cell for detection by routine methods

but enough to be demonstrable by meth-

odssuchasflowcytometry,enzyme-

linked antiglobulin tests, solid phase,

PEG, direct Polybrene, column agglutina-

tion, or concentrated eluate.

Nonroutine Reagents

The causative antibody may be IgM or IgA

not detected by routine AHG reagents.

Anti-IgG, anti-C3d, and the combined

anti-C3b, -C3d reagents are the only li-

censed products available in the United

States for use with human red cells. AHG

reagents that react with IgA, IgM, or C4

are available commercially but have been

prepared for use with endpoints other

than agglutination. These must be used

cautiously and their hemagglutinating re-

activity carefully standardized by the

user.3Quality control must be rigorous

because agglutination with AHG reagents

is more sensitive than precipitation; a se-

rum that appears to be monospecific by

precipitation tests may react with several

different proteins when used in agglutina-

tion tests.

Antigen Depression

Patients with autoantibodies of Kell, Rh,

LW, Ge, Sc, Lu, and Lan specificities may

have depressed red cell expression of the

respective antigens. When this occurs, an-

tibody may be detected in the serum and

eluate, but the DAT may be negative or very

weakly positive. This may provide in-vivo

protection of autologous cells. Donor cells

of common specific antigen type may be

destroyed, but cells lacking the corre-

sponding antigen (usually high-incidence)

may survive well. When the autoantibody

subsides, autologous cells again express

normal amounts of antigen.6(p228),24

468 AABB Technical Manual

Serologic Problems with

Autoantibodies

In pretransfusion tests on patients with auto-

antibodies, the following problems may

arise:

1. Cold-reactive autoantibodies can cause

autoagglutination, resulting in erro-

neous determinations of ABO and

Rh type.

2. Red cells strongly coated with globu-

lins may undergo spontaneous ag-

glutination with high-protein, anti-

Rh, blood-typing reagents, and occa-

sionally even with low-protein re-

agents.41

3. Thepresenceoffreeautoantibodyin

theserummaymakeantibodyde-

tection and crossmatching tests dif-

ficult to interpret. If time permits, the

presence or absence of unexpected,

clinically important alloantibody(ies)

should be determined (see Methods

4.9 through 4.12) before blood is trans-

fused.

Although resolving these serologic prob-

lems is important, delaying transfusion in

the hope of finding serologically compati-

ble blood may cause greater danger to the

patient in some cases. Only clinical judg-

ment can resolve this dilemma; therefore,

dialogue with the patient’s physician is im-

portant.

Resolution of ABO Problems

There are several approaches to the reso-

lution of ABO typing problems associated

with cold-reactive autoagglutinins. Often,

it is only necessary to maintain the blood

sample at 37 C immediately after collec-

tion and to wash the red cells with warm

(37 C) saline before testing. It is helpful to

perform a parallel control test, using 6%

to 10% bovine albumin in saline, to deter-

mine if autoagglutination persists. If the

control test is nonreactive, the results ob-

tained with anti-A and anti-B are usually

valid. If autoagglutination still occurs, it

may be necessary to treat the red cells

with sulfhydryl reagents.

Because cold-reactive autoagglutinins

are almost always IgM and sulfhydryl re-

agents denature IgM molecules, reagents

such as 2-mercaptoethanol (2-ME) or

dithiothreitol (DTT) can be used to abolish

autoagglutination (see Method 2.11). Treat-

ing the red cells with ZZAP reagent as in the

preparation for adsorptions can also be used

(see Method 4.10). Appropriate controls are

essentialforalltests.

When the serum agglutinates group O

reagent red cells, the results of serum tests

may be unreliable. Repeating the tests us-

ing prewarmed serum and group A, B, and

O red cells at 37 C will often resolve any dis-

crepancy, but weak anti-A and/or -B in

some patients’ sera may not react at 37 C.

Alternatively, adsorbed serum (either auto-

adsorbed or adsorbed with allogeneic

group O red cells) can be used. Because

rabbit red cells express a B-like antigen,

sera adsorbed with rabbit red cells or

stroma may not contain anti-B, and sera

adsorbed in this manner should not be

used for ABO serum tests.

Resolution of Rh Problems

Autoagglutination of red cells by cold- or

spontaneous agglutination of red cells by

warm-reactive autoantibodies may also

cause discrepant Rh typing. The same

procedures described for the resolution of

ABO problems, with the exception of us-

ing ZZAP-treated red cells, may be useful.

Also, IgG antibody can be dissociated from

the cells by treatment with chloroquine

diphosphate (Method 2.13), or by glycine-

HCl/EDTA (Method 2.14), methods that

leave red cells intact for subsequent typ-

ing. IgM-coated cells can be treated with

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction 469

sulfhydryl reagents (such as 2-ME or DTT,

Method 2.11) to circumvent autoagglu-

tination and spontaneous agglutination.

Detection of Alloantibodies in the

Presence of Warm-Reactive

Autoantibodies

If the patient who has warm-reactive

autoantibodies in the serum needs trans-

fusion, it is important to evaluate the

possible simultaneous presence of allo-

antibodies to red cell antigens. Some

alloantibodies may make their presence

known by reacting more strongly or at dif-

ferent phases than the autoantibody, but

quite often studies may not suggest the

existence of masked alloantibodies.29 It is

helpful to know which of the common red

cell antigens are lacking on the patient’s

red cells, to predict which clinically signif-

icant alloantibodies the patient may have

produced or may produce. Antigens ab-

sent from autologous cells could well be

the target of present or future alloanti-

bodies. When the red cells are coated with

IgG, antiglobulin-reactive reagents can-

not be used to test IgG-coated cells unless

the IgG is first removed (see Methods 2.13

and 2.14). Low-protein antisera (eg, mono-

clonal reagents) that do not require an

antiglobulin test may be helpful in typing

the DAT-positive red cells. Cell separation

procedures (see Methods 2.15 and 2.16)

may be necessary if the patient has been

transfused recently.

Methods to detect alloantibodies in the

presence of warm-reactive autoantibodies

attempt to remove, reduce, or circumvent

the autoantibody. Antibody detection meth-

ods that use PEG, enzymes, column aggluti-

nation, or solid-phase red cell adherence

generally enhance autoantibodies. Testing

LISS- or saline-suspended red cells may

avoid autoantibodies but allow detection of

most significant alloantibodies. Other pro-

cedures involve adsorption, the principles

of which are discussed in Chapter 19. Two

widely used approaches are discussed be-

low.

Autologous Adsorption

In a patient who has not been recently

transfused, autologous adsorption (see

Method 4.9) is the best way to detect allo-

antibodies in the presence of warm-reac-

tive autoantibodies. The adsorbed serum

canbeusedintheroutineantibodyde

tection procedure.

Autoadsorption generally requires some

initial preparation of the patient’s red cells.

At 37 C, in-vivo adsorption will have oc-

curred and all antigen sites on the patient’s

own red cells may be blocked. It may be

necessary, therefore, to remove autoanti-

body from the red cells to make sites avail-

able for adsorption. A gentle heat elution at

56 C for 5 minutes can dissociate some of

the bound IgG. This can be followed by

treatment of the autologous red cells with

proteolytic enzymes to increase their ca-

pacity to adsorb autoantibody. Treatment

of the red cells with ZZAP, a mixture of

papain or ficin and DTT (see Method 4.9)

accomplishes both of these actions in one

step; the sulfhydryl component makes the

IgG molecules more susceptible to the pro-

tease and dissociates the antibody mole-

cules from the cell. Multiple sequential

autoadsorptions with new aliquots of red

cells may be necessary if the serum con-

tains high levels of autoantibody. Once the

autoantibody has been removed, the ad-

sorbed serum is examined for alloantibody

activity.

If the patient is to be transfused, it can

be advantageous to collect and save addi-

tional aliquots of pretransfusion cells, to be

used for later adsorptions.

Autologous adsorption is not recom-

mended for patients who have been re-

470 AABB Technical Manual

cently transfused, because they may have

an admixture of transfused red cells that

might adsorb alloantibody. Red cells nor-

mally live for about 110 to 120 days. In pa-

tients with AIHA, autologous and trans-

fused red cells can be expected to have

shortened survival. However, determining

how long transfused red cells remain in cir-

culation in patients who need repeated

transfusions is not feasible. It has been

demonstrated that very small amounts

(<10%) of antigen-positive red cells are ca-

pable of removing alloantibody reactivity in

in-vitro studies42; therefore, it is recom-

mended to wait for 3 months after transfu-

sion before autologous adsorptions are per-

formed.

Allogeneic Adsorption

The use of allogeneic red cells for adsorp-

tion may be helpful when the patient has

been recently transfused or when insuffi-

cient autologous red cells are available.

The goal is to remove autoantibody and

leave the alloantibody in the adsorbed se-

rum. The adsorbing cells must not have

the antigens against which the alloanti-

bodies react. Because alloantibody speci-

ficity is unknown, red cells of different

phenotypes will usually be used to adsorb

several aliquots of the patient’s serum.

Given the number of potential alloanti-

bodies, the task of selecting cells may ap-

pear formidable. However, the selected

cells need only demonstrate those few allo-

antibodies of clinical significance likely to

be present. These include the common Rh

antigens (D, C, E, c, and e), K, Fyaand Fyb,

Jkaand Jkb, and S and s. Cell selection is

made easier by the fact that some antigens

can be destroyed by appropriate treatment

(eg, with enzymes) before use in adsorption

procedures. Antibodies to high-incidence

antigens cannot be excluded by allogeneic

adsorptions because the adsorbing cells

will almost invariably express the antigen

and adsorb the alloantibody along with

autoantibody.

Patient’s Phenotype Unknown. When

thepatient’sphenotypeisnotknown,

group O red cell samples of three different

Rh phenotypes (R1R1,R

2R2, and rr) should

be selected (see Method 4.10). One should

lack Jkaand another Jkb.Iftreatedwith

ZZAP, these cells would also lack all anti-

gens of the Kell system and enzyme-sensi-

tive antigens (see Table 19-3). If ZZAP is not

available, cells treated only with proteolytic

enzyme can be used, but at least one of the

adsorbing cells must be K– because Kell

system antigens will not be destroyed. Un-

treated cells may be used, but antibody

maybemoredifficulttoremoveandthe

adsorbing cells must, at a minimum, in-

clude at least one negative for the S, s, Fya,

Fyb, and K antigens in addition to the Rh

andKiddrequirementsabove.

Each aliquot may need to be adsorbed

two or three times. The fully adsorbed

aliquots are tested against reagent red cells

knowneithertolackortocarrycommon

antigensoftheRh,MNS,Kidd,Kell,and

Duffy blood group systems. If an adsorbed

aliquot is reactive, that aliquot (or an addi-

tional specimen similarly adsorbed) should

be tested to identify the antibody. Adsorb-

ing several aliquots with different red cell

samples provides a battery of potentially

informative specimens. For example, if the

aliquot adsorbed with Jk(a–) red cells sub-

sequently reacts only with Jk(a+) red cells,

thepresenceofalloanti-Jk

acan confidently

be inferred.

Patient’s Phenotype Known. If the pa-

tient’s Rh and Kidd phenotypes are known

or can be determined, adsorption can be

performed with a single sample of allo-

geneic ZZAP-treated red cells of the same

Rh and Kidd phenotypes as the patient (see

Method 4.11).

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction 471

Problems Encountered. Occasionally

autoantibody will not be removed by three

sequential adsorptions. Further adsorptions

canbedone,butmultipleadsorptionshave

the potential to dilute the serum. If the ad-

sorbing cells do not appear to remove the

antibody, the autoantibody may have an

unusual specificity that does not react with

the cells used for adsorption. For example,

autoantibodies with Kell, LW, or EnaFS

specificity would not be removed by ZZAP-

treated cells (see Table 19-3 for a list of anti-

gens altered by various agents).

Autoantibodies Mimicking Alloantibodies

Sometimes, autoantibodies have patterns

of reactivity that are easily mistaken for

alloantibody. For example, the serum of a

D– patient may have apparent anti-C and

-e reactivity. The anti-C reactivity may re-

flect warm-reactive autoantibody even if

the patient’s cells lack C. The autoantibody

nature of the reactivity can be demon-

strated by autologous and allogeneic ad-

sorption studies. In this case, the appar-

ent alloanti-C would be adsorbed by C–

red cells, both autologous and allogeneic.

This is quite unlike the behavior of a true

alloanti-C, which would be adsorbed only

by C+ red cells. In one study,43 the serum

prepared from an initial autoadsorption

would often retain autoantibodies that

mimicked alloantibodies in addition to

the true alloantibody(ies) present. Serum

prepared from an initial alloadsorption

most often contains only alloantibodies.

The differences in the auto- or alloanti-

body nature of specificities detected in

the autoadsorbed serum as compared to

the alloadsorbed serum reflect an ineffi-

ciency of autologous adsorption. This is

primarily due to limited volumes of auto-

logous cells available for removing all

autoantibody reactivity from the serum.43

Detection of Alloantibodies in the

Presence of Cold-Reactive Autoantibodies

Cold-reactive autoagglutinins rarely mask

clinically significant alloantibodies if se-

rum tests are conducted at 37 C and if

IgG-specific reagents are used for the

antiglobulin phase. In rare instances, it

may be necessary to perform autoadsorp-

tion at 4 C (see Method 4.6). Achieving the

complete removal of potent cold-reactive

autoagglutinins is very time-consuming

and is usually unnecessary. Removal of

sufficient cold autoagglutinins may be fa-

cilitated by treating the patient’s cells with

enzymes or ZZAP before adsorption.

Drug-Induced Immune

Hemolytic Anemia

Drugs sometimes induce the formation of

antibodies, either against the drug itself

or against intrinsic red cell antigens, that

may result in a positive DAT, immune red

cell destruction, or both. Some of the an-

tibodies produced appear to be depend-

ent on the presence of the drug (ie, drug

dependent) for their detection or destruc-

tive capability, whereas others do not (ie,

drug independent). In some instances, a

reactive DAT may result from nonim-

munologic effects of the drugs. Drugs that

have been reported to cause hemolytic

anemia and/or a positive DAT are listed in

Appendix 20-2.

Theories of the Immune Response and

Drug-Dependent Antibodies

Numerous theories have been suggested

to explain how drugs induce immune re-

sponses and what relation such responses

may have to the positive DAT and immune-

mediated cell destruction observed in some

patients. For many years, drug-associated

472 AABB Technical Manual

positive DATs were classified into four

mechanisms: drug adsorption (penicil-

lin-type), immune complex formation,

autoantibody production, and nonspe-

cific adsorption. Such classification has

been useful, but many aspects lacked de-

finitive proof. In addition, some drugs

created immune problems involving as-

pects of more than one mechanism. More

recent theories, still unproven, tend to-

ward a more comprehensive approach.44-48

Most drugs are probably capable of

binding loosely, or firmly, to circulating

cells, which can lead to an immune re-

sponse. Figure 20-1 illustrates this concept.

Antibodies can be formed to the drug itself

ortothedrugplusmembranecomponents.

When an antibody is formed to the drug

plus membrane components, the antibody

may recognize primarily the drug or primar-

ily the membrane. One or all three of these

antibody populations may be present.

Serologic and Clinical Classification

Drug-induced antibodies can be classi-

fied into three groups according to their

clinical and serologic characteristics.3In

one group, the drug binds firmly to the

cell membrane and antibody is appar-

ently largely directed against the drug it-

self. This was called the drug adsorption

mechanism. Antibodies to penicillin are

the best described of this group.

The second group of drug-dependent

antibodies reacts with drugs that do not

bind well to the cell membrane (eg, quini-

dine, ceftriaxone). The reactive mechanism

of these antibodies was previously thought

to be due to drug/antidrug immune com-

plex formation, but the theory has never

been proven.48 Antibodies in this group may

cause acute intravascular hemolysis and

may be difficult to demonstrate serologi-

cally. Testing for this type of drug antibody

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction 473

Figure 20-1. Proposed unifying theory of drug-induced antibody reactions (based on a cartoon by

Habibi as cited by Garratty23). The thicker darker lines represent antigen-binding sites on the F(ab) re-

gion of the drug-induced antibody. Drugs (haptens) bind loosely, or firmly, to cell membranes and anti-

bodies may be made to: a) the drug [producing in-vitro reactions typical of a drug adsorption (penicil-

lin-type) reaction]; b) membrane components, or mainly membrane components (producing in-vitro

reactions typical of autoantibody); or c) part-drug, part-membrane components (producing an in-vitro

reaction typical of the so-called immune complex mechanism).23(p55)

is still referred to as the “immune complex”

method.

Antibodies of the third group (eg, methyl-

dopa, procainamide, and fludarabine) have

serologic reactivity independent of the drug,

despite the fact that it was the drug that

originally induced the immune response.

Serologically, they behave as autoantibodies.

Drug-Dependent Antibodies Reactive with

Drug-Treated Red Cells: Penicillin-Type

Antibodies

The clinical and laboratory features of drug-

induced immune hemolytic anemia oper-

ating through this mechanism are:

1. The DAT is strongly positive due to

IgG coating. Complement coating may

also be present.

2. Antibody eluted from the patient’s

red cells reacts with drug-treated red

cells but not with untreated red cells.

3. The serum contains a high-titer IgG

antibody (especially when the target

is penicillin or cefotetan) reactive with

the drug-treated red cells but not with

the untreated red cells, unless the

patient also has alloantibodies to red

cell antigens.

4. For penicillin, the hemolysis-induc-

ing dose is millions of units daily for

a week or more; for other drugs, eg,

cefotetan, a single 1 to 2 g dose has been

implicated in immune hemolysis.

5. Hemolysis develops gradually but

may be life-threatening if the etiol-

ogy is unrecognized and drug ad-

ministration is continued.

6. Discontinuation of the drug is usu-

ally followed by increased cell sur-

vival, although hemolysis of decreas-

ing severity may persist for several

weeks.

Approximately 3% of patients receiving

large doses of penicillin intravenously (ie,

millions of units per day) will develop a

positive DAT; only occasionally will these

patients develop hemolytic anemia.6(p231) A

possible mechanism for the positive DAT is

given in Fig 20-2. The penicillin becomes

covalently linked to the red cells in vivo. If

the patient has antibodies to penicillin, they

bind to the penicillin bound to the red cells.

The result is that the penicillin-coated red

cells become coated with IgG. If cell destruc-

tion occurs, it takes place extravascularly,

probably in the same way that red cells

coated with IgG alloantibodies are de-

stroyed. Intravascular hemolysis is rare.

Many cephalosporins, which are related

to penicillins, behave in a similar manner.

The cephalosporins are generally classified

by “generations,” based on their effective-

ness against gram-negative organisms (see

Table 20-4). Approximately 4% of patients

receiving first- or second-generation cephalos-

porins develop a positive DAT.48 Dramati-

cally reduced red cell survival has been

associated with second- and third-genera-

tion cephalosporins.12,47,49-53 The prevalence

and severity of cephalosporin-induced im-

mune red cell destruction appear to be in-

creasing.3

Drug-Dependent Antibodies Reacting by the

“Immune Complex” Mechanism

Many drugs have been reported as caus-

ing hemolytic anemia by this mechanism.

Some of the second- and third-generation

cephalosporins react by this mechanism;

anti-ceftriaxone has been detected only

by the immune complex method.49 The

following observations are characteristic:

1. Complement may be the only globu-

lin easily detected on the red cells,

but IgG may be present.

2. The serum antibody can be either

IgMorIgG,orIgMwithIgG.

3. A drug (or metabolite) must be pres-

ent in vitro for demonstration of the

474 AABB Technical Manual

antibody in the patient’s serum. An-

tibodies may cause hemolysis, ag-

glutination, and/or sensitization of

red cells in the presence of the drug.

4. The patient need only take a small

amount of the drug.

5. Acute intravascular hemolysis with

hemoglobinemia and hemoglobinuria

is the usual presentation. Renal fail-

ure is quite common.

6. Once antibody has been formed, se-

vere hemolytic episodes may recur

after exposure to very small quanti-

ties of the drug.

Drug-Independent Antibodies: Autoantibody

Production

Some drugs induce autoantibodies that

appear serologically indistinguishable

from those of WAIHA. Red cells are coated

with IgG, and the eluate as well as the se-

rum react with virtually all cells tested in

the absence of the drug. Blood group spe-

cificity has been demonstrated at times,

similar to that seen in AIHA. The antibody

has no in-vitro activity with the drug, di-

rectly or indirectly.

The best studied of such cases are those

induced by α-methyldopa. A closely related

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction 475

Figure 20-2. The drug-adsorption mechanism. The drug binds tightly to the red cell membrane pro-

teins. If a patient develops a potent drug antibody, it will react with the cell-bound drug. Such red cells

will yield a positive result in the DAT using anti-IgG reagents. Complement is usually not activated and

lysis is primarily extravascular in nature. Penicillin-G is the prototype drug.

drug, L-dopa, has been implicated, as have

several drugs unrelated to α-methyldopa,

including procainamide, nonsteroidal

anti-inflammatory drugs (eg, mefenamic

acid), second- and third-generation cep-

halosporins, and fludarabine. In some

cases, drug-dependent antibodies are also

present.

Proof that a drug causes autoantibody

production is difficult to obtain. Sufficient

evidence would include: demonstration

that autoantibody production began after

drug administration; resolution of the im-

mune process after withdrawal of the drug;

and recurrence of hemolytic anemia or

autoantibodies if the drug is readmini-

stered. The last requirement is crucial and

the most difficult to demonstrate.

Nonimmunologic Protein Adsorption

The positive DAT associated with some

drugs is due to a mechanism independent

of antibody production. Hemolytic ane-

mia associated with this mechanism oc-

curs rarely.

Cephalosporins (primarily cephalothin)

are the drugs with which this was originally

associated. Red cells coated with cephalo-

thin (Keflin) and incubated with normal

plasma will adsorb albumin, IgA, IgG, IgM,

and C3 in a nonimmunologic manner. If

this occurs, a positive indirect antiglobulin

test will be seen with AHG reagents.

Other drugs that may cause nonimmuno-

logic adsorption of proteins and a positive

DAT include diglycoaldehyde, suramin,

cisplatin, clavulanate (in Timentin and

Augmentin), sulbactam in Unasyn,54 and

tazobactam (in Tazocin and Zosyn).55,56

Laboratory Investigation of Drug-Induced

Antibodies

The drug-related problems most com-

monly encountered in the blood bank are

those associated with a positive DAT. Typ-

476 AABB Technical Manual

Table 20-4. Some Cephalosporins

Generic Name Trade Name*

First Generation

cefadroxil Duricef

cefazolin Ancef, Kefzol,

Zolicef

cephalexin Keflex

cephalothin Keflin

cephapirin Cefadyl

cephradine Anspor

Second Generation

cefaclor Ceclor

cefamandole Mandol

cefmetazole Zefazone

cefonicid Monocid

cefotetan Cefotan

cefoxitin Mefoxin

cefuroxime Zinacef, Kefurox,

Ceftin

cefuroxime axetil Ceftin

Third Generation

cefixime Suprax

cefoperazone Cefobid

cefotaxime Claforan

ceftazidime Fortaz, Ceptaz,

Pentacef, Tazicef,

Tazidime

ceftizoxime Ceftizox

ceftriaxone Rocephin

Fourth Generation

cefepime Maxipime

*Several forms are marketed under other trade names.

This list is intended to be informative, not inclusive.

ical DAT results are shown in Table 20-3.

Recent red cell transfusions and/or dra-

matic hemolysis may result in a weak DAT

by the time hemolysis is suspected.

The patient’s serum should be tested for

unexpected antibodies by routine proce-

dures. If the serum does not react with un-

treated red cells, the tests should be re-

peated against ABO-compatible red cells in

the presence of the drug(s) suspected of

causing the problem. Techniques are given

in Method 4.14 and Method 4.15.

If the drug has already been reported as

causing hemolytic anemia, testing methods

maybeavailableinthecasereports.Ifsuch

information is not available, an initial

screening test can be performed with a so-

lution of the drug at a concentration of ap-

proximately 1 mg/mL in phosphate-buf-

fered saline at a pH optimal for solubility of

the drug.

If these tests are not informative, at-

tempts can be made to coat normal red cells

with the drug, and the patient’s serum and

an eluate from the patient’s red cells can be

tested against the drug-coated red cells.

This is the method of choice when penicil-

lin or cephalosporins are thought to be im-

plicated. Results definitive for a penicil-

lin-induced positive DAT are reactivity of

the eluate against penicillin-coated red

cells and absence of reactivity between the

eluate and uncoated red cells.

The immune response may be due to a

metabolite of a drug rather than the drug it-

self. If the clinical picture is consistent with

immune-mediated hemolysis and the above

tests are noninformative, it may be helpful

to test drug metabolites (see Method 4.16).

Normal sera commonly agglutinate and/

or sensitize cephalosporin-treated red cells

due to the nonspecific uptake of protein

discussed above. This problem can be over-

come by testing a 1 in 20 dilution of the pa-

tient’s serum and a normal serum control

against the cephalosporin-treated red cells.

During the testing of cefotetan-treated red

cells, a 1 in 100 dilution of the patient’s se-

rum should be tested because it has been

shown that some normal sera appear to

contain “naturally occurring” antibodies to

cefotetan, some of which still react weakly at

a1in20dilution.

57,58 Cefotetan antibodies

associated with drug-induced immune

hemolytic anemia have very high anti-

globulin titers (4000 to 256,000).49

Two other observations have been made

regarding the testing of cefotetan antibod-

ies: 1) the last wash from the eluate prepa-

ration may react with cefotetan-treated red

cells (possibly due to the high-titer anti-

bodies and/or the antibody affinity), and 2)

drug-independent antibodies may be detected

in the serum and eluate and hemolysis may

be inadvertently attributed to idiopathic

WAIHA.49

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penias. Transfus Med Rev 1993;7:215-29.

47. Christie DJ. Specificity of drug-induced im-

mune cytopenias. Transfus Med Rev 1993;7:

230-41.

48. Garratty G. Drug-induced immune hemolytic

anemia. In: Garratty G, ed. Immunobiology of

transfusion medicine. New York: Marcel

Dekker, 1994:523-51.

49. Arndt PA, Leger RM, Garratty G. Serology of

antibodies to second- and third-generation

cephalosporins associated with immune

hemolytic anemia and/or positive direct

antiglobulin tests. Transfusion 1999;39:1239-

46.

50. Gallagher NI, Schergen AK, Sokol-Anderson

ML, et al. Severe immune-mediated hemolytic

anemia secondary to treatment with cefote-

tan. Transfusion 1992;32:266-8.

51. Garratty G, Nance S, Lloyd M, Domen R. Fatal

immune hemolytic anemia due to cefotetan.

Transfusion 1992;32:269-71.

52. Stroncek D, Procter JL, Johnson J. Drug-in-

duced hemolysis: Cefotetan-dependent

hemolytic anemia mimicking an acute

intravascular immune transfusion reaction.

Am J Hematol 2000;64:67-70.

53. Viraraghavan R, Chakravarty AG, Soreth J.

Cefotetan-induced haemolytic anaemia. A re-

view of 85 cases. Adv Drug React Toxicol Rev

2002;21:101-7.

54. Garratty G, Arndt PA. Positive direct antiglo-

bulin tests and haemolytic anemia following

therapy with beta-lactamase inhibitor con-

taining drugs may be associated with non-

immunologic adsorption of protein onto red

blood cells. Br J Haematol 1998;100:777-83.

55. Broadberry RE, Farren TW, Kohler JA, et al.

Haemolytic anaemia associated with Tazobac-

tam (abstract). Vox Sang 2002;83(Suppl 2):

227.

56. Arndt PA, Leger RM, Garratty G. Positive di-

rect antiglobulin tests and haemolytic anae-

mia following therapy with the beta-lacta-

mase inhibitor, tazobactam, may also be

associated with non-immunologic adsorp-

tion of protein onto red blood cells (letter).

Vox Sang 2003;85: 53.

57. Arndt P, Garratty G. Is severe immune hemoly-

tic anemia, following a single dose of cefo-

tetan, associated with the presence of “natu-

rally-occurring” anti-cefotetan? (abstract)

Transfusion 2001;41(Suppl):24S.

58. Arndt PA. Practical aspects of investigating

drug-induced immune hemolytic anemia

due to cefotetan or ceftriaxone—a case study

approach. Immunohematology 2002;18:27-

32.

Suggested Reading

Dacie J. Historical review. The immune haemolytic

anaemias: A century of exciting progress in under-

standing. Br J Haematol 2001;114:770-85.

Engelfriet CP, Overbeeke MAM, von dem Borne

AEGKr. Autoimmune hemolytic anemia. Semin

Hematol 1992;29:3-12.

Garratty G. Novel mechanisms for immune destruc-

tion of circulating autologous cells. In: Silberstein

LE, ed. Autoimmune disorders of blood. Bethesda,

MD: AABB, 1996:79-114.

Garratty G. Autoantibodies induced by blood

transfusion (editorial). Transfusion 2004:445-9.

Mack P, Freedman J. Autoimmune hemolytic ane-

mia: A history. Transfus Med Rev 2000;14:223-33.

Petz LD, Garratty G. Immune hemolytic anemias.

2nd ed. Philadelphia: Churchill Livingstone, 2004.

Petz LD. A physician’s guide to transfusion in auto-

immune haemolytic anaemia. Br J Haematol 2004;

124:712-16.

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction 479

480 AABB Technical Manual

Appendix 20-1. An Example of an Algorithm for Investigating a Positive DAT (Excluding Investigation of HDFN)

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction 481

Appendix 20-2. Some Drugs Associated with Immune Hemolysis and/or Positive

DATs Due to Drug-Induced Antibodies

Drug Therapeutic Category Possible Mechanism

Acetaminophen Analgesic, antipyretic DD-IC

Aminopyrine Analgesic, antipyretic DD-IC

Amphotericin B Antifungal, antibiotic DD-IC

Ampicillin Antibacterial DD-IC

Antazoline Antihistamine DD-IC

Apazone (azapropazone) Anti-inflammatory, analgesic DI, DD-DA

Buthiazide (butizide) Diuretic, antihypertensive DD-IC

Carbenicillin Antibacterial DD-DA

Carbimazole Thyroid inhibitor DD-IC

Carboplatin Antineoplastic DD-DA, DD-IC

Carbromal Sedative; hypnotic DD-DA

Catergen Diarrheal astringent, treatment of hepatic disease DI

Cephalosporins Antibacterials

First generation NIA, DD-DA

Second generation DD-IC, DD-DA, DI

Third generation DD-IC, DD-DA, DI

Chaparral DI

Chlorpropamide Antidiabetic DD-IC

Chlorpromazine Antipsychotic DI, DD-IC

Cisplatin Antineoplastic NIA

Cladribine

(chlorodeoxyadenosine)

Antineoplastic DI

Clavulanate potassium β-lactamase inhibitor/antibacterial NIA

Cyanidanol DI, DD-DA, DD-IC

Cyclofenil Gonad-stimulating principle DI

Cyclosporine Immunosuppressive DI

Diclofenac Anti-inflammatory DI, DD-IC

Diethylstilbestrol Estrogen DD-IC

Diglycoaldehyde Antineoplastic NIA

Dipyrone Analgesic, antipyretic DD-IC, DD-DA

Elliptinium acetate Antineoplastic DD-IC

Erythromycin Antibacterial DD-DA

Etodolac Anti-inflammatory, analgesic IC

Fenfluramine Anorexic ?

Fenoprofen Anti-inflammatory, analgesic DI, DD-IC

Fludarabine Antineoplastic DI

Fluorescein Injectable dye DD-DA, DD-IC

Fluorouracil Antineoplastic DD-IC

Glafenine Analgesic DI, DD-IC

Hydralazine Antihypertensive DD-IC

Hydrochlorothiazide Diuretic DD-IC

Ibuprofen Anti-inflammatory DI

Insulin Antidiabetic DD-DA?, DD-IC

Interferon Antineoplastic, antiviral DI

Isoniazid Antibacterial, tuberculostatic DD-DA?, DD-IC

Levodopa Antiparkinsonian, anticholinergic DI

Mefenamic acid Anti-inflammatory DI

Mefloquine Antimalarial DD-IC (cont’d)

482 AABB Technical Manual

Appendix 20-2. Some Drugs Associated with Immune Hemolysis and/or Positive

DATs Due to Drug-Induced Antibodies (cont’d)

Drug Therapeutic Category Possible Mechanism

Melphalan Antineoplastic DD-IC

6-Mercaptopurine Antineoplastic DD-DA

Methadone Narcotic analgesic ?

Methicillin Antibacterial DD-DA

Methotrexate Antineoplastic, antimetabolite DD-IC

Methyldopa Antihypertensive DI

Moxalactam (latamoxef) Antibacterial DD-IC, DI

Nafcillin Antibacterial DD-DA

Nomifensine Antidepressant DI, DD-IC

p-Aminosalicylic acid Antitubercular DD-IC

Penicillin G Antibacterial DD-DA

Phenacetin Analgesic, antipyretic DI, DD-IC

Piperacillin Antibacterial DD-DA, DD-IC

Podophyllotoxin Antineoplastic, cathartic ?

Probenecid Uricosuric DD-IC

Procainamide Cardiac depressant, antiarrhythmic DI

Propyphenazone Analgesic, antipyretic, anti-inflammatory DD-IC

Pyramidon Analgesic, antipyretic DD-IC

Quinidine Cardiac depressant, antiarrhythmic DD-DA, DD-IC

Quinine Antimalarial DD-IC

Ranitidine Antagonist (to histamine H2receptors) ?

Rifampin (rifampicin) Antibacterial, antitubercular DD-IC

Sodium pentothal Anesthetic DD-IC

Stibophen Antischistosomal DD-IC

Streptomycin Antibacterial, tuberculostatic DI, DD-DA, DD-IC

Sulbactam sodium β-lactamase inhibitor/antibacterial NIA

Sulfonamides Antibiotics DD-IC

Sulfonylurea derivatives Antidiabetic DD-IC

Sulindac Anti-inflammatory DD-DA, DI

Suprofen Anti-inflammatory, analgesic DD-IC, DI

Suramin Antitrypanosomal, antifilarial NIA

Temafloxacin Antibacterial DD-IC

Teniposide Antineoplastic DI, DD-IC

Tetracycline Antibacterial, antirickettsial, antiamebic DD-DA?, DD-IC

Thiopental Anesthetic DD-IC

Tolbutamide Antidiabetic DD-DA

Tolmetin Anti-inflammatory DI, DD-IC

Triamterene Diuretic DD-IC

Trimellitic anhydride Used in preparation of dyes, resins, etc ?

Zomepirac Analgesic, anti-inflammatory DD-DA, DD-IC, DI

Mechanisms listed are based on descriptions in the literature.3,44-48

DAT = Direct antiglobulin test.

DD-DA = Drug-dependent. Drug adsorbed onto red cells; antibody reacts with drug on cells.

DD-IC = Drug-dependent. “Immune complex mechanism.” Requires drug, serum, and red cells for serologic

demonstration. For most of these drugs, there are only single or very few case reports.

DI = Drug-independent. Associated with autoantibodies similar to those in AIHA. Drug not required for in-vitro

demonstration. Mechanisms of autoantibody production may vary.

NIA = Nonimmunologic adsorption of proteins.

? = Mechanism unclear or unknown.

Chapter 21: Blood Transfusion Practice

Chapter 21

Blood Transfusion Practice

THE DECISION TO transfuse, like

any other therapeutic decision,

should be based on the risks, ben-

efits, and alternatives of treatment. Unfor-

tunately, data regarding the indications

for transfusion are frequently not available

and recipients run the risk of both over-

transfusion and undertransfusion. Trans-

fusions based solely on laboratory test

“triggers,” in particular, are problematic.

Consensus statements on the use of blood

components such as those produced by

the National Institutes of Health (NIH)

help guide therapy but cannot substitute

for clinical judgment.

Red Blood Cell Transfusion

Physiologic Principles

The primary indication for transfusion of

Red Blood Cells (RBCs) is to restore or main-

tain oxygen-carrying capacity to meet tis-

sue demands; transfusion to replace red

cells destined for destruction in hemoly-

tic disease of the newborn is discussed in

Chapter 24. Because demand for oxygen

varies greatly among different individuals

in different clinical circumstances, mea-

surement of only the hematocrit or the

hemoglobin concentration (“the hemo-

globin”) cannot accurately assess the need

for transfusion.1-3

Normal Oxygen Supply and Demand

Tissues at rest have a baseline demand for

oxygen, particularly the heart, kidneys,

brain, liver, and gastrointestinal tract;

consumption by muscle is very low at

rest. The oxygen content of blood (mL

O2/mL blood) is determined by the hemo-

globin, the binding coefficient of hemo-

globin for oxygen, the oxygen saturation

of hemoglobin, and a small quantity of

483

oxygen dissolved in the plasma. This is

described as:

O2content = (Hb ×1.39 ×%sat)

+(pO

2×0.003)

Tissue oxygen consumption is calculated

as the difference between oxygen delivery

in the arterial blood and oxygen return by

the venous blood:

O2consumption = Cardiac output ×

Hb ×1.39 ×(%satarterial –%sat

venous)/100

which is expressed as

(mL O2/minute) =

L/minute×g/L×mLO

2/g

The oxygen saturation of arterial and ve-

nous hemoglobin varies with the partial

pressure of oxygen. Under normal circum-

stances the pO2falls from 100 mm Hg in the

arteries to 40 mm Hg in the veins as the tis-

sues extract oxygen, and hemoglobin satu-

ration falls from near 100% in the arteries to

approximately 75% in the veins; thus, the

oxygen extraction ratio is 0.25. That is, the

hemoglobin “gives up” only 25% of its oxy-

gen. When tissue demand for oxygen in-

creases or the supply of oxygen decreases,

the tissues extract a greater fraction of oxy-

gen from the plasma and from hemoglobin;

this results in a lower venous pO2and de-

creased oxygen saturation of the venous

blood. Studies in primates suggest that a

critical point of limited oxygen delivery is

reached when the oxygen extraction ratio

approaches twice normal or 0.50.2

Under normal resting conditions, the

body has a large reserve of oxygen supply

relative to demand. In the average adult,

approximately 1000 mL/minute is available

to the tissues and only 250 mL/minute is

consumed as follows:

O2supply (Calculation assumes a hemo-

globin of 140 g/L and a pO2of 100)

= Cardiac output × O2contentarterial

= 5 L/minute × [(140 × 1.39 × 100%)

+ (100 × 0.003)]

= 5 L/minute × 200 mL O2/L

= 1000 mL O2/minute

O2consumption = Cardiac output × (O2

contentarterial –O

2contentvenous)

= 5 L/minute × (200 mL O2/L –

150 mL O2/L)

=5L/minute×50mLO

2/L =

250 mL O2/minute

Compensation for Anemia

The above equations demonstrate that any

decrease in oxygen content due to anemia

can be compensated for by an increase in

cardiac output.1,2 This occurs because of

increased cardiac work and also because

anemia decreases blood viscosity, and

thus peripheral vascular resistance. The

increase in oxygen supply provided by in-

creased cardiac output is augmented by

increased oxygen extraction. Oxygen ex-

traction is augmented acutely by a de-

crease in tissue oxygen tension, acting at

the steep portion of the hemoglobin-oxy-

gendissociationcurve

1(see Fig 8-1), and

by acidosis, which promotes oxygen dis-

sociation from hemoglobin. Over time, an

increase in red cell 2,3-diphosphogly-

cerate (2,3-DPG) concentration also has a

significant positive effect on oxygen un-

Measuring the Adequacy of Oxygen Supply

As shown above, multiple factors deter-

mine oxygen delivery to the tissues, ex-

cept at the lower extremes. Therefore,

measurements in addition to the hemo-

globin must be used to guide most trans-

fusion decisions. The adequacy of the

oxygen supply depends on the partial

484 AABB Technical Manual

pressure of inspired oxygen, gas exchange

in the lungs, the patient’s cardiac perfor-

mance, hemoglobin, oxygen-hemoglobin

affinity, and current oxygen demand; all

but the oxygen-hemoglobin affinity are

subject to substantial variation. For pa-

tients in an intensive care unit or in the

operating room, direct measurement of

the cardiac output and mixed venous oxy-

gentension,inassociationwithhemoglo

bin level, may serve as more physiologic

guides for transfusion decisions than he-

moglobin alone. Nonetheless, most such

decisions will continue to be made based

on hemoglobin and standard clinical as-

sessment.

Determinants of cardiac performance

include the patient’s intravascular volume,

the anemia-related reduction in peripheral

vascular resistance, the presence of coro-

nary artery disease or other forms of heart

disease, and the patient’s age. Tissue oxy-

gen debt results when oxygen demand ex-

ceeds supply; tissues convert to anaerobic

metabolism and produce increased quanti-

ties of lactic acid. Metabolic acidosis, in

turn, impairs cardiac performance, further

decreasing perfusion and tissue oxygen de-

livery, leading to greater tissue hypoxia in a

vicious cycle.

Treating Inadequate Oxygen Supply

RBC transfusion is the most direct means

of raising the hemoglobin concentration.

Other ways to improve oxygen supply rel-

ative to demand include increasing tissue

perfusion (maximizing cardiac perfor-

mance), increasing oxygen saturation

with supplemental oxygen or mechanical

ventilation, and decreasing tissue oxygen

demands with bed rest, antipyretics, and

avoidance of hypertension.

For example, a patient with a hemoglo-

bin of 6 g/dL and a cardiac index of 5 L/

minute/m2has decreased oxygen delivery.

Assuming that oxygen extraction remains

constant, oxygen delivery could be normal-

ized by an increase in the hemoglobin con-

centration to 9 g/dL, or by an increase in

the cardiac index to 7 L/minute/m2.A

smaller increase in hemoglobin would

blunt the required increase in cardiac in-

dex.

Red-Cell-Containing Components

Whole Blood

Whole Blood provides oxygen-carrying

capacity, stable coagulation factors (the

concentrations of Factors V and VIII de-

crease during storage), and blood volume

expansion. Thus, it is useful for patients

with concomitant red cell and volume

deficits, such as actively bleeding pa-

tients, and will help support coagulation

in appropriate clinical settings such as

liver transplantation.4In fact, Whole Blood

is rarely available for allogeneic transfu-

sion; RBCs and asanguinous solutions

have become the standard for most cases

of active bleeding in trauma and surgery,

with supplementation of hemostatic ele-

ments as needed. The major use of Whole

Blood in the United States today is for

autologous transfusion (see Chapter 5).

Red Blood Cells

Red cell components are indicated for the

treatment of patients who require an in-

crease in oxygen-carrying capacity and

red cell mass.5The transfusion of red cells

increases oxygen-carrying capacity with

less expansion of blood volume per unit

than Whole Blood. This is important for

patients who are at risk for circulatory

overload (eg, neonates or patients with

congestive heart failure). In a typical adult

in the absence of bleeding, hemolysis, or

major fluid shifts, one RBC unit is ex-

pected to raise the hemoglobin concen-

Chapter 21: Blood Transfusion Practice 485

trationbyapproximately1g/dL,orthe

hematocrit by 3%.

Selection of Whole Blood and Red Cell

Components

Whole Blood must be ABO identical. RBC

units need not be ABO identical but must

be compatible with ABO antibodies in the

recipient’s plasma. Rh-negative recipients

should receive Rh-negative Whole Blood

or red cell components. In cases of

trauma or massive transfusion, it may be

necessary to use Rh-positive components,

as discussed below. See Table 21-1 for

blood group selection in red cell transfu-

sion. Selection of RBC units for patients

with blood group alloantibodies is dis-

cussed in Chapter 19.

Indications for Transfusion

Several organizations have published guide-

lines for RBC transfusion.3,6,7 Reviews of

indications for transfusion are also avail-

able.1,8,9

Blood Loss and Perioperative Transfusion

For actively bleeding patients, the goal of

initial treatment should be to prevent the

development of hypovolemic shock by

stoppingthebleedingandrestoringintra

vascular volume. Efforts to restore volume

by the infusion of crystalloid or colloid so-

lutions take precedence over restoration

of oxygen-carrying capacity and should

be started immediately. In situations of

acute bleeding, guidelines suggest trans-

fusing patients who have lost 30% to 40%

of their blood volume in conjunction with

other measures to correct and maintain

total blood volume.3,6 Patients with car-

diac or other disease may require replace-

ment sooner. Healthy resting adults have

been demonstrated to tolerate acute iso-

volemic hemodilution to hemoglobin

concentrations as low as 5 g/dL without

demonstrating evidence of inadequate

oxygenation.10 In patients who refuse

transfusion on religious grounds and un-

dergo surgery, mortality increases pro-

gressively below postoperative hemoglo-

bin levels of 5 or 6 g/dL, particularly for

those with cardiovascular disease.11

Perioperative transfusion accounts for

55% to 65% of red cell component use.12

Randomized trials have demonstrated the

safety of a “transfusion trigger” of 8 g/dL of

hemoglobin in patients undergoing cardio-

vascular surgery,13,14 orthopedic surgery,15

and acute gastrointestinal bleeding.16 Even

before most of this evidence was available,

an NIH consensus conference on periopera-

tive red cell transfusion7emphasized that a

hemoglobin of 10 g/dL was inappropriate

as a guideline or transfusion trigger in the

perioperative setting and suggested a he-

486 AABB Technical Manual

Table 21-1. Suggested ABO Group Selection Order for Transfusion of RBCs

Recipient

ABO Group

Component ABO Group

1st Choice 2nd Choice 3rd Choice 4th Choice

AB AB A B O

AAO

BBO

moglobin of 7 g/dL as a level at which

transfusion was frequently required in oth-

erwise healthy individuals with acute ane-

mia. Factors to be considered in making an

individual transfusion decision included

“the duration of the anemia, the intravas-

cular volume, the extent of the operation,

the probability of massive blood loss, and

thepresenceofcoexistingconditionssuch

as impaired pulmonary function, inade-

quate cardiac output, myocardial ischemia,

or cerebrovascular or peripheral circulatory

disease.”7The guidelines also emphasized

that transfusion does not improve wound

healing, which depends on pO2rather than

total oxygen content of the blood.

A guideline by an American Society of

Anesthesiologists task force3cited a hemo-

globin below 6 g/dL as “almost always” in-

dicating transfusion, a hemoglobin above

10 g/dL as rarely indicating transfusion,

and the range in between as the realm of

clinical judgment. A task force of the Col-

lege of American Pathologists reached a

similar conclusion and proposed several

objective measures that might indicate red

cell transfusion for hemoglobin levels in the

range of 6 to 10 g/dL, including tachycardia

or hypotension in the face of normovole-

mia, a mixed venous pO2of <25 torr, an ox-

ygen extraction ratio >50%, or a total oxy-

gen consumption of <50% of baseline.6

In the past, there was some concern that

transfusions of a single RBC unit were likely

to represent unnecessary intervention. How-

ever, if transfusion of a single unit will

achieve the desired clinical outcome, then

only one unit should be transfused. Trans-

fusing additional units in this setting will

increase the risk of transfusion without any

additional benefit.

Anemia

Among medical patients, those with car-

diovascular and malignant diseases ac-

count for a large proportion of those re-

ceiving RBC units.13 In a prospective ran-

domized trial of red cell transfusion, ane-

mic but euvolemic patients in the

intensive care unit (ICU) were assigned to

either “restrictive” or “liberal” transfusion

regimens that maintained the hemoglo-

bin between 7 and 9 g/dL or between 10

and 12 g/dL, respectively.17 The mortality

rate during hospitalization (but not at 30

days) was significantly lower in the re-

strictive-strategy group (22.3% vs 28.1%, p

= 0.05). No difference in mortality rate

was seen among all patients with clini-

cally significant cardiac disease. One third

of the restrictive group avoided transfu-

sion, and total red cell use was half that of

the liberal group. These authors con-

cluded that a restrictive strategy of red

cell transfusion is at least as effective as,

and possibly superior to, a liberal transfu-

sion strategy in critically ill patients, with

the possible exception of the subset of pa-

tients with acute myocardial infarction

(MI) and unstable angina. Reanalysis of

the patients in this study with cardiovas-

cular disease showed a trend, albeit not

statistically significant, toward increased

mortality in the restrictive group among

patients with MI and unstable angina.18

A retrospective study of a large number

of elderly (>65 years old) patients hospital-

ized with acute MI divided into groups ac-

cording to admission hematocrit compared

30-day mortality rates in patients who re-

ceived transfusion and those who did not.19

Transfusion appeared beneficial in patients

with a hematocrit <30%. This result per-

sisted when data were adjusted for multiple

clinical and institutional factors.

Patients with chronic anemia tolerate a

low hemoglobin better than those with

acute anemia because of cardiovascular

compensation and increased oxygen ex-

traction. Moreover, patients at bed rest

who are not febrile, who do not have con-

Chapter 21: Blood Transfusion Practice 487

gestive heart failure, and who are not

hypermetabolic have low oxygen require-

ments and may tolerate anemia remark-

ably well. However, the high oxygen needs

of cardiac muscle may precipitate angina

in patients with cardiac disease and ane-

mia. A hemoglobin concentration of 8 g/

dL adequately meets the oxygen needs of

most patients with stable cardiovascular

disease.

Although it is desirable to prevent un-

necessary transfusions, anemic patients

who are symptomatic should receive ap-

propriate treatment. Anemia may cause

symptoms of generalized weakness, head-

ache, dizziness, disorientation, breathless-

ness, palpitations, or chest pain, and signs

include pallor (not cyanosis) and tachycar-

dia. Elwood and coworkers20 could not cor-

relate symptoms with the hemoglobin level

in patients with chronic iron deficiency

anemia and a hemoglobin as low as 8 g/dL.

A study of the use of erythropoietin demon-

strated improvement in symptoms as he-

moglobin is raised to 10 g/dL, but no

change above that level.21 Patients with

chronic hypoproliferative anemia who are

known to be transfusion dependent should

be maintained at a level that prevents

symptoms by establishing a transfusion

schedule and then adjusting it as needed.

Platelet Transfusion

Physiologic Principles

Hemostasis occurs in four phases: the

vascular phase, the formation of a platelet

plug, the development of fibrin clot on

the platelet plug, and the ultimate lysis of

the clot. Platelets are essential to the for-

mation of the primary hemostatic plug

and provide the surface upon which fibrin

forms. Deficiencies in platelet number

and/or function can have unpredictable

effects that range from clinically insignifi-

cant prolongation of the bleeding time to

major life-threatening hemorrhage. Pla-

telet plug formation results from the com-

bined processes of adhesion, activation and

release, aggregation, and procoagulant

activity.22 Platelet adhesion to damaged

endothelium is mediated largely by the

von Willebrand factor (vWF), which binds

to the surface glycoprotein (GP) receptor

GPIb-IX-V complex. The process of acti-

vation and release causes a dramatic

change in platelet shape, with extension

of pseudopod-like structures, a change in

the binding properties of membrane acti-

vation proteins, secretion of internal gran-

ule contents, and activation of several

metabolic pathways. These changes have

many effects, including the recruitment of

additional platelets, which aggregate with

the help of fibrinogen or vWF binding to

platelet surface glycoproteins. Finally, the

platelet membrane procoagulant activity

localizes and directs formation of fibrin.

Assessing Platelet Function

Decreased platelet numbers may result

from decreased production, increased de-

struction, or splenic sequestration. Plate-

let function may be adversely affected by

such factors as drugs, liver or kidney dis-

ease, sepsis, fibrin(ogen) degradation

products, cardiopulmonary bypass, and

primary marrow disorders. Platelet hemo-

stasis is assessed by the medical history,

physical examination, and laboratory

tests including platelet count and bleed-

ing time or in-vitro platelet function as-

says (eg, PFA100). Patients with inadequate

platelet number or function may demon-

strate petechiae, easy bruising, mucous

membrane bleeding, nose and gum bleed-

ing, and hematuria. Preprocedure platelet

counts have predicted bleeding in some

studies23,24 but not in others.25-27

488 AABB Technical Manual

Bleeding time measures both the vascu-

lar phase and the platelet phase of hemo-

stasis. Although the bleeding time may be a

useful diagnostic test in the evaluation of

patients with known or suspected abnor-

malities of platelet function, it is a poor pre-

dictor of surgical bleeding28 and is not a re-

liable indicator of the need for platelet

transfusion therapy.29 The in-vitro measure-

ment of platelet function is useful but, like

the bleeding time, is a poor predictor of

bleeding.30

Platelet Life Span and Kinetics

Platelets normally circulate with a life span

of 10.5 days,31 and platelets that have been

properly collected and stored have a near

normal residual mean life span of 4 to 5

days when reinfused into the original do-

nor. Conditions that shorten platelet life

span include splenomegaly, sepsis, drugs,

disseminated intravascular coagulation

(DIC), auto- and alloantibodies, endothe-

lial cell activation, and platelet activation

(eg, cardiopulmonary bypass or intra-aor-

tic balloon pumps). Because a relatively

constant number of platelets (7,000-

10,000/µL/day) are consumed by routine

plugging of minor endothelial defects, the

fraction of the circulating platelet pool re-

quired for maintenance functions increases

as the total number of platelets declines.

Therefore, the life span of native and

transfused platelets decreases with pro-

gressive thrombocytopenia.31 The re-

sponse to platelet transfusion is best as-

sessed by observing whether bleeding

stops and by measuring the posttransfu-

sion platelet increment. The posttransfu-

sion increment is generally measured

between 10 minutes and 1 hour after

completion of the transfusion and is ex-

pressed as a corrected count increment

(CCI) or percent recovery, as outlined in

Chapter 16. Two consecutive poor re-

sponses suggest platelet refractoriness.

Platelet Components

Platelets

A single unit of Platelets prepared from an

individual unit of Whole Blood may be

adequate for transfusion to neonates or

infants, but, for adults, 4 to 6 units are or-

dinarily pooled for transfusion to achieve

a dose greater than 3.0 × 1011 platelets.

This should increase the platelet count by

30,000 to 60,000/µL.

Platelets Pheresis

Units of platelets prepared by apheresis

technology (“single-donor platelets”) have

a platelet content similar to that of pooled

platelets from four to six donors and, de-

pending on the equipment used, may

have a reduced leukocyte content. The

fact that a single such unit can provide an

entire transfusion facilitates provision of

compatible platelets to recipients who are

refractory because of alloimmunization.

Transfusion of recipients who are refrac-

tory to platelet transfusions is discussed

in Chapter 16.

Selection of Platelets

ABO Matching

Because ABO antigens are present on the

platelet surface, recovery of group A pla-

telets transfused into group O patients is

somewhat decreased,32 but this effect is

not usually clinically significant (see

Chapter 16). Transfusion of ABO-incom-

patible plasma present in platelet compo-

nents may also result in a blunted post-

transfusion platelet count increment.33

Hemolysis occurs rarely in this setting but

it frequently causes a positive direct

antiglobulin test (DAT), which may in-

crease costs and charges to the patient for

Chapter 21: Blood Transfusion Practice 489

serologic investigation. Moreover, a retro-

spective analysis suggested that survival

after marrow transplantation was signifi-

cantly reduced in patients who received

substantial amounts of ABO-incompati-

ble plasma from platelet transfusion,34

and it has been suggested that infusion of

soluble A and B antigen in platelet or

plasma components may have a similar

adverse effect mediated by immune com-

plex function.35 Therefore, it may be pru-

dent to use ABO-matched platelets, par-

ticularly for patients requiring repeated

transfusions. However, urgently needed

transfusions should not be delayed in or-

der to obtain them.

For infants, it is desirable to avoid ad-

ministration of plasma that is incompatible

with the infant’s red cells; if platelets con-

taining compatible plasma are not avail-

able, the plasma can be reduced (see

Method 6.15). This is rarely necessary in

adults or older children, although signifi-

cant hemolysis has been reported after

transfusion of group O Platelets Pheresis

with high-titer anti-A or B.36 If transfused

ABO antibodies are detectable in the recipi-

ent, it may become necessary to use group

ORBCunits.

Matching for Rh

The D antigen is not detectable on plate-

lets, and posttransfusion survival of plate-

lets from Rh-positive donors is normal in

recipients with anti-D. However, platelet

components contain small numbers of

red cells so Rh-negative individuals may

become alloimmunized by platelet com-

ponents from Rh-positive donors. For im-

munocompetent normal Rh-negative fe-

males of childbearing potential, it is

especially desirable to avoid administra-

tion of platelets from Rh-positive donors;

however, if this is unavoidable, Rh Im-

mune Globulin (RhIG) should be admin-

istered. If hematoma formation is an is-

sue, an intravenous form of RhIG is avail-

able. A full dose of RhIG, which is consid-

ered immunoprophylactic for up to 15 mL

of Rh-positive red cells, should protect

against the red cells in a minimum of 30

unitsofRh-positivePlateletsor7unitsof

Rh-positive Platelets Pheresis.

Therapeutic Platelet Transfusion

Significant bleeding due to thrombo-

cytopenia or abnormal platelet function is

an indication for “therapeutic” platelet

transfusion.37 The decision to transfuse

platelets depends on the cause of bleed-

ing, the patient’s clinical condition, and

the number and function of the circulat-

ing platelets.3,38-40 Platelet transfusions are

most likely to be of benefit when thrombo-

cytopenia is the primary hemostatic de-

fect. The goal is to maintain counts

>50,000/µL. Other blood components

may also be required in patients with

multiple defects. Bleeding due to the de-

fects in platelet function that follow

cardiopulmonary bypass surgery or to the

ingestion of aspirin-containing com-

pounds, glycoprotein IIb/IIIa antagonists

(eg, abciximab), and P2 inhibitors (eg,

clopidigrel and ticlopidine) often re-

sponds to platelet transfusion. Other de-

fects such as those found in uremia or von

Willebrand disease respond less well be-

cause the transfused platelets tend to ac-

quire the same defect.

Prophylactic Platelet Transfusion

Indications for prophylactic platelet

transfusion are more controversial than

for therapeutic rationales. A threshold of

20,000/µL or less for patients with chemo-

therapy-induced thrombocytopenia has

been used by many physicians, but pro-

spective randomized trials have shown

that a threshold of 10,000/µLinstablepa

490 AABB Technical Manual

tients is equally safe and results in signifi-

cant decreases in platelet usage.41-43 A

higher transfusion trigger is often used for

patients with fever, evidence of rapid con-

sumption, high white cell counts, coagu-

lation defects, and intracranial lesions.38

In contrast, many stable thrombocyto-

penic patients can tolerate platelet counts

as low as 5000/µL.41

Despite the widespread use of prophy-

lactic platelet transfusions, few studies have

documented their clinical benefit. One

study comparing patients given prophylac-

tic transfusions with patients transfused

only for clinically significant bleeding dem-

onstrated a significant decrease in bleed-

ing, but the number of patients in the study

was too small to show a difference between

the groups in overall survival or in deaths

due to bleeding.44 Of interest, the prophy-

lactic group received twice as many platelet

transfusions, and there was a suggestion

that refractoriness developed more often in

this group. This observation raises the ca-

veat that prophylactic platelet transfusion

may be most relevant to patients in whom

thrombocytopenia is expected to be a tem-

porary condition.38 If thrombocytopenia or

platelet dysfunction will be prolonged, the

development of refractoriness may limit the

response to platelets, particularly if im-

mune function is normal as it is in patients

with aplastic anemia or congenital thrombo-

cytopathies.

Patients with severe preoperative thrombo-

cytopenia are generally assumed to benefit

from prophylactic platelet transfusion, but

this has not been demonstrated in experi-

mental studies. The threshold for such pro-

phylaxis is typically set at platelet counts

between 50,000 and 100,000/µL.3Prophy-

lactic transfusion of platelets has been in-

vestigated in circumstances in which

thrombocytopenia is expected to develop

intraoperatively, either because of dilution45

or cardiopulmonary bypass46;inbothcases,

prophylaxis was ineffective. Although it en-

dorsed the logic of prophylactic platelet

transfusion for thrombocytopenic patients

undergoing surgery, the NIH consensus

panel38 suggested that such transfusions

were most appropriate for patients in

whom hemorrhage could not be observed

or in whom it occurred at a site where it

could be critical in small amounts (eg, in

the central nervous system). Published

guidelines3,39 suggest a platelet transfusion

trigger of 50,000/µL for most major surgery,

with counts “near” 100,000/µL possibly re-

quired for patients undergoing neurosur-

gery or ophthalmic procedures.39 Prophy-

lactic platelet transfusion may also be

useful for patients who are having surgery

and who have a platelet function defect,39

including that due to treatment with

abciximab.47

Refractoriness to Platelet Transfusion

Platelet refractoriness, defined as a poor

increment following a dose of platelets, can

result from either immune or nonimmune

mechanisms and is discussed in detail in

Chapter 16 and other reviews.48,49 The anti-

bodies that cause immune refractoriness

may have either allo- or autoreactivity, with

alloantibodies most commonly directed

against Class I HLA antigens. Autoantibodies

occur in immune thrombocytopenic pur-

pura (ITP) (see Chapter 16). Nonimmune

causes of the refractory state include infec-

tion, splenomegaly, drugs (particularly

amphotericin B), and accelerated platelet

consumption (see Table 16-3).

Contraindications to Platelet Transfusion

There are several conditions for which

platelet transfusions may be requested

but are contraindicated. Relative contra-

indications include conditions in which

the likelihood of benefit is remote; trans-

fusion in this setting merely wastes a valu-

Chapter 21: Blood Transfusion Practice 491

able component. Examples include pro-

phylactic platelet transfusions in stable

patients with ITP50 or platelet refractoriness.

For ITP patients undergoing splenectomy,

transfusion of platelets should be delayed

until the vascular pedicle is clamped.

Platelet transfusion should be avoided for

patients with thrombotic thrombocyto-

penic purpura (TTP) or active heparin-in-

duced thrombocytopenia except in life- or

organ-threatening hemorrhage. These

conditions are associated with platelet

thrombi, and major thrombotic compli-

cations may follow platelet transfusions.51

Granulocyte Transfusion

The use of granulocyte transfusions for

adult recipients is rare. New antibiotics,

adverse effects attributable to granulocyte

transfusions, the advent of recombinant

growth factors, and difficulty demonstrat-

ing efficacy have contributed to this de-

cline. Nevertheless, in selected patients,

transfused granulocytes may produce

clinical benefits,52 particularly with the

larger granulocyte doses available from

donors treated with granulocyte colony-

stimulating factor.53 Attention to HLA

compatibility is also required for allo-

immunized recipients.54 The preparation,

storage, and pretransfusion testing of

granulocytes are discussed in Chapter 6,

andtheiruseinneonatesisdiscussedin

Chapter 24.

Indications and Contraindications

The goals of granulocyte transfusion should

be clearly defined before a course of ther-

apy is initiated. In general, the patient

should meet the following minimum con-

ditions:

1. Neutropenia (granulocyte count less

than 500/µL).

2. Fever for 24 to 48 hours, positive

bacterial or fungal blood cultures, or

progressive parenchymal infection

unresponsive to appropriate antibi-

otic therapy.

3. Myeloid hypoplasia.

4. A reasonable chance for recovery of

marrow function.

Patients with documented granulocyte

dysfunction, such as those with chronic

granulomatous disease, may also be candi-

dates to receive granulocyte transfusions

during life-threatening episodes of infec-

tion or while awaiting hematopoietic pro-

genitor cell transplantation.

Other Considerations

Granulocyte components contain signifi-

cant amounts of red cells, which must be

crossmatch compatible and Rh specific,

particularly for females with childbearing

potential. Granulocytes should be irradi-

ated to avoid the risk of graft-vs-host dis-

ease (GVHD). If cytomegalovirus (CMV)

transmission is an issue, its risk can be re-

duced by the use of a CMV-seronegative

donor (leukocyte reduction filters are

contraindicated). For alloimmunized re-

cipients, donors should be matched by HLA

typing or leukocyte crossmatching.52,54

Special Cellular Blood

Components

Leukocyte Reduction

The approximate leukocyte content of com-

mon blood components is summarized in

Table 21-2.55-59 Leukocyte reduction has

been used for some time for select groups

of patients. Current federal guidelines57,58

and AABB Standards for Blood Banks and

Transfusion Services59(pp25,28,29,31,32) define a

leukocyte-reduced component as one

with <5 ×106residual donor leukocytes

492 AABB Technical Manual

per final product (this includes RBCs;

Platelets Pheresis; and pooled Platelets).

AABB Standards59(p31) requires <8.3 ×105

leukocytes in Platelets Leukocytes Re-

duced, which are prepared from a single

unit of Whole Blood, to achieve the re-

quirement for a pool of 6 platelet units.

Draft guidance from the Food and Drug

Administration (FDA) recommends qual-

ity control to indicate with 95% confi-

dence that more than 95% of blood units

meet these criteria. By comparison, Euro-

pean guidelines define leukocyte-reduced

components as those with <1 ×106resid-

ual leukocytes per unit and require that

thereshouldbenomorethana10%fail

urerateintheprocess.

Published data demonstrate that leuko-

cyte reduction reduces the risk of:

1. Febrile nonhemolytic reactions (see

Chapter 27).

2. CMV transmission.60

3. HLA alloimmunization that may lead

to patients becoming refractory to

platelet transfusions.61

Controversial and unproven indications

for leukocyte reduction include:

1. Reduction of immunomodulation that

may lead to an increased risk of can-

cer recurrence or bacterial infections.62

2. Reductionintheriskofpriondisease.

3. ReductionintheriskofYersinia entero-

colitica contamination of RBC units.63

Many studies have investigated the pos-

sibility that leukocyte reduction can reduce

the incidence of clinical outcomes due to

transfusion-related immunomodulation,

but the results are contradictory.62 One such

proposal was that savings related to re-

duced immunomodulation could offset the

costs of leukocyte reduction, but this was

not demonstrable in a large prospective

randomized trial.64 Nonetheless, several

countries have converted to a leukocyte-re-

duced blood supply, and this subject re-

mains controversial.65

Irradiation

Irradiation of a cellular blood component

is the only accepted method to prevent

GVHD. GVHD has been reported after

transfusion of leukocyte-reduced compo-

nents.66 For more details on GVHD and in-

dications for irradiation, see Chapter 27.

Replacement of Coagulation

Factors

Physiologic Principles

Coagulation results from a complex but

ordered enzyme cascade occurring on the

surface of platelets and cells that express

tissue factor (see Fig 21-1). The coagula-

tion cascade is typically divided into the

intrinsic and the extrinsic pathways, the

Chapter 21: Blood Transfusion Practice 493

Table 21-2. Approximate Leukocyte

Content of Blood Components (per

Unit)55-59

Whole Blood 109

RBCs 108

RBCs Washed 107

RBCs Deglycerolized 106-107

RBCs Leukocytes Reduced

(by filtration)*

<5×10

Platelets Pheresis 106-108

Platelets 107

Platelets Pheresis

Leukocytes Reduced*

<5×10

Platelets Leukocytes Reduced <8.3 × 105

Platelets Pooled

Leukocytes Reduced

<5×10

FFP Thawed <0.6 × 106-

1.5×10

*Leukocyte reduction with third-generation leukocyte ad-

sorption filter.

in-vitro activity of which can be measured

by the activated partial thromboplastin

time (aPTT) and prothrombin time (PT),

respectively, but, in vivo, the cascades are

interdependent.67 The central procoagu-

lant enzyme is thrombin, which is acti-

vated by both pathways.

Minimal levels of coagulation factors (see

Table 21-3) are required for normal forma-

tion of fibrin and hemostasis, so normal

plasma contains coagulation factors in ex-

cess, a reserve that usually allows patients

to tolerate replacement of one or more

blood volumes of red cells and crystalloid

without needing Fresh Frozen Plasma (FFP).

Patients with liver disease have less physio-

logic reserve and are more susceptible to

dilutional coagulopathy.

Monitoring Hemostasis

The PT, aPTT, and measurement of

fibrinogen level are commonly used to

monitor coagulation. Results should be

interpreted with four considerations in

mind: 1) mild prolongations of the PT or

aPTT occur before the residual factor con-

centration falls below the level normally

needed for hemostasis; 2) conversely, the

PT and aPTT are relatively insensitive to

low fibrinogen levels; 3) significant defi-

ciencies of coagulation factors (or the

presence of coagulation factor inhibitors)

cause clearly prolonged values for the PT

or aPTT; and 4) an infusion of FFP that in-

creases the concentration of factors by 20%

will have a far greater impact on a greatly

494 AABB Technical Manual

Figure 21-1. (A) The classic cascade model of coagulation reactions was based on in-vitro experimen-

tal data in cell-free systems. The term extrinsic reflects the fact that tissue factor does not circulate in

plasma. (B) More recent evidence emphasizes that the coagulation reactions occur on the surfaces of

tissue factor-bearing cells at the site of injury and on the surface of platelets that are subsequently re-

cruited. HK = high-molecular-weight kininogen, PK = prekallikrein, TF = tissue factor, TFPI = tissue

factor pathway inhibitor. (Adapted with permission from Roberts et al.67)

Roberts HR, Monroe DM III, Hoffman M. Molecular biology and biochemistry of the coagulation factors and pathways of

hemostasis. In: Beutler E, Lichtman MA, Coller BS, et al. Williams’ hematology. 6th ed. New York: McGraw-Hill, 2001:1409-34.

Chapter 21: Blood Transfusion Practice 495

Table 21-3. Coagulation Factors

Factor Name

In-vivo

Half-life

In-vitro, 4 C

Half-life

%ofNormal

Needed for

Hemostasis

% In-vivo

Recovery Initial Therapeutic Dose

I Fibrinogen 3-6 days Years 12-50 50-70 1 bag cryoprecipitate/7 kg body weight

II Prothrombin 2-5 days >21 days 10-25 50 10-20 units/kg body weight

V Labile factor, Proaccelerin 4.5-36 hours 10-14 days 10-30 ~80 10-20 mL plasma/kg body weight

VII Stable factor, Proconvertin 2-5 hours >21 days >10 100 10-20 units/kg body weight

VIII Antihemophilic factor 8-12 hours 7 days 30-40 60-70 See Table 21-6

IX Plasma thromboplastin

component, Christmas factor

18-24 hours >21 days 15-40 20 See Table 21-6

X Stuart-Prower factor 20-42 hours >21 days 10-40 50-95 10-20 units/kg body weight

XI Plasma thromboplastin

antecedent (PTA)

40-80 hours >28 days 20-30 90 10-20 mL/kg body weight

XIII Fibrin stabilizing factor 12 days >21 days <5 50-100 500 mL plasma every 3 weeks

AT Antithrombin 60-90 hours >42 days 80-120 50-100 40-50 IU/kg body weight

Notes:

1. All dosings are provided as a general guideline for initial therapy; the exact loading dose and maintenance intervals should be individualized for each patient.

2. One unit of coagulation factor is present in each mL of Fresh Frozen Plasma.

3. DDAVP is the treatment of choice for patients with hemophilia A who are responders.

4. Composite data from the following references:

a. Beutler E, Lichtman MA, Coller BS, Kipps TL, eds. Williams’ hematology. 5th ed. New York: McGraw-Hill, 1995:1413-58, 1657.

b. Mollison PL, Engelfriet CP, Contreras M. Blood transfusion in clinical medicine. 10th ed. Oxford: Blackwell Scientific Publications, 1997:459-88.

c. Huestis DW, Bove JR, Case J, eds. Practical blood transfusion. 4th ed. Boston, MA: Little Brown and Co, 1988:319.

d. Counts RB, Haisch C, Simon TL, et al. Hemostasis in massively transfused trauma patients. Ann Surg 1979;190:91-9.

e. Package inserts.

prolonged PT or aPTT than on a mildly

prolonged PT or aPTT. For example, the

infusion of two units of FFP in a patient

with a PT of 14.5 seconds (normal: 11 to

13 seconds) is unlikely to provide any clin-

ical benefit and is also unlikely to correct

the PT to the normal range.

Guidelines typically cite a PT 1.5 times

the upper limit of normal3or the midpoint

of the normal range39 and an aPTT 1.5 times

the upper limit of normal3,39 as thresholds at

which therapeutic or prophylactic replace-

ment may be indicated in an appropriate

clinical setting. Of note, however, studies

have consistently shown that the PT and

aPTT, even when elevated to this degree,

have little predictive value for bleeding

complications of invasive procedures in-

cluding paracentesis or thoracentesis,25 liver

biopsy,26,27 angiography,23 or central venous

catheter placement.24

Components and Products Available for

Coagulation Factor Replacement

The plasma components that are avail-

able differ according to the timing of

freezing and/or thawing and variations in

preparation (see Chapter 8). FFP contains

all the clotting factors, including labile

Factors V and VIII. Other forms of plasma

have lower levels of labile factors but

could substitute for FFP for most of the

indications for which the latter is trans-

fused. Plasma Cryoprecipitate Reduced

has a decreased content of vWF and is

used specifically for treatment of TTP (see

Chapter 6). Pooled Plasma, Solvent/De-

tergent-Treated is no longer available in

the United States.

Cryoprecipitated AHF (CRYO) is a con-

centrate of high-molecular-weight plasma

proteins that precipitate in the cold, includ-

ing vWF, Factor VIII, fibrinogen, Factor XIII,

and fibronectin (see Chapter 8).

Plasma derivatives are concentrates of

specific plasma proteins from large pools of

plasma or cryoprecipitate. Cohn fraction-

ation, which relies on the precipitation of

various plasma proteins in cold ethanol-

water mixtures, was developed during

World War II and is still used with some

modifications.68 After fractionation, deriva-

tives undergo further processing to purify

and concentrate the proteins and inactivate

contaminating viruses. Virus-inactivation

procedures include heat treatment, micro-

filtration, the use of chemical solvents and

detergents, and affinity column purifica-

tion. Factors VIII, IX, VIIa, and anti-

thrombin are also produced by recombi-

nant DNA technology. These products

appear to be efficacious and are not known

to carry any infectious risk.

The specific activity (factor units/mg

protein) of presently available concentrates

has been dramatically increased in concen-

trates prepared with affinity columns or by

recombinant technology. Moreover, HIV,

HBV, and HCV transmission appear to be

absent in patients with hemophilia treated

exclusively with new preparations.69 Unfor-

tunately, their cost has also increased due

to the increased complexity of manufactur-

ing and the protein losses resulting from

extensive manipulation. Coagulation factor

concentrates are supplied in lyophilized

form and the factor activity is stated on the

label.69 Currently available products for re-

placement of Factors VIII and IX are listed

in Table 21-4.

Selection of ABO-Compatible Plasma

Because of its long shelf life, group-spe-

cificorcompatibleplasma(seeTable

21-5) is typically available. (Note that

platelet transfusions usually contain a

volume of plasma equivalent to one unit

of FFP, and limitations in availability may

496 AABB Technical Manual

Chapter 21: Blood Transfusion Practice 497

Table 21-4. Available Coagulation Factor Concentrates

Product Type Product Approved Indications Comment

Factor VIII, fractionated Humate-P Factor VIII and vWF repl. Contains vWF

Koate-HP Factor VIII replacement Contains vWF

Koate-DVI Factor VIII replacement Contains vWF

Factor VIII, affinity puri-

fied

Alphanate Factor VIII replacement Contains vWF

Factor VIII, immunaffinity

purified

Hemofil-M Factor VIII replacement

Monarc M Factor VIII replacement

Monoclate-P Factor VIII replacement

Factor VIII, recombinant Kogenate FS

Helixate FS

Factor VIII replacement Contains trace amounts

of human albumin

Recombinate

(Bioclate)

Factor VIII replacement

ReFacto Factor VIII replacement Does not contain human

albumin

Factor VIII, porcine Hyate C Factor VIII inhibitor tx.

Factor IX, affinity

purified

AlphaNine-SD Factor IX replacement

Factor IX, immunaffinity

purified

Mononine Factor IX replacement

Factor IX, recombinant BeneFIX Factor IX replacement Does not contain human

albumin

Factor IX complex Bebulin VH Factor IX replacement Contains Factor II,

Factor X, trace

Factor VII

Konyne 80 Factor IX replacement

Factor VIII inhibitor tx.

Warfarin reversal

Contains Factor II,

Factor X, some

Factor VII

Profilnine SD Factor IX replacement Contains Factor II,

Factor X, some

Factor VII

Proplex T Factor IX and Factor VII

replacement

Factor VIII inhibitor tx.

Contains Factor II,

Factor VII, and

Factor X

Factor IX complex,

activated

Autoplex-T Factor VIII inhibitor tx. Contains Factor lIa,

Factor VIIa, and

Factor Xa

FEIBA VH Factor VIII inhibitor tx. Contains Factor IIa,

Factor VIIa, and

Factor Xa

Factor VIIa,

recombinant

NovoSeven Factor VIII inhibitor tx. Does not contain human

albumin

require infusion of incompatible plasma

in this context.)

Indications for FFP

Guidelines exist for the appropriate use of

FFP.3,39,70 FFP is the only approved compo-

nent for clinically significant deficiency of

Factors II, V, X, and XI. Plasma is most of-

tenusedinpatientswithmultiplefactor

deficiencies, including those with liver

disease, dilutional and consumption

coagulopathy, or a need for rapid reversal

of warfarin treatment. It is of limited clini-

cal benefit in patients with inhibitors to

any coagulation factor. Plasma Cryopreci-

pitate Reduced may be more effective

than FFP for some patients receiving

plasma exchange treatment for TTP or

hemolytic-uremic syndrome71 (see Chap-

ter 6).

Vitamin K Deficiency and Warfarin Reversal

The most common cause of multiple co-

agulation factor abnormalities among

hospitalized patients is deficiency of the

vitamin-K-dependent factors due to treat-

ment with the vitamin K antagonist

warfarin (Coumadin) or nutritional defi-

ciency.67 Vitamin K is a fat-soluble vitamin

required for hepatocellular synthesis of

coagulation Factors II, VII, IX, and X, as

well as the anticoagulant proteins C and

S. Body stores of vitamin K last only 2

weeks, so deficiency may occur in hospi-

talized patients unable to tolerate normal

food intake. Absorption of vitamin K re-

quires precursor metabolism by bacteria

in the intestine and the action of bile salts;

therefore, deficiencies can occur with anti-

biotic use, obstructive jaundice, and fat

malabsorption syndromes.

VitaminKdepletionorwarfarinusually

cause a prolongation of the PT that is out of

proportion to the aPTT because Factor VII,

which has the shortest half-life of the vita-

min-K-dependent factors, has little effect

on the aPTT. Deficiency of vitamin K is best

managed by treatment of the underlying

condition and by administration of vitamin

K.67 If liver function is adequate, coagulation

factors will return to effective levels in

about 12 hours.

Although most patients with vitamin K

deficiency do not require plasma, plasma

transfusion is occasionally needed to treat

active bleeding or to prepare for emergency

invasive procedures.67 Transfusion of 10 to

15 mL of plasma per kg of body weight will

generally achieve hemostatic coagulation

factor levels in patients with warfarin-in-

duced coagulopathy. One form of Factor IX

complex concentrate is licensed for warfa-

rin reversal (see Table 21-4) but carries a sig-

nificant risk of thrombotic complications and

is rarely used for this indication. Concurrent

vitamin K supplementation should also be

given unless only transient correction is de-

498 AABB Technical Manual

Table 21-5. Suggested ABO Group Selection Order for Transfusion of Plasma

Recipient

ABO Group

Component ABO Group

1st Choice 2nd Choice 3rd Choice 4th Choice

AB AB (A) (B) (O)

AAAB(B)(O)

BBAB(A)(O)

OOABAB

(Blood groups in parentheses represent choices with incompatible plasma, listed in “least incompatible” order.)

sired. Although the International Normalized

Ratio can provide useful information about

response to therapy, the need for additional

treatment should be guided by the clinical re-

sponse and not by the results of laboratory

tests. As discussed above, it is rarely neces-

sary to correct the PT or aPTT to normal to

achieve adequate hemostasis.

Liver Disease

Patients with liver disease have multiple

derangements that contribute to an in-

creased bleeding tendency. These abnor-

malities include portal hypertension and

engorgement of systemic collateral ve-

nous shunts; splenomegaly with second-

ary thrombocytopenia; decreased synthe-

sis of all coagulation factors except Factor

VIII; dysfibrinogenemia; decreased clear-

ance of fibrin, fibrinogen degradation

products, and fibrinolytic activators; and

decreased synthesis of inhibitors of the

fibrinolytic system. As in vitamin K defi-

ciency, the short half-life of Factor VII

causes the PT to be prolonged more than

the aPTT, and, for the same reason, FFP

infusion corrects the PT for only about 4

hours.72 Because the defect in hepato-

cellular disease is in primary protein syn-

thesis, supplemental vitamin K will not

usually correct the abnormality. However,

because it is one of the few treatable causes

of coagulopathy in liver disease, a trial of

replacement vitamin K may be indicated.

FFP corrects coagulation factor deficien-

cies found in severe liver disease, but is of-

ten used inappropriately. The most com-

mon error is to attribute all bleeding to

coagulopathy and to give systemic treat-

ment when the cause is localized bleeding.

For example, bleeding at the operative site

after cardiac surgery usually responds

better to local hemostatic measures than to

intravenous infusion of FFP. A second com-

mon error in treating liver-associated

coagulopathy is overdependence on the PT.

Again, a normal PT is rarely, if ever, re-

quired for the cessation of serious bleeding,

and the goal of FFP therapy in severe liver

disease should be to correct or prevent

bleeding complications. If FFP is to be used

prophylactically before an invasive proce-

dure, it should be given immediately before

the procedure.

Patients with liver disease may also have

abnormalities of platelet plug formation

and fibrinolysis. In addition, severe spleno-

megaly may impair the response to platelet

transfusions. Platelet function in some pa-

tients with liver disease can be enhanced by

administration of 1-deamino-8-D-arginine

vasopressin (desmopressin, DDAVP).73 Cryo-

precipitated AHF should be given if there is

severe hypofibrinogenemia or bleeding re-

lated to dysfibrinogenemia. The increase in

systemic fibrinolysis associated with severe

liver disease may not respond to FFP alone,

and antifibrinolytic agents in combination

with plasma therapy can be useful in these

patients (see below).

Dilutional Coagulopathy

Massive blood loss and replacement with

crystalloid and/or colloid solutions may

produce a dilutional coagulopathy,74 but

most patients can tolerate loss and re-

placement of at least one blood volume

without developing impaired hemo-

stasis.75 Shock accompanying traumatic

hemorrhage also contributes to the coa-

gulopathy (see Chapter 27). In the setting

of trauma, thrombocytopenia generally

develops before plasma clotting factors are

diluted to the point of causing impaired

hemostasis, and adequate platelet re-

placement generally has priority.75 However,

in elective surgical patients, coagulation

factor deficits may predominate.74 FFP may

be beneficial if the PT and/or aPTT are

greater than 1.5 times normal.3,39 If surgi-

Chapter 21: Blood Transfusion Practice 499

cal hemostasis has not been achieved and

significant continued bleeding is expected,

FFP may be indicated.39

Patients undergoing plasmapheresis

without plasma replacement develop a va-

riety of coagulation factor deficits, particu-

larly hypofibrinogenemia, depending on

thevolumeandfrequencyoftheex

changes.76 Although these changes can be

striking, most authors have concluded that

routine supplements with FFP in patients

with normal liver function are unnecessary,

particularly for alternate-day regimens.

Other Conditions

Plasma exchange is lifesaving in TTP (see

Chapter 6). FFP may be an adjunct to treat-

ment of DIC. Hereditary angioneurotic

edema results from a congenital defi-

ciency of C1-esterase inhibitor, an inhibi-

tory protein that regulates complement

activation. Patients with this condition

develop localized edema and may experi-

ence lifethreatening obstruction of the

upper respiratory tract following comple-

ment activation. FFP or Liquid Plasma

contain normal levels of C1-esterase in-

hibitor and FFP transfusion appeared to

prevent attacks at the time of oral surgery

in one study.77 There are rare anecdotal re-

ports of exacerbation of angioneurotic

edema with FFP administration. The need

for treatment of isolated deficiency of

Factors II, V, VII, X, or XI is uncommon;

guidelines for initial therapy are given in

Table 21-3 (however, for a more complete

treatment, refer to one of the standard he-

matology or coagulation texts).

Plasma can also be used to replace pro-

teins C and S, and antithrombin; these are

discussed separately below.

Misuse of Fresh Frozen Plasma

Plasma should not be used as a volume

expander, as a nutritional source, or to

enhance wound healing.38,70,75 Transfusing

plasma for volume expansion carries a

risk of infectious disease transmission and

other transfusion reactions (eg, allergic)

that can be avoided by using crystalloid or

colloid solutions. Plasma is also not a

suitable source of immunoglobulins for

patients with severe hypogammaglobu-

linemia because more effective prepara-

tions exist (immunoglobulin for intrave-

nous or intramuscular use).

FFP is often given prophylactically to pa-

tients with mild to moderate prolongation

of the PT or aPTT before invasive proce-

dures, but there is little or no evidence that

this prevents bleeding complications. Be-

cause these tests do not accurately predict

the risk of bleeding when mildly prolonged,

there is little logic for a transfusion in-

tended to “improve” the results.

Cryoprecipitated AHF

Transfusion

CRYO is the only concentrated fibrinogen

product currently available for systemic

use, and intravenous supplementation of

fibrinogen is its primary clinical use, par-

ticularly in DIC. A second major use has

been in patients with severe von Wille-

brand disease, but there are Factor VIII

concentrates that contain vWF and are

more appropriate if available (see Table

21-4). CRYO can be used in isolated Fac-

tor XIII deficiency and to ameliorate the

platelet dysfunction associated with ure-

mia. It is also used topically as a fibrin

sealant, although a commercial prepara-

tion is available. CRYO is seldom used for

patients with hemophilia because Factor

VIII concentrates are available commer-

cially and have been processed to reduce

or eliminate the risk of blood-borne viral

infection; CRYO is used as a last resort for

this indication. Because CRYO contains

500 AABB Technical Manual

ABO antibodies, consideration should be

given to ABO compatibility when the in-

fused volume will be large relative to the

recipient’s red cell mass.

Calculating the CRYO Dose for Fibrinogen

Replacement

On average, one unit of CRYO contains

approximately 250 mg of fibrinogen; the

minimum required by AABB Standards is

150 mg.59(p30) The amount of transfused

CRYO required to correct the fibrinogen

level depends upon the nature of the

bleeding episode and the severity of the

initial deficiency and can be calculated as

follows:

1. Weight (kg) ×70 mL/kg = blood vol-

ume (mL).

2. Blood volume (mL) ×(1.0–hemato-

crit) = plasma volume (mL).

3. Mg of fibrinogen required = (Desired

fibrinogen level in mg/dL – initial

fibrinogen level in mg/dL) ×plasma

volume (mL) ÷ 100 mL/dL.

4. Bags of CRYO required = mg of fibrino-

gen required ÷ 250 mg fibrinogen/

bag of CRYO

This calculation assumes that 100% of

administered fibrinogen is recovered as

measurable intravascular fibrinogen, but,

because the content of CRYO is variable,

further refinements are unproductive.

von Willebrand Syndromes

von Willebrand syndromes are the most

common major inherited coagulation ab-

normalities.78 These conditions are usu-

ally autosomal dominant and represent a

collection of quantitative and qualitative

abnormalities of vWF, the most important

protein mediating platelet adhesion to

damaged endothelial surfaces. The pro-

tein also transports Factor VIII. As a re-

sult, patients with von Willebrand syn-

dromes have varying degrees of abnormal

platelet plug formation and partial defi-

ciency of Factor VIII. The former may

manifest as a prolonged bleeding time

and the latter as a prolonged aPTT, but

these abnormalities vary between syn-

dromes. vWF exists in the plasma as a

family of multimeric molecules with a

wide range of molecular weights. The

high-molecular-weight species of vWF are

the most hemostatically effective. Labora-

tory evaluation demonstrates a specific de-

ficiency in the level of vWF, often mea-

sured as ristocetin cofactor activity because

vWF is required for the platelet-agglutinat-

ing effect of ristocetin in vitro.

Mild cases of von Willebrand syndrome

can often be treated with DDAVP, which

causes a release of endogenous stores of

Factor VIII and vWF. Many Factor VIII con-

centrates do not contain therapeutic levels

of vWF, but several with satisfactory levels

are commercially available and one is li-

censed for this indication (see Table 21-4).

In the absence of a suitably therapeutic vi-

rus-inactivated concentrate, severe von

Willebrand syndrome can be treated with

CRYO. The quantity of CRYO required to

treat bleeding episodes or to prepare for

major surgery varies greatly among patients

with von Willebrand syndromes. In addi-

tion to the clinical response of the patient,

the template bleeding time, the level of Fac-

tor VIII, or the ristocetin cofactor activity

may help to guide therapy.

Fibrinogen Abnormalities

Hypofibrinogenemia may occur as a rare

isolated congenital deficiency, may be ac-

quired as part of the DIC syndrome, or may

be due to obstetric complications such as

abruptio placentae. Dysfibrinogenemias

may be congenital or acquired and repre-

sent conditions in which fibrinogen is

present as measured by immunoassays

but functionally defective as measured by

Chapter 21: Blood Transfusion Practice 501

the thrombin time. Patients with severe

liver disease frequently exhibit a dys-

fibrinogenemia.

Disseminated Intravascular Coagulation

DIC occurs when circulating thrombin in-

duces widespread fibrin formation in the

microcirculation and consumption of pla-

telets and coagulation factors, particularly

fibrinogen,prothrombin,FactorV,and

Factor VIII. Fibrin strands in the micro-

circulation may cause mechanical damage

to red cells, a condition called microangio-

pathic hemolysis, manifest as schistocytes

(fragmented red cells) in the circulation.

Widespread microvascular thrombi pro-

mote tissue ischemia and release of tissue

factor, which further activates thrombin.

Lysis of microvascular fibrin causes in-

creased quantities of fibrin degradation

products to enter the bloodstream.

Several clinical conditions can initiate

DIC, including shock, tissue ischemia, sep-

sis, hemolytic transfusion reactions, dis-

seminated cancer (particularly adeno-

carcinoma), acute promyelocytic leukemia,

tumor lysis syndrome, and obstetric com-

plications such as amniotic fluid embolism.

The common precipitating event is a pro-

coagulant signal for thrombin production that

exceeds the normal physiologic defenses

against disseminated thrombin activity.

Treatment of DIC depends on correcting

the underlying problem and preventing

further hypotension and tissue ischemia.

Replacement therapy focuses on the build-

ing blocks of the thrombus (platelets and

fibrinogen), and secondarily on other coag-

ulation factors, including Factors VIII, XIII,

V, and II. Thus, in bleeding patients, platelet

transfusion is indicated when the platelet

count falls below 50,000/µL, and cryopre-

cipitate is supplemented if the fibrinogen

level is below 100 mg/dL. FFP is indicated

in the setting of hemorrhage that results

from DIC once the fibrinogen is above 100

mg/dL.

Topical Use

The fibrinogen in CRYO has been used

during surgery as a topical hemostatic

preparation, so-called fibrin sealant or fi-

brin glue, made from one or two units of

CRYO, which may be of autologous origin.

The fibrinogen is converted to fibrin by

the action of bovine thrombin at the site

that is bleeding or to be “glued.” Commer-

cial preparations of fibrin sealant are

available that have a higher fibrinogen

concentration than that of CRYO and in-

clude human thrombin and bovine apro-

tinin (see below) to decrease the lysis of

the resulting fibrin. These pooled, virus-

inactivated products have been licensed

for the reduction of bleeding in cardiovas-

cular surgery,79 for repairing splenic

trauma, and for colostomy closure. Fibrin

sealants have also been used for a variety

of indications in which it is desired to

bind two tissue surfaces together, includ-

ingrepairoftheduramateroreardrum.

Useofbovinethrombincanstimulate

the formation of antibodies against thrombin

and other contaminant proteins including

Factor V.80 These antibodies can cross-react

with human thrombin and Factor V, caus-

ing abnormal clotting times and, in some

cases, bleeding. For this reason, it has been

suggested that use of “homemade” fibrin

sealants be replaced by use of the commer-

cial product.80

Hemophilia A

Each unit of CRYO prepared from a single

blood donation should contain a minimum

of 80 international units (IU) of Factor

VIII.59(p30) Although no longer the compo-

nent of choice, CRYO can serve as replace-

502 AABB Technical Manual

ment therapy for patients with hemo-

philia A if virus-inactivated Factor VIII

concentrates are unavailable.39 If CRYO is

used, the amount required to provide a

therapeutic dose of Factor VIII is based on

calculations similar to those used for AHF.

Hemophilia A is a sex-linked recessive

trait (ie, affected males, carrier females)

causing deficiency of Factor VIII (anti-

hemophilic factor, AHF).81 The responsible

genes usually produce a protein with re-

duced activity, so immunologic measure-

ment of Factor VIII antigen gives normal re-

sults despite deficient Factor VIII coagulant

activity. In contrast, antigen is typically de-

pressed in von Willebrand disease. Charac-

teristic laboratory findings include a pro-

longed aPTT, normal PT and template

bleeding time, and decreased Factor VIII

activity.

The severity of hemophilia A depends on

the patient’s level of Factor VIII activity, and

this varies; patients with severe hemophilia

have Factor VIII levels below 1%, whereas

those with moderate disease typically have

1% to 5% activity, and severity is mild with

levels of 6% to 30%.81 One unit of Factor VIII

activity is defined as the Factor VIII content

of 1 mL of fresh, citrated, pooled normal

plasma. The measured level of Factor VIII

can be expressed as a concentration, as

percent activity, or as a decimal fraction.

For example, a patient with mild hemo-

philia with one-tenth the normal activity of

Factor VIII can be said to have a Factor VIII

level of 10 units/dL or 0.1 unit/mL or 10%

activity.

Patients with mild-to-moderate hemo-

philia can often be managed without re-

placement therapy.81 Careful attention to lo-

cal hemostasis and the use of topical

antifibrinolytics may prevent the need for

further replacement. Systemic levels of Fac-

torVIIIcanberaisedinmildhemophilia

with the use of DDAVP, which stimulates

the release of endogenous Factor VIII from

storage sites. However, DDAVP is ineffective

in patients with severe hemophilia A; in

such cases, Factor VIII replacement is re-

quired. The amount of Factor VIII infused

depends upon whether therapy is intended

to prevent bleeding or, if bleeding has oc-

curred, the nature of the bleeding episode

and the severity of the initial deficiency (see

Table 21-6). For example, treatment for

hemarthrosis ordinarily requires more Fac-

tor VIII than epistaxis.

Calculating the Dose of Factor VIII

When the desired result is determined

(see Table 21-6), the amount of Factor VIII

required for transfusion can be calculated

by one of the following formulas:

Factor VIII dose (IU/kg) =

Desired factor increase (%) ×0.5 (1

IU/kg typically raises the Factor VIII

level by 2%)

Total dose =

(Patient mass ×70mL/kg) ×(1 – Hct) ×

(Desired activity – current activity)

Example: A 70-kg patient with severe he-

mophilia has an initial Factor VIII level of

2% (0.02 unit/mL) and a hematocrit of 40%.

How many units of Factor VIII concentrate

should be given to raise his Factor VIII level

to 50% (0.5 unit/mL)?

(70 ×70) ×(1 – 0.4) ×(0.5 – 0.02) =

1411 units

The therapy of choice for severe hemo-

philia A is a Factor VIII concentrate (see Ta-

ble 21-4). CRYO could be used to supply

1411 units of Factor VIII, but, at 80 IU per

bag, this would require at least 18 bags (and

18 allogeneic donor exposures). The half-

life of Factor VIII is about 12 hours, so infu-

sions are repeated at 8- to 12-hour intervals

Chapter 21: Blood Transfusion Practice 503

to maintain hemostatic levels. The duration

of treatment with Factor VIII infusions de-

pends upon the type and location of the

hemorrhage or the reason for prophylaxis,

and the clinical response of the patient (see

Table 21-6). After major surgery, the Factor

VIII level should be maintained above 40%

to 50% for at least 10 days. When elective

surgery is planned, Factor VIII assays

should be made available to serve as a

guide to therapy.

Treatment of Inhibitors of Factor VIII

About 10% to 35% of patients with hemo-

philia A, typically, those with severe dis-

ease or genetic defects involving large

portions of the molecule, develop a de-

tectable inhibitor to human Factor VIII.68,81

These antibodies are directed against the

active site of Factor VIII, rendering the pa-

tient relatively unresponsive to infusion.

Patients having an elective invasive pro-

cedure should be screened for such inhi-

bitors. Management is difficult; ap-

proaches have included attempts to

overwhelm the inhibitor with very large

doses of human Factor VIII; use of por-

cine Factor VIII, which has low cross-reac-

tivity with human Factor VIII antibody81;

use of Factor VIII bypassing agents in-

504 AABB Technical Manual

Table 21-6. General Factor Replacement Guidelines for the Treatment of Bleeding

in Hemophilia

Indication

Initial

Minimum

Desired

Factor Level

(%)

Factor VIII

Dose*

(IU/kg)

Factor IX

Dose*

(IU/kg)

Duration

(days)

Severe epistaxis, oral

mucosal bleeding†

20-30 10-15 20-30 1-2

Hemarthrosis, hematoma,

persistent hematuria,‡

gastrointestinal tract

bleeding, retroperitoneal

bleeding

30-50 15-25 30-50 1-3

Trauma without signs of

bleeding, tongue/

retropharyngeal

bleeding†

40-50 20-25 40-50 2-4

Trauma with bleeding,

surgery,§intracranial

bleeding§

100 50 100 10-14

Data from USP.69

*Dosing intervals are based on a half-life for Factor VIII of 8 to 12 hours (2 to 3 doses/day) and a half-life for Factor IX

of 18 to 24 hours (1 to 2 doses/day). Maintenance doses of one half the initial dose may be given at these intervals. The

frequency depends on the severity of bleeding, with more frequent dosing for serious bleeding.

†In addition to antifibrinolytics.

‡Painless spontaneous hematuria usually requires no treatment. Increased oral or intravenous fluids are necessary to

maintain renal output.

§Continuous factor infusion may be administered. Following the initial loading dose, a continuous infusion at a dose of 3

IU/kg per hour is given. Subsequent doses are adjusted according to the plasma factor levels.

cluding Factor IX complex, activated Fac-

tor IX complex, and activated Factor VII

(see Table 21-4); and desensitization ther-

apy. The latter includes large daily doses

of AHF in conjunction with cortico-

steroids or Immunoglobulin Intravenous

(IGIV) and cyclophosphamide. Success rates

of 50% to 80% are reported. If hemorrhage

is life-threatening, intensive plasma-

pheresis to remove the inhibiting anti-

body, coupled with immunosuppression,

as well as infusions of Factor VIII and pos-

sibly antifibrinolytic therapy (see below),

can be employed.

Hemophilia B

Factor IX deficiency (hemophilia B, Christ-

mas disease) is clinically indistinguish-

able from Factor VIII deficiency in that

both are sex-linked disorders that cause a

prolonged aPTT in the presence of a nor-

mal PT and bleeding time.81 The disorder

is confirmed by specific measurement of

Factor IX activity. Factor IX complex con-

centrate has been used for treatment of

hemophilia B for the past 2 decades, but

the new, more pure forms of Factor IX

concentrate (see Table 21-4) are preferred

because they carry much less risk of in-

ducing thrombosis.82

The formula for calculating of Factor IX

dosage is similar to that for Factor VIII, but

the units to be given should be doubled be-

causeonlyhalfoftheinfusedFactorIX

dose is recovered in the vascular space. The

biologic half-life is 18 to 24 hours, so doses

are given 1 or 2 times/day. As with hemo-

philia A, recommended dose and treatment

schedules vary with the severity and type of

bleeding (see Table 21-6).

Immunoglobulin, Intravenous

IGIV is prepared from modified Cohn frac-

tion II and subjected to virus inactivation.

Preparations intended for intramuscular

administration contain aggregates that

may activate the complement and kinin

systems and produce hypotensive and/or

anaphylactoid reactions if administered

intravenously, but the intravenous prod-

uct contains almost exclusively mono-

meric IgG molecules.83

The indications for the use of IGIV are

evolving.83,84 Some conditions in which IGIV

is used are listed in Table 21-7. Infusion of

IGIV can induce such reactions as head-

ache, vomiting, volume overload, allergic

reactions, renal failure, and pulmonary re-

actions, but they can usually be prevented

by infusing slowly and pretreating with

diphenhydramine and/or hydrocortisone.

Passively transferred blood group allo- and

autoantibodies and/or therapy-induced

hypergammaglobulinemia may cause a

positive DAT result in recipients, but signif-

icant hemolysis is rarely noted.

Antiprotease Concentrates

Antithrombin, also known as heparin co-

factor, is a serine protease inhibitor syn-

thesized in the liver.85 It circulates in nor-

mal plasma at a concentration of 15 mg/

dL but is typically measured as % activity,

with a normal range of 84% to 116%. Anti-

thrombin inactivates serine proteases in-

cluding thrombin, and Factors IXa, Xa,

XIa, and XIIa by covalently bonding at the

serine site, followed by a conformation

change.86 This activity is greatly acceler-

ated by heparin, which induces a confor-

mation change in antithrombin and helps

approximate thrombin and antithrombin

as well.

Patients who are deficient in anti-

thrombin are prone to thromboembolic

complications.87 Such deficiency can be

congenital or acquired. Acquired deficiency

occurs in a wide variety of disease states,

including decreased synthesis due to liver

Chapter 21: Blood Transfusion Practice 505

disease or malnutrition; losses due to

nephroticsyndromeandgastrointestinal

states; accelerated consumption as in DIC,

surgery or trauma, and preeclampsia; and

associated with pharmacotherapy includ-

ing heparin, L-asparaginase, and oral

contraceptives.

Antithrombin is stable in plasma so defi-

ciency can be treated with FFP or with Liq-

uid or Thawed Plasma. A heat-treated con-

centrate of antithrombin is also available;

recombinant and transgenic sources are

under investigation.

Clinical uses of antithrombin have re-

cently been reviewed.87 Antithrombin is ap-

proved for use in hereditary antithrombin

deficiency as part of the treatment for

thromboembolic episodes and for prophy-

lactic use in perioperative, postoperative,

and peripartum settings. Several off-label

uses of antithrombin exist, eg, patients with

low antithrombin levels and DIC, neonates

born to mothers with antithrombin defi-

ciency, and liver transplant recipients.

The half-life of purified antithrombin is

long,approximately60to90hours,

88 but is

abbreviated when replacement is for con-

sumptive states. The dose is calculated on

the basis of an expected increment of 1.4%

per U per kg, with an initial target of 120%.

Other available concentrates of antipro-

teases include alpha-1-proteinase inhibitor

(alpha-1-antitrypsin). C1-esterase inhibitor

is not available in the United States.

506 AABB Technical Manual

Table 21-7. Potential Indications and Clinical Uses for Intravenous Immunoglobulin

Preparations

Congenital immune deficiencies

Hypogammaglobulinemia and agammaglobulinemia

Selective antibody deficiency

IgG subclass deficiency and recurrent infection

Premature newborns

Acquired antibody deficiency

Malignancies with antibody deficiency and recurrent infection: multiple myeloma,

chronic lymphocytic leukemia

Protein-losing enteropathy

Drug- or radiation-induced humoral immunodeficiency

Prophylaxis or treatment of bacterial and viral diseases

Pediatric HIV infection for prevention of bacterial and secondary viral infections

Cytomegalovirus infection in transplant recipients

Neonatal sepsis

Other

HIV-related immune thrombocytopenic purpura

Immune cytopenias (ITP, NAIT, PTP, WAIHA)

Kawasaki syndrome

Guillain-Barré syndrome and chronic inflammatory demyelinating neuropathy

Acquired Factor VIII inhibitors

Myasthenia gravis

Multiple sclerosis

Protein C and Protein S

Protein C and protein S are vitamin-K-de-

pendent proteins with anticoagulant ef-

fects.86 Protein S is a cofactor for activated

protein C, which, in turn, inactivates Fac-

tor Va and Factor VIIIa. Patients with defi-

ciencies of protein C or protein S have a

predisposition to thrombotic complications

and are often treated with anticoagulants.85

Warfarin treatment, however, can cause

these vitamin-K-dependent proteins to

decrease to dangerously low levels, lead-

ing to skin necrosis and exacerbating

thrombosis. Transfusion of FFP can serve

as an immediate source of supplemental

protein C or protein S for patients with se-

vere deficiencies, and a human protein C

concentrate is under development.

Patients with heterozygous protein C de-

ficiency have plasma levels 40% to 60% of

normal and characteristically have minimal

symptoms, rarely requiring treatment with

protein C supplementation.85 If treatment is

needed for a thrombotic episode, anticoag-

ulants suffice. Homozygous protein C defi-

ciency causes neonatal purpura fulminans,

which requires immediate administration of

protein C, along with complex regulation of

the rest of the coagulation cascade. The

half-life of infused protein C is 6 to 16 hours.

Colloid Solutions

Human albumin (5% and 25%) and plasma

protein fraction (PPF) provide volume ex-

pansion and colloid replacement without

risk of transfusion-transmitted viruses.89

PPF has a greater concentration of non-

albumin plasma proteins than 5% albumin.

Pharmacologic agents such as hydroxyethyl

starch or dextran are also commonly used

for volume expansion.

Physiology of Albumin

The total body albumin mass is about 300

g, of which 40% (120 g) is in the plasma.

Daily albumin synthesis in a normal adult

approximates 16 g. Albumin has complex

roles in normal physiology and disease in

addition to its obvious one of mainte-

nance of intravascular volume.90 Hypo-

albuminemia resulting from decreased

synthesis, increased catabolism or losses,

and shifts between different fluid com-

partmentsiscommoninacuteandchronic

illness.

Replacement

Albumin solutions are effective volume

expanders, with the promise of better

intravascular fluid retention than simpler

and less expensive crystalloids (eg, nor-

mal saline or lactated Ringer’s solution).

Because hypoalbuminemia is involved in

the pathogenesis of many disease states

and may correlate with their prognosis, it

has been tempting to try to alter their

course by exogenous supplementation,

particularly in view of the perceived low

risk status of albumin solutions. However,

this low-risk is in question.

Indications for albumin approved by a

consensus panel91 include:

1. Volume replacement in nonhemor-

rhagic shock unresponsive to crystal-

loid or in the presence of capillary

leak syndromes.

2. Volume replacement after the first

day in patients with extensive burns

(>50%) unresponsive to crystalloid.

3. Replacement after removal of large

volumes (>4 L) of ascitic fluid in pa-

tients unresponsive to crystalloid.

4. Replacement of ascitic fluid or post-

operative treatment of ascites and

peripheral edema in hypoalbumine-

mic liver transplant recipients.

5. Replacement during large-volume

plasma exchange.

6. Volume replacement in patients with

severe necrotizing pancreatitis.

Chapter 21: Blood Transfusion Practice 507

7. Diarrhea (>2 L/day) in hypoalbum-

inemic (<2.0 g/dL) patients on enteral

feedings, unresponsive to short chain

peptide supplementation.

Nonalbumin colloid solutions were con-

sidered less expensive first alternatives in

several of these situations.

In spite of the conceptual attractiveness,

the long history of albumin replacement,

and the consensus on its use, a number of

prospective randomized trials have sug-

gested that albumin use was ineffective or

increased mortality. A meta-analysis of 30

randomized trials including 1419 patients

grouped according to indication (namely,

hypovolemia, burns, and hypoproteinemia)

demonstrated higher mortality with albu-

min treatment for each of the groups.92 In

response to this outcome, the authors of the

meta-analysis called for an immediate re-

view of albumin use in critically ill patients.

Special Transfusion

Situations

Thalassemia

Thalassemia and sickle cell disease are in-

herited syndromes characterized by defi-

cient or abnormal hemoglobin structures

and anemia. Thalassemia is caused by a

deficiency in alpha or beta chain produc-

tion that ranges from mild to severe. Total

absence of synthesis of one of the alpha

chains is lethal in utero; absence of beta

chain synthesis (thalassemia major) re-

sults in a progressive anemia in the new-

born period. In an attempt to compensate

for significant degrees of anemia, hemato-

poietic tissue expands, causing character-

istic bone abnormalities and enlargement

of the liver and spleen. Tissue iron accu-

mulates as a result of increased adsorp-

tion and transfusion (see Chapter 27). The

only current cure for thalassemia is hemato-

poietic transplantation, but because the

anemia can be controlled with red cell

transfusions and concurrent iron chela-

tion therapy, the use of this expensive and

potentially hazardous therapy is contro-

versial. Transfusion of thalassemia pa-

tients is discussed in Chapter 24.

Sickle Cell Disease

Sickle cell disease (SCD) results from a

single base substitution in the gene for

the beta chain of hemoglobin. The hemo-

globin of individuals homozygous for this

abnormality can irreversibly polymerize

and cause red cells to deform or “sickle.”

Such cells may initiate blockage of the

microvasculature directly or in associa-

tion with endothelial damage and throm-

bosis. They also have a decreased life

span, so SCD patients have a variably

compensated hemolytic anemia. Sickling,

which can be triggered by fever, infection,

or hypoxia, can lead to a variety of com-

plications or “crises,” including pain crisis

due to musculoskeletal or other tissue

ischemia, splenic or pulmonary seques-

tration crisis (chest syndrome), aplastic

crisis due to transient marrow suppression

by viruses (particularly parvoviruses), leg

ulcers, priapism, tissue infarction, and

stroke.

Most patients with sickle cell disease are

asymptomatic most of the time and do not

require routine transfusion. Although

sickling can be prevented or reversed by

maintaining the level of normal hemoglo-

bin above 50% to 70%, the risks from allo-

immunization, iron overload, and disease

transmission outweigh the benefits of pro-

phylactic transfusion in most patients.

Moreover, uncomplicated pain crises do not

respond well to simple transfusion. Simple

transfusion is indicated for symptomatic

anemia, aplastic crises, and blood loss.

Sometimes, patients with a history of stroke

508 AABB Technical Manual

or pulmonary or cardiac disease are some-

times treated with a hypertransfusion proto-

col or chronic red cell exchange program to

maintain their hematocrit at 25% to 30%

and the proportion of hemoglobin S below

about 30%. Care should be taken to avoid

raising the hematocrit above 35% because

of the risks of hyperviscosity. Red cell ex-

change is used to manage and/or prevent

life- or organ-threatening complications,

particularly stroke and pulmonary crisis.

Red cell exchange has also been used to

prepare patients for surgery, but a random-

ized controlled trial did not support this

measureoversimpletransfusiontoahe

moglobin of 10 g/dL.93

The clinical management of sickle cell

disease is complex and the reader is re-

ferred to recent reviews for more details.94,95

Patients with thalassemia and sickle cell

disease can receive standard red cell com-

ponents. However, leukocyte reduction is

generally offered to avoid febrile, non-

hemolytic transfusion reactions. Pheno-

typing the patient’s red cells and providing

antigen-matched units for transfusion

helps reduce alloimmunization to red cell

antigens and delayed hemolytic transfusion

reactions, although the cost and logistics of

such a program may be impractical for

many institutions. A frequent compromise

is to match for Rh system and K antigens.

Patients with SCD should be given hemo-

globin-S-negative RBC units. For more

details, see Chapter 24.

Transfusing Known Incompatible Blood

Clinicians must occasionally transfuse a

patient for whom no serologically com-

patible RBC units are available. This most

often occurs in patients with autoanti-

bodies, which typically react with all red

cells; however, once alloantibodies are

ruled out, the transfused cells are ex-

pected to survive as long as autologous

cells. Other situations in which all units

appear incompatible include the presence

of alloantibodies to high-incidence anti-

gens and multiple antibody specificities.

If serologic testing fails to resolve the

problem, or if the problem is identified but

time is not sufficient for acquisition of

compatible units, the physician must weigh

the risks and benefits of transfusion and

consider what alternative therapies are

suitable. If the need is sufficiently urgent,

incompatible red cells of the patient’s ABO

and Rh type may have to be given. Depend-

ing on the alloantibody, incompatible

transfusion does not always result in imme-

diatehemolysis,andtheincompatiblecells

mayremaininthecirculationlongenough

to provide therapeutic benefit.96

If time permits and if equipment is avail-

able, the survival of a radiolabeled aliquot

of the incompatible cells can be deter-

mined. Alternatively, an “in-vivo cross-

match” can be performed by cautiously

transfusing 25 to 50 mL of the incompatible

cells, watching the patient’s clinical re-

sponse, and checking a 30-minute post-

transfusion specimen for hemoglobin-

tinged serum. Such assessment does not

guarantee normal survival, but it can indi-

cate whether an acute reaction will occur. If

no adverse symptoms or hemolysis are ob-

served, the remainder of the unit can be

transfused slowly with careful clinical mon-

itoring. If the transfusion need is life-

threatening, RBC units may sometimes be

given without special testing, but clinical

staff should be prepared to treat any

reaction that may result.

Transfusing Patients with Autoimmune

Hemolytic Anemia

Because of the serologic difficulties that

accompany autoimmune hemolytic ane-

mia and the expected short red cell sur-

vival, a conservative approach to trans-

Chapter 21: Blood Transfusion Practice 509

fusion is recommended. The presence of

underlying alloantibodies should be in-

vestigated before beginning transfusions,

time permitting. It is very helpful to es-

tablish the patient’s phenotype before

transfusion in order to simplify subse-

quent investigation for the presence of

possible alloantibodies. Chapter 20 con-

tains a more complete discussion.

Massive Transfusion

Massive transfusion is defined as replace-

ment approximating or exceeding the pa-

tient’s blood volume within a 24-hour in-

terval. The most important factor in

supporting tissue oxygenation is mainte-

nance of adequate blood flow and blood

pressure by infusing a sufficient volume

of crystalloid or blood components to

correct or prevent hypovolemic shock.

Emergency Issue

The transfusion service should establish a

standard operating procedure for emer-

gency provision of blood. Immunohema-

tologic testing is relatively time-consum-

ing, so it may have to be abbreviated in

trauma cases or other hemorrhagic emer-

gencies.BecauseABO-compatiblecom

ponents are entirely compatible in the

vast majority of cases, particularly if the

patient has never been transfused before,

group O RBCs are often used for emer-

gency transfusion before completion of

any compatibility tests. In this situation,

Rh-negativeRBCsshouldbeusedforfe

males of childbearing potential because

of the concern for immunizing such indi-

viduals and possibly causing hemolytic

disease of the newborn in the future. For

women beyond their childbearing years or

men, only the current presence of anti-D

is a concern. Because this antibody is no

more common than certain other blood

group alloantibodies, Rh-positive RBCs can

be used with similar safety.

The use of so-called “universal donor”

RBCs, as discussed above, has several draw-

backs. Group O RBCs are typically in short

supply because of demographic differences

between the donor and recipient popula-

tions in the United States, and such univer-

sal donor practices accentuate this prob-

lem. ABO typing can be performed very

quickly, and, in most emergencies, there is

time for ABO typing of the recipient and

provision of ABO-specific components.

Second, transfusion of large quantities of

group O RBCs before a recipient blood

sample is obtained may obscure subse-

quent immunohematologic testing. There-

fore, even if universal donor RBCs are to be

used, a blood sample should be obtained

before transfusion; this is possible in all but

the most dire cases of exsanguinating hem-

orrhage. Finally, unexpected blood group

antibodies can cause fatal transfusion reac-

tions, particularly in multitransfused pa-

tients such as those with SCD or liver dis-

ease who present to an emergency room

with severe anemia or bleeding. In such

situations, a call to another hospital trans-

fusion service at which the patient was pre-

viously transfused can be life-saving. Indi-

viduals caring for such patients must

understand the above principle concerning

the priority of volume deficits over anemia.

Emergency situations should not be

construed as justification for exceptions to

strict identification of recipient blood sam-

ples. On the contrary, increased attention to

patient identification is warranted, and one

indication for use of universal donor RBCs is

in situations when multiple trauma victims

arrive concurrently, when there is a high

risk of recipient misidentification.

AABB Standards requires physicians who

order transfusion before completion of

standard compatibility tests to document the

need by signing some type of “emergency

510 AABB Technical Manual

release form.”59(p46) This documentation should

be required only after the emergency is

over, typically within 24 hours, and signa-

tures on such forms should not be con-

strued as a release of the transfusion ser-

vice’s responsibility. The transfusion service

must insist on strict identification of sam-

ples, documentation of unit disposition,

and documentation of the emergency sta-

tus of the transfusion. However, it also has

the responsibility to perform testing on a

STAT basis, provide consultation, and avoid

unnecessarily restrictive practices.

Changing Blood Types

The transfusion service should establish

guidelines for switching blood types dur-

ing massive transfusion. An alternative to

ABO-identical RBCs is the use of ABO-

compatible units (see Table 21-1). The age

and sex of the patient are important con-

siderations. For example, when transfus-

ingayounggroupB,Rh-negativewoman,

it is preferable to switch to group O, Rh-

negative RBCs before switching to group

B, Rh-positive cells. The clinical situation

should be evaluated by the transfusion

service’s physician. If the continuing

transfusion requirement is expected to

exceed the available supply of Rh-nega-

tive blood, evaluation of the change to

Rh-positive blood should be made early,

to conserve blood for other recipients. Once

the patient receives one or more Rh-posi-

tive units, there may be little advantage in

returning to Rh-negative blood.

Coagulation Support During Massive

Transfusion

Massive transfusion is often associated with

coagulation abnormalities that may man-

ifest as microvascular bleeding (MVB) in

theformofoozingfrommultipleIVsites,

failure of blood shed into body cavities to

clot, and bleeding from tissue surfaces on

which hemostasis was previously obtained.

These situations have been attributed to

the dilution of platelets or coagulation

factors, but consumptive coagulopathy

also plays a role (see Chapter 27). Inade-

quate volume resuscitation and poor tis-

sue perfusion not only promote the re-

lease of tissue procoagulant material

leading to DIC but also result in lactic aci-

dosis, acidemia, and poor myocardial

performance. If MVB occurs, the results of

platelet counts, fibrinogen level, PT, and

aPTT ideally should guide the need for

hemostatic components. Empiric therapy

with platelets and/or plasma may be initi-

ated immediately after specimens are ob-

tained. Additional tests may be indicated

to evaluate the possibility of DIC. In this

situation, a platelet count less than

50,000/µL and a fibrinogen level less than

100 mg/dL are better predictors of hem-

orrhage than the PT and aPTT.97 PT results

below 1.5 times normal are usually associ-

ated with adequate hemostasis during

surgery.3In most adult patients, these lev-

els are encountered only after transfusion

of 15 to 20 RBC units (1.5 to 2 red cell vol-

umes). FFP or platelets should not be ad-

ministered in a fixed ratio to the number

of RBC units given.

Hypothermia, Tissue Oxygenation, and

2,3-DPG

Hypothermia as a complication of trans-

fusion is discussed in Chapter 27. In hypo-

volemic shock, the underlying patho-

physiologic defect is inadequate tissue

oxygenation. Oxygen supply to the tissues

is determined by many factors, the most

important of which are blood flow (perfu-

sion) and hemoglobin concentration. The

level of 2,3-DPG decreases in stored RBCs,

and this decrease has been suggested as a

potential cause of poor tissue oxygena-

tion after massive transfusion. Low 2,3-DPG

Chapter 21: Blood Transfusion Practice 511

levels have not been shown to be detri-

mental to massively transfused patients,

although for infants undergoing exchange

transfusion, blood with near-normal

2,3-DPG levels is frequently requested.

Within 3 to 8 hours after transfusion, pre-

viously stored red cells regenerate 50% of

normal 2,3-DPG levels.98

Pharmacologic Alternatives

to Transfusion

Concern over the risks and limitations of

transfusion has led to examination of

pharmacologic alternatives. Such alterna-

tives might: 1) stimulate increased pro-

duction or release of blood elements that

otherwise would require replacement (eg,

erythropoietin); 2) substitute for a blood

component (eg, colloid solutions or oxy-

gen-carrying solutions including chemi-

cally modified hemoglobins); or 3) alter

physiologic mechanisms to reduce the

need for replacement (eg, fibrinolytic in-

hibitors).99

Recombinant Growth Factors

Growth factors are low-molecular-weight

protein hormones that regulate hemato-

poiesis by specific interaction with recep-

tors found on progenitor cells. The use of

growth factors to stimulate endogenous

blood cell production is an important al-

ternativetotheuseofblood.

100

Erythropoietin

Recombinant erythropoietin (EPO) is a

growth factor that stimulates RBC pro-

duction.100 It has been approved for pre-

surgical administration to increase preop-

erative hemoglobin and hematocrit levels;

typical dose regimens range from 300 to

600 U/kg by subcutaneous injection weekly.

The use of EPO has markedly reduced the

need for transfusion in patients with end-

stage renal disease and is indicated for

the treatment of anemia in patients in-

fected with human immunodeficiency vi-

rus. Intensive EPO treatment (40,000 units

weekly) reduced the transfusion require-

ment of ICU patients.101 It may also have a

role in treating anemia due to chronic dis-

ease or to receipt of other medications

that suppress the marrow.

Other Blood Cell Growth Factors

Granulocyte-macrophage colony-stimu-

lating factor (GM-CSF) and G-CSF stimu-

late marrow production of granulocytes.100

G-CSF is approved for the treatment of

chemotherapy-induced neutropenia, for

patients undergoing peripheral blood

progenitor cell collection and therapy,

and for patients with chronic neutropenia.

The use of these stimulants decreases the

duration of neutropenia, increases toler-

ance to cytotoxic drugs, and decreases the

need for granulocyte transfusions. GM-

CSF is approved for use in patients under-

going autologous marrow transplanta-

tion. Another potential use for GM-CSF

and G-CSF is support of patients under-

going allogeneic marrow transplantation

or patients receiving antiviral agents that

suppress the marrow. Recombinant inter-

leukin-11 is licensed for cancer patients

with thrombocytopenia, but other activa-

tors for thrombopoietin receptors have

been disappointing and none are avail-

able at this time.

Oxygen Therapeutics (Carriers)

Stroma-free hemoglobin solution, in which

free hemoglobin has been separated from

cell membranes, has several characteris-

tics that render it unsuitable as a blood

substitute, including a low p50, short cir-

culation time, high oncotic pressure, and

vasopressor/nephrotoxic properties.102,103

512 AABB Technical Manual

However, chemical modifications of he-

moglobin solutions may successfully

overcome these disadvantages and such

products are in Phase III clinical trials at

thetimeofthiswriting.Patientsinthese

trials have survived very severe anemia

(residual cellular hemoglobin as low as 1

g/dL) without significant toxicity when

supported by these agents.104 Bovine he-

moglobin is approved for veterinary use.

Hemoglobin produced by recombinant

DNA techniques has also been investi-

gated. Finally, fluorocarbon products that

bind oxygen have been extensively inves-

tigated.103 One of the latter, Fluosol, was

approved by the FDA for use during per-

cutaneous transluminal angioplasty, but

it is no longer available in the United

States.

DDAVP

DDAVP is a synthetic analogue of the hor-

mone vasopressin that lacks significant

pressor activity.99,105 First used in the treat-

ment of diabetes insipidus, DDAVP is also

useful in promoting hemostasis because

of its ability to cause the release of endog-

enous stores of high-molecular-weight

vWF from the vascular subendothelium

and the concomitant increase in Factor

VIII. Because of its effect on Factor VIII

and vWF, DDAVP is used as a hemostatic

agent in patients with mild-to-moderate

hemophilia A and in patients with some

von Willebrand syndromes. Because

platelet adhesion and the subsequent for-

mation of a platelet plug depend upon

vWF, DDAVP may also be beneficial in a

wide variety of platelet function disor-

ders, including uremia, cirrhosis, drug-in-

duced platelet dysfunction (including as-

pirin), primary platelet disorders, and

myelodysplastic syndromes.99,105,106

DDAVP can be administered intrave-

nously, subcutaneously, or intranasally. It is

usually given as a single injection (0.3 to 0.4

µg/kg) to treat bleeding or for prophylaxis

before a procedure. Doses are not usually

repeated within a 24- to 48-hour period be-

cause of tachyphylaxis (the loss of biologic

effect with repeated administration of an

agent) and the induction of water retention

and hyponatremia. Some patients experi-

ence facial flushing or mild hypotension,

but side effects are rare. Its effect on vWF

occurs within 30 minutes and lasts 4 to 6

hours.105

Fibrinolytic Inhibitors

Epsilon aminocaproic acid (EACA) and

tranexamic acid—synthetic analogues of

lysine—competitively inhibit fibrinolysis

by saturating the lysine binding sites at

which plasminogen and plasmin bind to

fibrinogen and fibrin. The drugs can be

used locally or systemically and can be

given orally. Aprotinin is a polypeptide

prepared from bovine lung that inhibits

proteinases including plasmin, kallikrein,

trypsin, and, to some extent, urokinase.

Thus, it has an antifibrinolytic action but

may also inhibit coagulation because

kallikrein activates Factor XII. Aprotinin is

used intravenously. Because it is a poly-

peptide, hypersensitivity reactions can

occur.99,107

Antifibrinolytic agents have been used

successfully in cardiac surgery, prostatec-

tomy, and liver transplantation. EACA and

tranexamic acid can also be used locally at

sites where fibrinolysis contributes to

bleeding, as from mucosal lesions of the

mouth and gastrointestinal tract, and are of

benefit in the control of hemorrhage fol-

lowing dental extractions in patients with

hemophilia and in the control of gastroin-

testinal bleeding. EACA and tranexamic

acid may be helpful in controlling bleeding

due to severe thrombocytopenia. Systemic

Chapter 21: Blood Transfusion Practice 513

administration of fibrinolytic inhibitors has

been associated with serious thrombotic

complications, including ureteral obstruc-

tion due to clot formation and thrombosis

of large arteries and veins. When used in

excessive doses, fibrinolytic inhibitors can

prolongthebleedingtime.Thesedrugs

should be employed by physicians with

experience in their use.

All three of these antifibrinolytic agents,

as well as DDAVP, have been used in an at-

tempt to decrease blood use in cardiac sur-

gery, and meta-analyses have shown a

decrease in the proportion of patients re-

ceiving allogeneic RBCs, the number of units

transfused,108,109 estimated blood losses,108

and the number of patients requiring re-

operation for bleeding.108,109 Of the three

agents, a much larger data base exists for

aprotinin, which is the most frequently

used. DDAVP was not effective overall but

may be useful in patients taking aspirin.108

Of concern has been a trend toward in-

creased thrombotic complications (myo-

cardial infarction and graft thrombosis)

with aprotinin, but they do not appear to

be statistically significant.107,108 Unfortu-

nately, very large studies would be required

to demonstrate whether the risk profile of

these agents is superior to that of blood,

particularly in view of the decreasing risks

of transfusion.

Oversight of Transfusion

Practice

Of the various institutions that regulate or

accredit aspects of transfusion, the Joint

Commission for Accreditation of Health-

care Organizations (JCAHO) has histori-

cally emphasized oversight of transfusion

practice as a requirement for accredita-

tion. As part of its performance improve-

ment standards, the JCAHO requires col-

lection of data regarding the use of blood

and scores the institution on the appro-

priateness of the selected performance

measures and the size of the data sam-

ple.110 Moreover,theJCAHOstandardsre

quire that the medical staff take the lead-

ership role in measurement, assessment,

and improvement of clinical processes re-

lated to the use of blood and blood com-

ponents. This assessment process must

include peer review, and its findings must

be communicated to involved staff mem-

bers as well as being a part of the renewal

of their clinical privileges.

Typically, this function has been dele-

gated to a medical staff committee, often a

dedicated “Transfusion Committee.” The

medical director of the transfusion service

should be a member of the committee. This

committee should review blood bank activ-

ities and statistics, blood ordering, and

transfusion practices and should have a

process to review records of patients trans-

fused with blood or components. The com-

mittee should monitor significant develop-

ments in transfusion medicine that would

affect patients in the health-care institution

and take appropriate action regarding these

developments.

The College of American Pathologists

(CAP) laboratory accreditation program

also requires transfusion oversight. This is

mandated by its general standard on qual-

ity control and improvement, which states

that the blood bank director must evaluate

the appropriateness of any laboratory’s out-

put in a multidisciplinary fashion.111 More-

over, the CAP accreditation checklist for

blood banks seeks documentation that “. . .

the transfusion service medical director ac-

tively participates in establishing criteria

and in reviewing cases not meeting transfu-

sion audit criteria.”112

Finally, the AABB Standards requires that

there be a peer-review program that moni-

tors appropriateness of use of blood com-

ponents.59(p86)

514 AABB Technical Manual

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51. GordonLI,KwaanHC,RossiEC.Deleterious

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52. Strauss RG. Granulocyte transfusion. In:

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54. Vamvakas EC, Pineda A. Determinants of the

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55. Gresens CJ, Paglieroni TG, Moss CB, et al.

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56. Stringham JC, Bull DA, Fuller TC, et al. Avoid-

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59. Silva MA, ed. Standards for blood banks and

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61. The trial to reduce alloimmunization to pla-

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62. Vamvakas EC, Dzik WH, Blajchman MA. Del-

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63. KrishnanLA,BrecherME.Transfusiontrans

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64. Dzik WH, Anderson JK, O’Neill EM, et al. A

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68. vanAkenWG.Preparationofplasmaderiva

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104. Gould S, Moore ED, Hoyt DB, et al. The life-

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Chapter 21: Blood Transfusion Practice 519

Chapter 22: Administration of Blood and Components

Chapter 22

Administration of Blood and

Components

GOOD TRANSFUSION PRACTICE

requires that comprehensive poli-

cies and procedures for blood ad-

ministration be designed to prevent errors.

The development of these policies should

be a collaborative effort between the medi-

cal director of the transfusion service, the

directors of the clinical services, both nurs-

ing and medical, and all personnel involved

in blood administration. Policies and pro-

cedures must be accessible, periodically

reviewed for appropriateness, and moni-

tored for compliance. In addition to blood

administration policies and procedures,

this chapter discusses pretransfusion pre-

paration, issuing of blood components,

the equipment used in blood administra-

tion, and compatible intravenous solutions.

Pre-Issue Events

Patient Education and Consent

Patients who are aware of the steps in-

volved in a transfusion will experience less

anxiety. This is important not only for an

adult but also for any child who has the

ability to understand the process. In the

latter situation, it is appropriate to edu-

cate the parents, so that they are better

prepared to support their child through-

out the transfusion. The transfusionist

should explain how the transfusion will be

given, how long it will take, what the ex-

pected outcome is, what symptoms to re-

port, and that vital signs will be taken. The

physician has a responsibility to explain

the benefits and risks of transfusion ther-

apy as well as the alternatives in a fashion

that the patient can comprehend. Other

than in emergency situations, the patient

shouldbegivenanopportunitytoask

questions, and his or her informed choice

should be documented. State and local

laws govern the process of obtaining and

documenting the consent of the patient.

Some states have specific requirements

for blood transfusion consent. Institu-

tions should be careful to ensure that

their individual processes and procedures

comply with applicable laws.

521

Individual institutions have different re-

quirements for obtaining and documenting

this interaction, as well as different policies

about how often it is necessary. Some facili-

tiesrequiretheuseofaformalconsent

form, which provides information in un-

derstandable language, signed by the pa-

tient. Others expect the physicians to make

a note in the medical record stating that the

risksof,andalternativesto,bloodtransfu

sions were explained and that the patient

consented. If a patient is unable to give

consent, a responsible family member

should be asked. If no family member is

available or if the emergency need for

transfusion leaves no time for consent, it is

prudent to note this in the medical record.1-3

Prescription and Special Instructions

There must be documentation of the or-

der by the physician for the blood compo-

nent(s).4Although a telephone order may

be acceptable during urgent situations, this

must be followed by a written request.

Special instructions should be indicated

regarding the transfusion relating to the:

■Component—eg, washed, irradiated,

leukocyte-reduced, cytomegalovirus

negative

■Patient—eg, premedication, timing

■Process—flowrates,rateofinfusion,

use of a blood warmer or electrome-

chanical pump

■Need for emergency release

Component Considerations

Some components require special prepa-

ration before release for transfusion. Be-

cause these steps are time-consuming and

may significantly shorten the shelf life of

the component, preparation should be

carefully coordinated with the anticipated

time of transfusion. The transfusion ser-

vice must strive to make every effort to

ensure that the component is ready when

needed, but not so early that it expires be-

fore administration.

Medical and nursing staffs need to be

aware of special requirements for prepara-

tion and to understand that these times

cannot be significantly shortened, even in

urgent situations. Close communication is

required. Some examples of pretransfusion

processing procedures are given in Table

22-1.

Patient Considerations

Patients with a history of recurrent aller-

gic transfusion reactions may benefit from

premedication with antihistamines or by

slowing the rate of transfusion. Routine

premedication with antipyretics should

be discouraged because delaying a rise in

temperature may mask one sign of a

hemolytic reaction and partly because

they may be ineffective.5-7

Antipyretics typically do not mask other

clinical features of hemolysis, such as

changes in blood pressure, pulse, or respi-

ration. Premedication orders should be

carefully timed with the anticipated admin-

istration of the unit. Medication ordered in-

travenously may be given immediately be-

fore the start of the transfusion, but orally

administered drugs need to be given 30 to

60 minutes before the start of the transfu-

sion.

Process Considerations

Blood warmers and electromechanical

pumps need to be available if required for

the transfusion.

Emergency Release

Blood may be released without complet-

ing pretransfusion testing if it is urgently

needed for a patient’s survival, provided

that: 1) the records properly document

522 AABB Technical Manual

the emergency request and 2) the issued

units are of an ABO group unlikely to

cause immediate harm to the recipient.

Venous Access

To avoid any delay in transfusion and po-

tential wastage of blood components, ve-

nous access should be established before

the component is issued. If a pre-existing

line is to be used, it should be checked for

patency; signs of infiltration, inflamma-

tion, or infection; and the compatibility of

any intravenous solutions (see below). Many

venous access devices can be used for

blood component transfusion. Selection

depends on the location, size, and integ-

rity of the patient’s veins; the type of med-

ication or solution to be infused; the type

of component to be transfused; the vol-

ume and timing of the administration; the

possibility of interactions among paren-

teral solutions; and expected duration of

intravenous therapy.

The lumen of needles or catheters used

for blood transfusion should be large

enough to allow appropriate flow rates

without damaging the vein. There are no

strict guidelines limiting the size of the

catheter or needles used for transfusion. An

18-gauge catheter provides good flow rates

for cellular components without excessive

discomfort to the patient, but patients with

small veins require much smaller catheters.

High-pressure flow through needles or

catheters with a small lumen may damage

red cells8-10 unless the transfusion compo-

nent is sufficiently diluted.10 Undiluted

preparations of red cells flow very slowly

through a 23-gauge needle, but dilution

with saline to increase the flow rate may

cause unwanted volume expansion. Even

in patients with cardiac disease or volume

expansion, transfusions should be able to

be given safely within 4 hours. For rare pa-

tients unable to tolerate a transfusion with-

in 4 hours, local policy should be developed

regarding whether to split units or to dis-

card the unused portion. Specific models of

infusion pumps have been approved for

use in blood transfusion. These pumps

maintain a constant delivery of blood, and

studies have indicated no significant evi-

dence of hemolysis as the needle size var-

ies.11

Chapter 22: Administration of Blood and Components 523

Table 22-1. Component Preparation Times

Component Minimum Time* Shelf Life

RBCs: saline-washed 45 minutes 24 hours

RBCs: thawed-deglycerolized 75 minutes 24 hours or 2 weeks†

Fresh Frozen Plasma: thawed 30 minutes 24 hours

Thawed Plasma — 5 days

Platelets: pooled 15 minutes 4 hours

CRYO: thawed (single unit) 15 minutes 6 hours

CRYO: pooled 15 minutes 4 hours

*Will vary with institutional procedures.

†Depends on the method used.

RBCs = Red Blood Cells; CRYO = Cryoprecipitated AHF.

Central venous catheters are used for

medium- and long-term therapy or for the

administration of solutions potentially toxic

to a peripheral vein to allow the dilution

achieved with high-volume blood flow.

Some catheters are placed via special intro-

ducing needles and guide-wires; others are

surgically implanted. Catheters with a

multilumen design have separate infusion

ports for each lumen, permitting the simul-

taneous infusion of fluids without intermix-

ture in the infusion line, thereby avoiding

the potential for hemolysis from incompat-

ible fluids.

Need for Compatibility Testing

The transfusion service personnel deter-

mine what pretransfusion testing is re-

quired. Compatibility testing must be per-

formed for transfusion of Whole Blood,

components with a clinically significant

red cell content, and all red cell compo-

nents. For test and specimen require-

ments, refer to Chapter 18. Compatibility

testing other than ABO and Rh typing is

not required for platelet and plasma com-

ponents, but most facilities require that

the recipient’s ABO and D types be known

(onfile)beforesuchcomponentsarese

lected for issue. Whenever possible, plasma-

containing components should be com-

patible with the patient’s red cells (see

Chapter 21).

Blood Issue and

Transportation

Delivering Blood to the Patient Area

Institutions should define blood pick-up

and delivery policies and appropriate

training programs for the staff assigned to

these functions. Blood is not routinely

dispensed from the controlled environ-

ment of the blood bank until all testing is

completed, the patient is properly pre-

pared, and the transfusionist is ready to

begin the procedure. There must be a

mechanism to identify the intended re-

cipient and the requested component at

the time of issue. It is optimal to identify

the transporter.

Transfusion service personnel will re-

view identifying information, inspect the

appearance of the component before re-

lease, and ensure there is a system to main-

tain proper storage temperature during

transport.Thesafestpracticeistoissueone

unit to one patient at a time. For patients

who are rapidly bleeding or who have mul-

tiple venous access sites, multiple units

may be issued to a single patient. It is not

recommended that blood for two or more

patients be issued simultaneously to one

transporter because this could increase the

chance of transfusion error. However, logis-

tical considerations may make this imprac-

tical. The transporter should transport the

blood to the intended site of transfusion

without delay—preferably to the transfu-

sionist. It is preferable to place the unit of

blood in a protective container that would

contain any spillage in the event of inadver-

tent breakage during transport.

The responsibility for accurately identi-

fying a transfusion component rests with both

the transfusion service personnel who issue

the blood and the transfusionist who re-

ceives it. Before a unit of blood is issued,

transfusion service personnel complete the

following steps:

1. The records that identify the in-

tended recipient and the requested

component are reviewed.

2. The identifiers (Standards requires

at least two) of the intended recipi-

ent, the ABO and D type of the recip-

ient, the component unit number,

the ABO and D type of the donor

unit, and the interpretation of com-

patibility tests (if performed) are re-

524 AABB Technical Manual

corded on a transfusion form for

each unit. This form, or a copy, be-

comes a part of the patient’s perma-

nent medical record after comple-

tion of the transfusion. In some

institutions, the transfusion form is

attached to the unit and, therefore,

serves as the tie tag that is required

and described below. This form typi-

cally will have fields to identify the

transfusionist and co-identifier (if re-

quired) and other information, such

as pre- and posttransfusion vital signs,

amount of blood given, whether a re-

action occurred, etc.

3. A tie tag or label with the name and

identification number of the in-

tended recipient, the component unit

number, and the interpretation of

compatibility tests (if performed) must

be securely attached to the blood

container.

4. The appearance of the unit is checked

before issue and a record is made of

this inspection.

5. The expiration date (and time if ap-

plicable) is checked to ensure that the

unit is suitable for transfusion.

6. The name of the person issuing the

blood and the date and time of issue

are recorded. Recording the name of

the transporter to whom the blood is

issued is optimal.

Delay in Starting the Transfusion

Ideally, blood should be requested from

the blood bank only at the time when it is

intended to be administered. If the trans-

fusion cannot be initiated promptly, the

blood should be returned to the blood

bank for storage, unless the transfusion to

the originally intended recipient can be

completed within 4 hours. It should not

be left at room temperature or stored in

an unmonitored refrigerator. Units re-

turned to the blood bank after a period

outside monitored refrigeration will be

unsuitable for reissue if the sterility of the

container is compromised or if the tem-

perature has risen to 10 C or above, which

is generally considered to happen in less

than 30 minutes. If the units have been

kept in suitable conditions, such as iced

coolers that have been validated for sev-

eral hours of storage, longer periods are

acceptable. Requirements for blood col-

lected intraoperatively differ. See Table

5-8. Units that have been entered (punc-

tured) after release from the blood bank

cannot be accepted into general inven-

tory for later reissue.

Pre-Administration Events

Identifying the Recipient and Donor Unit

Accurate identification of the transfusion

component and the intended recipient

may be the single most important step

in ensuring transfusion safety.12-15 Most

hemolytic transfusion reactions and

deaths occur because of inadvertent ad-

ministration of ABO-incompatible red

cells.14-15 Plasma and platelets are also ca-

pable of causing serious transfusion reac-

tions.16 Identification and labeling of do-

nor blood are discussed in Chapter 7;

procedures to identify the patient’s speci-

men used for compatibility testing are

discussed in Chapter 18. The most impor-

tant steps in safe transfusion administra-

tion are clerical and occur when the

transfusion service issues blood for a spe-

cific patient and when the blood is ad-

ministered.

The transfusionist who administers the

blood represents the last point at which

identification errors can be detected before

transfusion of the component is initiated.

The transfusionist must check all identify-

ing information immediately before begin-

Chapter 22: Administration of Blood and Components 525

ning the transfusion and record that this in-

formation has been checked and found to

be correct, typically, on the transfusion

form. Any discrepancy must be resolved

before the transfusion is started. It is com-

mon practice in many institutions that a

second person (co-identifier) confirms the

identity of the blood unit and of the patient.

Some institutions may require that the

transfusionist check for documentation of

patient consent before blood is given. It is

also common practice to check the ABO

and D type as written in the transfusion

form with a record of ABO and D type in

the patients’ chart. The following informa-

tion, however, must be reviewed and found

to be correct:

1. Physician’s order. The nature of the

blood or component should be

checked against the physician’s writ-

ten order to verify that the correct

component and dose (number of

units) are being given. All identifica-

tion attached to the container must

remain attached until the transfu-

sion has been completed.

2. Recipient identification. The patient’s

identifiers on the patient’s identificat-

ion band must be identical with the

identifiers attached to the unit. It is de-

sirable to ask the patient to state his or

her name, if capable of so doing, be-

cause the information on the identifi-

cation band may be in error.17

3. Unit identification. The unit identifi-

cation number on the blood con-

tainer, the transfusion form, and the

tie tag attached to the unit (if not the

same as the latter) must agree.

4. ABO and D. The ABO and D type on

the primary label of the donor unit

must agree with those recorded on

the transfusion form. The recipient’s

ABO and D type must be recorded

on the transfusion form. The pa-

tient’stypeandthetypeofthecom

ponent may not be identical (see

Chapter 21), but the information on

the transfusion form and that on the

container label must be the same.

5. Expiration. The expiration date and

time of the component should be

verified as acceptable.

6. Compatibility. The interpretation of

compatibility testing (if performed)

must be recorded on the transfusion

form and on the tag attached to the

unit (if not the same). If blood was

issued before compatibility tests were

completed, this must be conspicu-

ously indicated.

In certain clinical situations, such as the

operating room (OR), emergency room, or

in the outpatient setting, an identifying

band may not be attached to all patients at

thetimeoftransfusion.Wristbandsarefre-

quently removed in the OR for arterial line

insertions and identity confirmation may

not be possible in this manner. Further-

more, a co-identifier confirming identity,

although always desirable, may not func-

tion for all circumstances in this setting.

Institutions should address such clinical

situations, and site-specific transfusion

protocols should be developed.18

Starting the Transfusion

After checking all the identifying informa-

tion, the transfusionist (and co-identifier)

must indicate in the medical record that

the identification was correct (such as by

signing the transfusion form) to docu-

ment who started the transfusion and to

recordthedateandtime.Vitalsigns

should be taken and recorded, if not done

previously. A record of the date and time

of transfusion, the name and volume of

the component, and vital signs may be re-

quired on other parts of the medical re-

cord, such as intake/output records, anes-

526 AABB Technical Manual

thesia records, or intensive care flow

sheets, depending on the institution’s pol-

icy. This may suffice for documentation

purposes.

Several reports have documented the oc-

currence of errors at the point of blood ad-

ministration.19-22 In particular, Baele and

colleagues19 studied the charts and records

of 808 patients who received 3485 units of

blood over a period of 15 months, to deter-

mine if there had been errors in blood ad-

ministration. They detected 165 errors oc-

curring after blood units had left the blood

bank, 15 of which were considered to be

major. Seven of the major errors involved

patient misidentification that resulted in

blood being given to patients for whom it

was not intended, constituting 0.74% of pa-

tients and 0.2% of units. One error resulted

in an ABO-incompatible hemolytic reaction

that was not reported to the blood bank.

Eight other major errors occurred in four

patients (0.5%), including the administra-

tion of five allogeneic units to a patient for

whom autologous blood was available, and

the transfusion of one anemic patient

whose doctor had ordered only a cross-

match. The remaining 150 errors included

misrecording (n = 61), mislabeling (n = 6),

and failure to adequately document the

transfusion(n=83).

Both mechanical barrier systems23 and

electronic means of patient and product

identifications are marketed to supplement

(but not substitute for) the paper identifica-

tion process. In electronic systems, a bar

code or radio frequency identifiers are at-

tached to the blood component (and trans-

fusion form) that, when scanned/read, will

match the patient’s information on the

identification band.24 It is likely that these

electronic means will find more widespread

use in the future. Empiric experience with

medication administration, however, sug-

gests that unanticipated side effects of such

procedures may need consideration.25

Compliance with institutional blood ad-

ministration policies requires a Quality

Assurance/Continuous Improvement pro-

gram in which continued monitoring and

re-education of staff occur when variance

with procedures is observed. Such an ap-

proach has been initiated in some institu-

tions, resulting in improvement in transfu-

sion practice.26 Direct observation of

administration27 or educational videos28

may also be useful.

Administration

Infusion Sets

Any blood component must be adminis-

tered through a filter designed to retain

blood clots and particles potentially harm-

ful to the recipient.4,29(p48) All filters and in-

fusion devices must be used according to

the manufacturer’s directions.

Standard Sets

Standard blood infusion sets have inline

filters (pore size: 170 to 260 microns), drip

chambers, and tubing in a variety of con-

figurations. Sets should be primed ac-

cording to the manufacturer’s directions,

using either the component itself or a so-

lution compatible with blood (see the sec-

tion on Compatible IV Solutions). For op-

timal flow rates and performance, filters

should be fully wetted. Drip chambers

should be half-filled to allow observation

of blood flow.

Many institutions have a policy of chang-

ing sets after every transfusion or of limit-

ing their use to several units or several

hours in order to reduce the risks of bacte-

rial contamination. A reasonable time limit

is 4 hours; this is consistent with the 4-hour

outdate that the Food and Drug Adminis-

tration (FDA) places on blood in an open

system held at room temperature. Most

Chapter 22: Administration of Blood and Components 527

standard filters are designed to filter 2 to 4

units of blood, but if the first unit required 4

hours for infusion, the filter should not be

reused. The filter traps cell aggregates, cel-

lular debris, and coagulated proteins, re-

sulting in a high protein concentration at

the filter surface. The high protein milieu

and room temperature conditions promote

growth of any bacteria that might be pres-

ent. Accumulated material also slows the

rate of flow.30

Special Sets

High flow sets for rapid transfusion have

large filter surface areas, large-bore tub-

ing, and may have an inline hand pump.

Sets designed for rapid infusion devices

also may have “prefilters” to retain parti-

cles over 300 microns in diameter and to

extend the life of standard blood filters

“downstream.” Gravity-drip sets for the

administration of platelets and cryopreci-

pitate have small drip chamber/filter ar-

eas, shorter tubing, and smaller priming

volumes. Syringe-push sets for compo-

nent administration have the smallest

priming volumes and an inline blood fil-

ter that may be inconspicuous.

Microaggregate Filters

Microaggregate filters are designed for the

transfusion of red cells. Microaggregates

(smaller than 170 microns in size) pass

through standard blood filters. Screen- or

depth-type filters have an effective pore

size of 20 to 40 microns and trap the

microaggregates composed of degenerat-

ing platelets, leukocytes, and fibrin strands

that form in blood after 5 or more days of

refrigerated storage.

Microaggregate filters may be used for

other components if this use is mentioned

in the manufacturer’s instructions; how-

ever, the large volume required for priming

causes a significant portion of these com-

ponents to be lost if the set is not flushed

with saline afterward. Depth-type micro-

aggregate filters, or any filters capable of re-

moving leukocytes, must not be used for

the transfusion of granulocyte concen-

trates.29(p48) Hemolysis of red cells has been

reported with microaggregate filters.30

Leukocyte Reduction Filters

Special “third-generation” blood filters

can reduce the number of leukocytes in

red cell or platelet components to less

than 5 × 106, a level that reduces the risk of

HLA alloimmunization and the transmis-

sionofcytomegalovirusaswellasthein-

cidence of febrile nonhemolytic transfu-

sion reactions31-35 (see Chapters 8, 21, and

27). These filters contain multiple layers

of synthetic nonwoven fibers that selec-

tively retain leukocytes but allow red cells

or platelets to pass, depending on the fil-

ter type. Selectivity is based on cell size,

surface tension characteristics, the differ-

ences in surface charge, density of the

blood cells, and, possibly, cell-to-cell in-

teractions and cell activation/adhesion

properties.36 Because filters for red cells

and filters for platelets do not use the

same technology for leukocyte removal

and may have strict priming and flow rate

requirements, they must be used only

with their intended component and only

according to the manufacturer’s direc-

tions.37

The use of these filters at the bedside is

more complex than the use of standard in-

fusion sets. The filters are expensive and

can be ineffective or may clog, if improp-

erly used.38-40 Those designed only for grav-

ity infusion should not be used with infu-

sion pumps or applied pressure. A quality

control program that measures the effec-

tiveness of leukocyte reduction is impor-

528 AABB Technical Manual

tant, but impractical, at the bedside; there-

fore, adherence to proper protocol is very

important.

Blood Warmers

It is desirable for the medical staff of the

transfusion service to participate in the

assessment and selection of transfusion

equipment and ensure that such items

are included in the facility’s quality assur-

ance program. The performance of de-

vices such as blood warmers or infusion

pumps must be validated before the

equipment is used and must be moni-

tored regularly throughout the facility to

identify malfunctions and ensure appro-

priate use. This characteristically requires

cooperation among personnel of several

hospital departments, including transfu-

sion medicine, nursing, anesthesiology,

quality assurance, and clinical engineer-

ing.

Patients who receive blood or plasma at

rates faster than 100 mL/minute for 30

minutes are at increased risk for cardiac ar-

rest unless the blood is warmed.41 Rapid in-

fusion of large volumes of cold blood can

lower the temperature of the sinoatrial

node to below 30 C, at which point an ar-

rhythmia can occur.

Transfusions at such rapid rates gener-

ally occur only in the OR or trauma settings.

There is no evidence that patients receiving

1 to 3 units of blood over several hours have

a comparable risk for arrhythmias; there-

fore, routine warming of blood is not rec-

ommended.42 Several types of blood

warmers are available: thermostatically

controlled waterbaths; dry heat devices

with electric warming plates; and high-vol-

ume countercurrent heat exchange with wa-

ter jackets.43 Blood warming devices must

not raise the temperature of blood to a level

that causes hemolysis.29(p6)

Devices should have a visible thermom-

eter and, ideally, an audible alarm that

sounds before the manufacturer’s desig-

nated temperature limit is exceeded. The

standard operating procedure for warming

blood should include guidelines on per-

forming temperature and alarm checks,

and instructions on what action to take

when warmers are out of range or the alarm

activates.44 Conventional microwave ovens

and microwave devices for thawing plasma

are not designed for warming other blood

components and can damage red cells.

Electromechanical Infusion Devices

Mechanical pumps that deliver infusions

at a controlled rate are useful, especially

for very slow rates of transfusion used for

pediatric, neonatal, and selected adult

patients. Some pumps use a mechanical

screw drive to advance the plunger of a

syringe filled with blood; others use roller

pumpsorotherformsofpressureapplied

to the infusion tubing. Although some can

be used with standard blood administra-

tion sets, many require special plastic

disposables or tubing supplied by the

manufacturer. Blood filters can be added

to the required setups upstream of the

pumps.

The manufacturer should be consulted

before blood is administered with an infu-

sion pump designed for crystalloid or

colloid solutions. Many induce hemolysis,

but of a magnitude that does not adversely

affect the patient.43 Red cells in components

with high hematocrit and high viscosity are

more likely to be hemolyzed when infused

under pressure than red cells in Whole

Blood or red cell components prepared in a

manner that reduces viscosity, such as ad-

ditive solutions.45 Platelets and granulocytes

appear to sustain no adverse effects when

infused with a pumping device.46,47 Proper

Chapter 22: Administration of Blood and Components 529

training of personnel and appropriate poli-

cies for maintenance and quality control

should reduce the chances of damage to

transfused components.

Pressure Devices

Urgent transfusion situations may require

flow rates faster than gravity can provide.

The simplest method to speed infusion is

to use an administration set with an inline

pump that the transfusionist squeezes by

hand. Pressure bags specially designed as

compression devices are also available.

These devices operate much like blood

pressure cuffs except that they completely

encase the blood bag and apply pressure

more evenly to the bag’s surface. Such de-

vices should be carefully monitored dur-

ing use because pressures greater than

300 mm Hg may cause the seams of the

blood bag to rupture or leak and air em-

bolism is a concern. Large-bore needles

are traditionally recommended for venous

access when the use of external pressure

is anticipated, but recent data question

this practice.11 Manually forcing red cells

through a small-gauge line has been

shown to cause hemolysis.45

Devices for intraoperative and postoper-

ative blood collection are discussed in

Chapter 5.

Compatible IV Solutions

AABB Standards for Blood Banks and

Transfusion Services29(p48) and the Circular

of Information for the Use of Human

Blood and Blood Components48 are explicit

in stating that medications must not be

added to blood or components. If red cells

require dilution to reduce their viscosity

or if a component needs to be rinsed from

the blood bag or tubing, normal saline

(0.9% sodium chloride injection, USP)

can be used. Red cells prepared with an

additive solution (AS) ordinarily do not

require dilution. These red cell compo-

nents have a hematocrit of approximately

60%. Other solutions intended for intrave-

nous use may be added to blood or com-

ponents or may come into contact with

blood in an administration set only if they

have been approved for this use by the

FDA or if there is documentation to show

that their addition to blood is safe and ef-

ficacious.29(p6),48 Calcium-free, isotonic

electrolyte solutions that meet the above

requirements also may be used, but they

usually are more expensive than saline

and offer little benefit in routine transfu-

sion. Lactated Ringer’s solution, 5% dex-

trose, and hypotonic sodium chloride so-

lutions should not be added to blood.

Dextrose solution may cause red cells to

clump in the tubing and, more important,

to swell and hemolyze as dextrose and as-

sociated water diffuse from the medium

into the cells. Lactated Ringer’s solution

contains enough ionized calcium (3

mEq/L) to overcome the chelating agents

in anticoagulant-preservative or additive

solutions, which results in clot develop-

ment.49-51

Patient Care During Transfusion

The transfusionist should either remain

with, or be in a position to closely observe,

the patient for at least the first 15 minutes

of the infusion. The transfusion should be

started slowly at a rate of approximately 2

mL/minute except during urgent restora-

tion of blood volume. Catastrophic reac-

tions from acute hemolysis, anaphylaxis,

transfusion-related acute lung injury, or

bacterial contamination can become ap-

parent after a very small volume enters

the patient’s circulation (see Chapter 27).

After the first 15 minutes, some institu-

tions record vital signs, but this is unneces-

530 AABB Technical Manual

sary if the patient’s condition is satisfactory.

Therateofinfusioncanbeincreasedto

that specified in the order or to be consis-

tent with institutional practice (approxi-

mately 4 mL/minute). The desirable rate of

infusion depends upon the patient’s blood

volume, cardiac status, and hemodynamic

condition. No experimental or clinical data

exist to support a specific time restriction;

however, the Circular of Information48 gives

4 hours as the maximum duration for an

infusion. Maximum time should not be

confused with recommended time. Most

RBC units are transfused within 1 to 2

hours, whereas platelet or plasma transfu-

sions are commonly administered over a

shorter period (30 to 60 minutes). However,

there is no physiologic reason to administer

compatible red cells more slowly than

plasma or platelets and rapid infusion of

these products may increase the risk of ad-

verse events. If rapid transfusion is needed,

blood can be infused as rapidly as the pa-

tient’s circulatory system will tolerate and

the type of vascular access will allow.

If it is anticipated that an infusion time

of longer than 4 hours may be required, the

physician covering the transfusion service

should be notified to assess the specific

clinical situation. Administration rates are

calculated by counting the drops per min-

ute in the drip chamber and dividing this

number by the “drop/mL” rating of the in-

fusion system. Blood may flow more slowly

than desired as a result of obstruction of

the filter or when there is excessive viscos-

ity of the component. Steps to investigate

and correct the problem include the follow-

ing:

■Elevate the blood container to in-

crease hydrostatic pressure.

■Check the patency of the needle.

■Examine the filter of the administra-

tion set for excessive debris.

■Consider the addition of 50 to 100

mL of saline to a preparation of red

cells, if there is an order permitting

such addition.

Clinical personnel should continue to

observe the patient periodically throughout

the transfusion (eg, every 30 minutes) and

up to an hour after completion.

Action for Suspected Reactions

Most transfusions proceed without com-

plication, but when adverse reactions do

occur, medical and nursing staff must be

prepared to deal with them immediately.

Different types of reactions, their etiology,

symptoms, treatment, and prevention are

discussed in Chapter 27. Because severity

can vary significantly and symptoms are

not specific, all transfusions must be care-

fullymonitoredandstoppedassoonasa

reaction is suspected. It may be helpful to

summarize common symptoms and the

immediate steps to take on the transfu-

sion form that accompanies the unit. This

eliminates the need to search for instruc-

tions and helps standardize patient care

in an urgent situation.

Post-Administration Events

After each unit of blood has been infused,

personnel should measure vital signs; re-

cord the time, the volume, and the com-

ponent given; record the patient’s condi-

tion; and record the identity of the person

who stopped the transfusion (if not the

transfusionist), made the observations,

and measured and recorded vital signs.

Many transfusion services require that a

copy of the completed transfusion form

be returned to the laboratory to docu-

ment the unit’s disposition for laboratory

records. The empty blood bag need not be

returned after uncomplicated transfu-

sions, but bags, tubing, and attached so-

lutions should be returned to the transfu-

sion service if a complication occurs.

Chapter 22: Administration of Blood and Components 531

Some transfusion services choose to have

all bags returned following all transfu-

sions in order to investigate reactions that

may not be evident at the time of transfu-

sion. Proper biohazard precautions

should be used in the handling of entered

containers and used administration sets.

The patient should remain under obser-

vation after the transfusion has been

completed, if this is practical. Because not

all severe reactions are immediately ap-

parent, all patients who receive transfu-

sionsinoutpatientorhomecaresettings,

or their caretakers, must be given clearly

written instructions outlining posttransfu-

sion care, a description of the signs and

symptoms of acute and delayed reactions,

and the appropriate action to take if such

are noted.

Quality Assurance

The process of blood administration should

begin and end with patient safety in

mind, starting with the generation of an

appropriate order; continuing through

collection of the patient’s pretransfusion

specimen, preparation and delivery of the

unit, identification of the unit with the re-

cipient, and selection and proper use of

equipment; and concluding with patient

care during the transfusion and mainte-

nance of appropriate records. Policies,

procedures, training, and assessment are

all critical to this process and must be

monitored as parts of blood usage review.

Periodic auditing of the blood adminis-

tration process and forms should be per-

formed in order to identify patterns of non-

conformance. Errors, regardless of clinical

outcome, should be subjected to root-cause

analysis because such errors are often in-

dicative of systematic problems.

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Afr Med J 1980;57:785-7.

51. Strautz RL, Nelson JM, Meyer EA, Shulman

IA. Compatibility of ADSOL-stored red cells

with intravenous solutions. Am J Emerg Med

1989;7:162-4.

534 AABB Technical Manual

Chapter 23: Perinatal Issues in Transfusion Practice

Chapter 23

Perinatal Issues in

Transfusion Practice

PREGNANCY PRESENTS SPECIAL

immunohematologic problems for

the transfusion service. The mother

may exhibit alloimmunization to antigens

on fetal cells, and the fetus may be affected

by maternal antibodies provoked by pre-

vious pregnancies, by previous or present

transfusions, or by the ongoing pregnancy.

This chapter discusses hemolytic disease

of the fetus and newborn (HDFN) and neona-

tal alloimmune thrombocytopenia (NAIT)—

the two primary immunohematologic

concerns during the perinatal period. Also

included is a brief discussion of neonatal

thrombocytopenia secondary to maternal

idiopathic thrombocytopenic purpura.

Hemolytic Disease of the

Fetus and Newborn

In HDFN, fetal red cells become coated

with IgG alloantibody of maternal origin,

directed against a paternally inherited an-

tigen present on the fetal cells that is

absent from maternal cells. The IgG-

coated cells may undergo accelerated de-

struction both before and after birth, but

the severity of the disease can vary from

serologic abnormalities detected in an

asymptomatic infant to intrauterine death.

Pathophysiology

Accelerated red cell destruction stimulates

increased production of red cells, many of

which enter the circulation prematurely as

nucleated cells, hence the term “erythro-

blastosis fetalis.” Severely affected fetuses

may develop generalized edema, called

“hydrops fetalis.” In HDFN resulting from

anti-D, erythropoiesis in the fetal liver

may be so extensive that portal circulation

is disrupted and albumin synthesis im-

paired, thereby reducing plasma colloid

osmotic pressure. The severe anemia may

cause cardiovascular failure, tissue hypo-

535

xia, and death in utero. Intrauterine trans-

fusion may be lifesaving in these circum-

stances. If live-born, the severely affected

infant exhibits profound anemia and

heart failure.1Less severely affected in-

fants continue to experience accelerated

red cell destruction, which generates

large quantities of bilirubin. Unlike HDFN

due to anti-D, HDFN due to anti-K1 re-

sults from suppression of fetal erythropo-

iesis in addition to causing peripheral red

cell destruction.2

Before birth severs the communication

between maternal and fetal circulation, fe-

tal bilirubin is processed by the mother’s

liver. At birth, the infant’s immature liver is

incapable of conjugating the amount of bil-

irubin that results from destruction of anti-

body-coated red cells. Unconjugated biliru-

bin is toxic to the developing central

nervous system (CNS), causing brain dam-

age referred to as “kernicterus.” For the

live-born infant with HDFN and rising lev-

els of unconjugated bilirubin, kernicterus

may pose a greater clinical danger than the

consequences of anemia.3Prematurity, aci-

dosis, hypoxia, and hypoalbuminemia in-

crease the risk of CNS damage. Decisions

about undertaking exchange transfusion

are based primarily on the bilirubin level,

the rate of bilirubin accumulation, and, to a

lesser degree, on the severity of the anemia.

Recently, the American Academy of Pediat-

rics has published guidelines aimed toward

preventing and managing hyperbiliru-

binemia in infants ≥35 weeks of gestation.4

Mechanisms of Maternal Immunization

HDFN is often classified into three cate-

gories, on the basis of the specificity of the

causative IgG antibody. In descending or-

der of potential severity, they are:

1. D hemolytic disease caused by anti-D

alone or, less often, in combination

with anti-C or anti-E. (The Rh blood

group is discussed in greater detail

in Chapter 14.)

2. “Other” hemolytic disease caused by

antibodies against other antigens in

the Rh system or against antigens in

other systems; anti-c and anti-K1 are

most often implicated.5

3. ABOHDFNcausedbyanti-A,Bina

group O woman or by isolated anti-A

or anti-B.

In all but ABO HDFN, maternal antibod-

ies reflect alloimmunization by pregnancy

or transfusion. Rising titers of antibody can

be documented, at least in the first affected

pregnancy, and the infant may be symp-

tomatic at birth as a result of effects on the

fetus in utero. In contrast, ABO fetomater-

nal incompatibility cannot be diagnosed

during pregnancy and the infant is rarely

symptomatic at birth.

Pregnancy as the Immunizing Stimulus

Pregnancy causes immunization when fe-

tal red cells, possessing a paternal antigen

foreign to the mother, enter the maternal

circulation as a result of fetomaternal he-

morrhage (FMH). FMH occurs in the vast

majority of pregnancies, usually during the

third trimester and during delivery.6De-

livery is the most common immunizing

event, but fetal red cells can also enter the

mother’s circulation after amniocentesis,

spontaneous or induced abortion, chori-

onic villus sampling, cordocentesis, rup-

ture of an ectopic pregnancy, and blunt

trauma to the abdomen.

Immunogenic Specificities. The antigen

that most frequently induces immunization

is D, but, in theory, any red cell antigen

present on fetal cells and absent from the

mother can stimulate antibody production.

One retrospective study determined that

there was a 0.24% prevalence of production

of clinically significant antibodies other

than anti-D during pregnancy. Because

536 AABB Technical Manual

other red cell antigens are less immuno-

genic than D, sensitization is more likely to

result from exposure to a large volume of

red cells, such as during blood transfusion.

Immunization to D, on the other hand, can

occur with volumes of fetal blood less than

0.1 mL.7

Frequency of Immunization. The proba-

bility of immunization to D correlates with

thevolumeofD-positiveredcellsentering

the D-negative mother’s circulation.6The

overall incidence of D sensitization in un-

treated D-negative mothers of D-positive

infantsisabout16%;1.5%to2%become

sensitized at the time of their first delivery,

an additional 7% become sensitized within

6 months of the delivery, and the final 7%

become sensitized during the second af-

fected pregnancy.8The sensitization during

the second affected pregnancy probably re-

flects primary immunization during the

first D-positive pregnancy and delivery that

happened without production of detectable

levels of antibody. The small numbers of

D-positive fetal red cells entering the ma-

ternal circulation early during the second

affected pregnancy constitute a secondary

stimulus sufficient to elicit overt production

of IgG anti-D. In susceptible women not

immunized after two D-positive pregnan-

cies, later pregnancies may be affected but

with diminished frequency. The incidence

of the more common genotypes in D-posi-

tive individuals can be found in Table 14-4.

This can be used to get a general idea of the

likelihood of how often an infant with a

D-positive father and D-negative mother

will express the D antigen.

Once immunization has occurred, suc-

cessive D-positive pregnancies often mani-

fest HDFN of increasing severity, particu-

larly between the first and second affected

pregnancies. After the second affected

pregnancy, the history is predictive of out-

come, although, in rare instances, some

women have a stable or diminishing pat-

tern of clinical disease in subsequent preg-

nancies.

Effect of ABO Incompatibility. Rh im-

munization of untreated D-negative women

occurs less frequently after delivery of an

ABO-incompatible D-positive infant than

when the fetal cells are ABO-compatible

with the mother. ABO incompatibility be-

tween mother and fetus has a substantial

but not absolute protective effect against

maternal immunization by virtue of the in-

creased rate of red cell destruction by anti-A

or anti-B. The rate of immunization is de-

creased from 16% to between 1.5% and 2%.7

Transfusion as the Immunizing Stimulus

It is extremely important to avoid trans-

fusing D-positive whole blood or red cells

to D-negative females of childbearing

potential because anti-D stimulated by

transfusion characteristically causes severe

HDFN in subsequent pregnancies with a

D-positive fetus. Red cells present in pla-

telet or granulocyte concentrates can con-

stitute an immunizing stimulus; if com-

ponents from D-positive donors are

necessary for young D-negative female

recipients, Rh immunoprophylaxis should

be considered. This is discussed further in

Chapter 21.

The risk of immunization to a red cell

antigen other than D after an allogeneic red

cell transfusion has been estimated to be

1% to 2.5% in the general hospital popula-

tion.7This will endanger the fetus only if the

antibody is IgG and directed against an an-

tigen that is also present on the fetal red

cells. For a couple planning to have chil-

dren, the woman should not be transfused

with red cells from her sexual partner or his

blood relatives. This form of directed dona-

tion increases the risk that the mother will

be immunized to paternal red cell, leuko-

cyte, and/or platelet antigens, which could

cause alloimmune cytopenias in future

Chapter 23: Perinatal Issues in Transfusion Practice 537

children who share the same paternal anti-

gens. Programs using parents as directed

donors for their sick newborns deserve spe-

cial consideration because of these unique

issues in the face of strong parental desires.9,10

ABO Antibodies

The IgG antibodies that cause ABO HDFN

nearly always occur in the mother’s circu-

lation without a history of prior exposure

to human red cells. ABO HDFN can occur

in any pregnancy, including the first. It is

restricted almost entirely to group A or B

infants born to group O mothers because

group O individuals make the IgG anti-

body, anti-A,B. Group A or B mothers with

an A- or B-incompatible fetus predomi-

nantly produce IgM antibody, with only

small amounts of IgG antibody capable of

crossing the placenta.

Prenatal Evaluation

Maternal History

Information about previous pregnancies or

blood transfusions is essential in evaluat-

ing fetal risk. Invasive tests, which carry

risk to the fetus, should be performed

only for pregnancies in which the fetus is

at risk for HDFN, by history and/or sero-

logic testing. For a woman with a history

of an infant with hydrops fetalis due to

anti-D, there is a 90% or more chance of a

subsequent fetus being similarly affected.5

In contrast, during the first sensitized

pregnancy, the risk of a hydropic fetus is

8% to 10%. Experience with other allo-

antibodies has not been as extensive as with

anti-D; in some series, anti-c and anti-K1

were by far the most common causes of

severe HDFN, other than anti-D.5,11

Serologic Studies

Alloantibodies capable of causing HDFN

can be detected during pregnancy. Initial

studies should be performed on all preg-

nant women as early in pregnancy as

possible; they should include tests for

ABO and D, and a screen for unexpected

red cell antibodies.12 If a woman’s red cells

are not directly agglutinated by anti-D,

testing for weak D is not required. When

testing for weak D is not performed,

women with weak D will be labeled as D

negative, although they are in fact D posi-

tive; the only potential negative outcome

is that these women will receive unneces-

sary Rh Immune Globulin (RhIG). Women

with some partial D phenotypes, such as

DVI, will also most likely type as D negative

in direct tests, and, in the absence of weak

D testing, these women are also candi-

dates for RhIG antenatal prophylaxis.

With the application of molecular tech-

niques, knowledge of the RHD gene is

evolving. Not all weak D red cells are the re-

sult of a decreased expression of the D anti-

gen, but, rather, some have altered RhD

proteins. Consequently, these patients are

at risk for immunization when exposed to

the D antigen, explaining formation of

anti-D in some patients classified as weak

D. As more is learned about the D antigen,

the distinction between partial D and weak

Disblurring.

13,14

Whether or not prophylaxis would be

successful in the setting of partial D remains

unknown.Theappropriatedoseisalsosub

ject to speculation. As a result, some practi-

tioners administer RhIG to these women,

whereas others consider it unnecessary.

Very rarely, a mother with partial D antigen

produces anti-D as a result of pregnancy.

If weak D testing is performed and the

test is clearly positive, the woman should

be regarded as D positive. If testing for

weak D is not performed, women whose

red cells do not react in direct tests with

anti-D can be considered candidates for

RhIG prophylaxis. Some laboratories con-

tinuetodoweakDtestingtoavoidconfu

538 AABB Technical Manual

sion in the interpretation of the FMH screen

during postpartum testing.12 Chapter 14

contains a more complete discussion of the

Rh blood group.

A woman should be classified as D posi-

tive if the test for either D or weak D is posi-

tive. If a D-negative woman has a negative

initial antibody screen, the test can be re-

peated at 28 weeks’ gestation before ad-

ministration of RhIG to detect immuniza-

tion that might have occurred before 28 weeks,

in accordance with AABB recommendations.

Because the incidence of immunization

during this period of pregnancy is extremely

low, the American College of Obstetricians

and Gynecologists (ACOG) points out that

no data exist that support the cost-effective-

ness of this practice.15 Repeat antibody

screening of D-positive women may be rec-

ommended if there is a history of clinically

significant red cell antibodies associated

with HDFN, previous blood transfusion, or

trauma to the abdomen.

Antibody Specificity. All positive screens

for red cell antibodies require identification

of the antibody.12 Themerepresenceofan

antibody, however, does not indicate that

HDFN will occur. Non-red-cell-stimulated

IgM antibodies, notably anti-Leaand anti-I,

are relatively common during pregnancy but

do not cross the placenta. In addition, the

fetal red cells may lack the antigen corre-

sponding to the mother’s antibody; the like-

lihood of fetal involvement can often be

predicted by typing the father’s red cell an-

tigens.16 The laboratory report on prenatal

antibody studies should include sufficient

information to aid the clinician in deter-

mining the clinical significance of the iden-

tified antibody.

Typing the Fetus. ThefetalDtypecanbe

established by using the polymerase chain

reaction (PCR) to amplify DNA obtained

from amniotic fluid, chorionic villus sam-

ples, or by serologic typing of fetal blood

obtained by cordocentesis.17 Chorionic villus

sampling is discouraged because it causes

FMH and has been associated with more

severe HDFN. Amniotic fluid samples are

recommended over cordocentesis because

cordocentesis has a fourfold or higher rate

of perinatal loss over amniocentesis.18 The

use of molecular techniques can help de-

tect variations in the RHD gene that might

go undetected using serology alone. It is

preferable that both paternal and maternal

blood samples accompany the fetal sam-

ples.19 Fetal DNA typing is also available for

Jka/Jkb,20 K1/K2,21 c,22 and E/e23 antigens. A

more recent development in fetal RhD typ-

ing involves the isolation of free fetal DNA

in the maternal serum. Although not rou-

tinely available in the United States at this

time, this will likely replace amniocentesis

for fetal genotyping in the near future.24

Maternal Antibody Titer

Antibody titrations can help in decisions

about the performance and the timing of

invasive procedures, especially if the anti-

body is anti-D. The antibody titer should

be established in the first trimester to

serve as a baseline, and the specimen

should be frozen for future comparisons

(see Method 5.3).7Because invasive tests

will not be undertaken before 16 to 18

weeks’ gestation, no further titration is in-

dicated until this time. The true signifi-

cance of an antibody titer in maternal se-

rum is controversial because some studies

have shown poor correlation between the

level of the titer and effects on the fetus.

For antibodies other than anti-D, critical

titers have not been identified, although a

critical titer similar to that used in cases

of anti-D alloimmunization is often uti-

lized.25 These techniques continue to be

performed because they represent a non-

invasive way to try to assess the presence

and severity of alloimmunization. When

performed, it is important that successive

Chapter 23: Perinatal Issues in Transfusion Practice 539

titrations use the same methods and test

cells of the same red cell phenotype. Test-

ing previously frozen serum samples in

parallel with a current specimen mini-

mizes the possibility that changes in the

titer result from differences in technique.

The critical titer for anti-D (the level be-

low which HDFN and hydrops fetalis are

considered so unlikely that no further in-

vasive procedures will be undertaken)

should be selected at each facility and is

usually 16 or 32 in the antihuman globu-

lin phase.26,27 Follow-up testing is recom-

mended for any titer greater than 8.26 The

critical titer for anti-K1 may be lower than

anti-D, typically a value of 8.25,28 Currently,

it is not recommended that gel technol-

ogy be used for prenatal antibody titra-

tion because of the lack of data showing a

correlation between gel and tube aggluti-

nation titers.12

Other Measures of HDFN Severity

Numerous laboratory procedures have been

investigated to improve the accuracy of

predicting the severity of hemolysis.29 The

antibody titer discussed above is not al-

ways reliable, nor is the serial change in

titer. Functional assays, including measures

of adherence, phagocytosis, antibody-de-

pendent cytotoxicity, and chemilumines-

cence have been investigated, but their use

has been limited and remains controver-

sial. These procedures are usually per-

formed in referral centers and may be use-

ful when additional information is required

to manage difficult and complex cases.

Amniotic Fluid Analysis

A good index of intrauterine hemolysis

and fetal well-being is the level of biliru-

bin pigment found in amniotic fluid ob-

tained by amniocentesis. Amniocentesis

is usually performed in alloimmunized

women who have a history of previously

affected pregnancies or have an antibody

titer at or above the critical titer.28 Because

fetal anemia secondary to K1 alloimmuni-

zation is not always associated with ele-

vated levels of bilirubin in amniotic fluid,

it has been recommended that fetal blood

sampling be used instead of serial amnio-

centesis when anti-K1 is detected in a

pregnant woman.2

Amniotic fluid is obtained by inserting a

long needle through the mother’s abdomi-

nal wall and uterus into the uterine cavity

under continuous ultrasound guidance.

The aspirated fluid is scanned spectropho-

tometrically at wavelengths of 350 to 700

nm. Peak absorbance of bilirubin is at 450

nm. An increase in optical density from the

projected baseline at 450 nm (∆OD450)isa

measure of the concentration of bile pig-

ments.30,31 The ∆OD450 value is plotted on a

graph against the estimated length of gesta-

tion, because bile pigment concentration

has different clinical significance at differ-

ent gestational ages. Liley’s system31 (Fig

23-1) of predicting the severity of fetal dis-

ease based on the ∆OD450 has been used for

decades. It delineates three zones to esti-

mate severity of disease: a top zone (zone 3)

indicates severe disease, the bottom zone

(zone 1) indicates mild or no disease, and

mid-zone (zone 2) values require repeat de-

termination to establish a trend. This

method is applicable to pregnancies from

27 weeks through term. Queenan et al32

have proposed a system for managing

D-immunized pregnancies based on the

∆OD450 from as early as 14 weeks’ gestation.

They identified four zones (Fig 23-2), with

early invasive intervention recommended if

∆OD450 values fall in the highest zone. With

both systems, the severity of HDFN is more

accurately predicted with serial ∆OD450

measurements than with a single observa-

tion, to evaluate whether readings are fall-

ing, rising, or stable. In general, the higher

the pigment concentration, the more se-

540 AABB Technical Manual

veretheintrauterinehemolysis.Itisimpor

tant to perform an ultrasound to establish

the correct gestational age so that the test

can be interpreted and the course of ther-

apy for a particular ∆OD450 will be appro-

priate.18 Alternatively, a ∆OD450 value in the

upper mid-zone of the Liley curve indicates

the need for fetal blood sampling.

Amniocentesis, particularly if the needle

goes through the placenta, or cordocentesis

may cause FMH, which can boost the titer

of existing red cell alloantibody, thereby in-

creasing the severity of HDFN, or inducing

immunization to additional antigens.18

Therefore, when amniocentesis or cordo-

centesis is performed for any reason on a

D-negative woman who does not have

anti-D, Rh immunoprophylaxis should be

given.

Percutaneous Umbilical Blood Sampling

In the early 1980s, the use of sophisticated

ultrasound equipment made it feasible to

direct a needle into an umbilical blood

vessel, preferably the vein at its insertion

into the placenta, and obtain a fetal blood

sample. Percutaneous umbilical blood

sampling (PUBS, or cordocentesis) allows

direct measurement of hematologic and

biochemical variables. Determination of the

fetal hematocrit provides an accurate as-

sessment of the severity of fetal hemolytic

disease.18 It is important to verify that the

sample has been obtained from the fetus.

Fetal and maternal red cells can be distin-

guished because of differences in red cell

size and red cell phenotyping, as well as

bythepresenceoffetalhemoglobin.

Chapter 23: Perinatal Issues in Transfusion Practice 541

Figure 23-1. Liley graph for collecting data from amniotic fluid studies. Intrauterine transfusion should

be done if the ∆OD450 value is in the top zone before 32 weeks’ gestation. After 34 weeks, top zone val-

ues indicate immediate delivery. Either intrauterine transfusion or immediate delivery may be indi-

cated for top zone ∆OD450 between 32 and 34 weeks, depending on studies of fetal maturity. Modified

from Liley.31

The fetal mortality of intrauterine fetal

blood sampling has been reported to be 1%

to 2%,34 and the procedure carries a high

risk of FMH. Its use is recommended only

for certain circumstances, such as when se-

rial amniotic fluid determinations indicate

severe HDFN, when hydrops is present,

when the D titer is high or rising, or when

HDFN occurred in previous pregnancies. In

addition to diagnosis, PUBS allows treat-

ment of the affected fetus.

Doppler Flow Studies

Because fetal anemia results in increased

cardiac output, several investigators have

measured various blood velocities in fetal

vessels using Doppler ultrasonography to

determine the clinical status of the fetus

in a noninvasive manner.18,35 Recent stud-

ies have found good correlation between

middle cerebral artery (MCA) peak veloc-

ity, fetal hemoglobin, and ∆OD450 read

ings.36 Many centers routinely use an MCA

peak systolic velocity value of greater than

1.5 multiples of the median to proceed

with cordocentesis to determine if the fe-

tus is anemic. In such centers, amniocen-

tesis is performed only after 35 weeks’

gestation when MCA Doppler is associ-

ated with a high false-positive rate for the

diagnosis of fetal anemia (Moise K, per-

sonal communication).

Suppression of Maternal

Alloimmunization

Several approaches to suppress maternal

alloimmunization have been attempted,

two of which have limited clinical benefit

542 AABB Technical Manual

Figure 23-2. Amniotic fluid OD450 management zones. (Reproduced with permission from Queenan et

al.32)

in reducing maternal antibody levels: in-

tensive plasma exchange and the admin-

istration of immunoglobulin (intravenous)

(IGIV).5,18 Plasma exchange can reduce an-

tibody levels by as much as 75%. Unfortu-

nately, rebound usually follows because

the IgG antibody is mostly extravascular

and antigen exposure may be ongoing.

Plasma exchange has been proposed as a

way to delay the need for fetal interven-

tion, particularly when there is a previous

pregnancy complicated by early hydrops.37

In this setting, plasma exchange can delay

the need for more invasive procedures un-

til the second trimester. The AABB and

the American Society for Apheresis (ASFA)

categorize plasma exchange as treatment

Category III because its efficacy and safety

have not been proven for this indication

(see Chapter 6). With the increasing safety

of intrauterine transfusion through ultra-

sound guidance and the decreasing inci-

dence of HDFN due to anti-D, the use of

plasma exchange as a treatment modality

has declined.

IGIV infusion has also been shown to

stabilize anti-D titers, with best results ob-

tained when started before 28 weeks’ gesta-

tion and when the fetus is not hydropic.38 In

a small study assessing the efficacy of IGIV,

it was found to be well tolerated and there

was a decrease in hemolysis.39 The mecha-

nism of IGIV effect is not clear, although it

may work by saturating placental Fc recep-

tors and inhibiting the transplacental trans-

fer of maternal antibody or by suppressing

ingestion of IgG-coated red cells by the fetal

reticuloendothelial system. An alternative

explanation is that the introduction of

anti-idiotype antibodies modifies maternal

antibody production. IGIV has also been

used with plasma exchange to reduce the

antibody rebound that has been seen fol-

lowing plasma exchange.18 Until larger stud-

ies assessing safety and efficacy can be per-

formed, intrauterine transfusion remains

the mainstay of standard therapy.

Intrauterine Transfusion

Intrauterine transfusion can be performed

by the intraperitoneal route (IPT) or the

direct intravascular approach (IVT) by the

umbilical vein. In many instances, IVT is

the procedure of choice, but there may be

problems of access that make IPT prefera-

ble; a combination may also be used to

minimize peaks and troughs of fetal hema-

tocrit between procedures. Intrauterine

transfusion is seldom feasible before the

20th week of gestation; once initiated,

transfusions are usually administered pe-

riodically until delivery. It is important

that blood is available and ready at the

time of the first diagnostic, and subse-

quent, cordocenteses. If fetal anemia is

detected, an intrauterine transfusion can

be performed at once, minimizing fetal

risks. The interval between transfusions

depends on the presence or absence of

hydrops, the gestational age, and the

amount of blood infused. Good outcomes

areachievedover80%ofthetimeand

94% of nonhydropic fetuses survive.40 Be-

cause intrauterine transfusion carries a 1%

to 2% risk of perinatal loss, it should be

performed only after careful clinical eval-

uation.18,34,41,42 Other perinatal conditions

that have been treated with intrauterine

transfusion include parvovirus infection,

large FMH, and alpha thalassemia.43

Techniques

IPT is performed through a needle passed,

with ultrasonographic monitoring, through

the mother’s abdominal wall into the

abdominal cavity of the fetus. Transfused

red cells enter the fetal circulation through

lymphatic channels that drain the peri-

toneal cavity. In IVT, the umbilical vein is

penetrated under ultrasound guidance,

Chapter 23: Perinatal Issues in Transfusion Practice 543

and a blood sample is taken to verify posi-

tioning in the fetal vasculature. Injection

of saline can also confirm correct place-

ment because it can be visualized by ul-

trasound. Blood is infused directly, as ei-

ther a simple transfusion or as a partial

exchange transfusion. IVT can be particu-

larly valuable for very severe cases of

HDFN associated with hydrops fetalis. In

hydropic infants, red cells administered

by IPT are not efficiently absorbed.

Selection of Red Cells

The red cells used should be group O,

D-negative, or negative for the antigen

corresponding to the mother’s antibody if

the specificity is not anti-D. Blood for

intrauterine transfusion should be irradi-

ated (see Chapter 27), and should be cyto-

megalovirus (CMV)-reduced-risk. It may

also be desirable to transfuse blood that is

knowntolackhemoglobinSinorderto

transfuse red cells with maximal oxygen-

transporting capacity, in the setting of low

oxygen tension. For optimal survival of the

transfused cells, blood used for intraute-

rine transfusion should be drawn as re-

cently as possible, generally less than 7

days old.

The hematocrit of the RBCs prepared for

exchange transfusion is usually high to

minimize the chance of volume overload in

the fetus. Washed, irradiated maternal

blood has also been used for intrauterine

transfusion.44 Toremovetheoffendinganti

body, the red cells are washed and resus-

pended in saline to a final hematocrit

between 75% and 85%. Washed or degly-

cerolized preparations have been used as a

means to remove plasma, anticoagulant/

preservative solutions, and excess electro-

lytes that might accumulate during pro-

longed storage. Blood for intrauterine

transfusions and all blood and cellular

components subsequently transfused in

the neonatal period should be irradiated to

prevent transfusion-associated graft-vs-host

diseasebecausethefetusisconsideredim

munologically naïve and tolerant.45,46

Volume Administered

Thevolumetransfusedvarieswiththe

technique used as well as the fetal size,

initial hematocrit, and gestational age.

For IPT, a volume calculated by the for-

mula V = (gestation in weeks – 20) ×10 mL

appears to be well tolerated by the fetus.

The volume of red cells transfused by IVT

can be calculated by the following for-

mula.47

Fetoplacental volume (mL) =

ultrasound estimated fetal weight

(g) ×0.14

Volume to transfuse (mL) =

Fetoplacental volume ×

(Hct after IVT – Hct before IVT)

Hct of donor cells

where Hct = hematocrit

Transfusion is repeated on the basis of

an estimated decline in fetal hematocrit of

approximately 1% per day in an effort to

maintain the fetal hematocrit in the range

of 27% to 30%.48

Postpartum Evaluation

It may be desirable to collect a sample of

cord blood, preferably by cannulation of

an umbilical vessel at delivery, from new-

bornswherethereisariskofHDFN(eg,

Rh-positive infants born to Rh-negative

mothers, type A and B infants born to

type O mothers). This sample should be

identified as cord blood and labeled in

the delivery suite with the mother’s name,

the date, and two unique forms of identi-

fication for the infant (eg, name and med-

ical record number).

544 AABB Technical Manual

In cases of suspected HDFN, samples of

both cord and maternal blood should be

tested. When the mother is known to have

antibodies capable of causing HDFN, the

hemoglobin or hematocrit and the biliru-

bin level of cord blood should also be de-

termined. If the mother is D negative and

the infant D positive, the mother’s blood

should be tested to determine the volume

of FMH, as discussed later. Testing cord

blood may present some special problems,

which are described below.

ABO Testing

ABO testing on newborns relies entirely on

red cell typing because ABO antibodies in

cord serum are nearly always of maternal

origin and are IgG. However, in the inves-

tigation of possible HDFN due to ABO in-

compatibility, cord serum should be tested

for antiglobulin-reactive ABO antibodies.

Also, if the infant will receive non-group-O

red cells, testing must be taken to the

antiglobulin phase.49(p42)

D Testing

Newborns who have had successful intra-

uterine transfusions often type at birth as

D negative or very weakly positive be-

cause over 90% of their circulating red

cells may be those of the donors. The ABO

and direct antiglobulin tests may also give

misleading results. If the infant’s red cells

are heavily coated with IgG antibodies, tests

with anti-D may give either false-positive

or false-negative results (see Chapter 14).

Antiglobulin Testing

The direct antiglobulin test (DAT) is usu-

ally strongly positive in HDFN resulting

from anti-D or antibodies to other blood

groups; reactions are much weaker or

even negative in HDFN resulting from

ABO antibodies. However, the strength of

the DAT does not correlate with the sever-

ity of hemolysis, especially in ABO HDFN.

Infants who have received an intrauterine

transfusion may have a weakly positive

DAT with a mixed-field pattern of aggluti-

nation. If the DAT on cord cells is positive,

the antibody can be eluted from the red

cells and tested for specificity. It is not

necessary to make and test an eluate if

the maternal serum has been shown to

contain a single red cell antibody. All clin-

ically significant red cell antibodies in the

maternal serum must be respected. (See

the section on Selection of Blood, later in

this chapter.) If the DAT is positive and

the maternal antibody screen is negative,

investigation should turn toward ABO an-

tibodies or HDFN caused by an antibody

directed against a low-incidence antigen

not present on reagent red cells.

Evaluation of ABO Antibodies. ABO

HDFN may be suspected on clinical

grounds even though the DAT is negative.

Testing the eluate from the cord cells

against A1and B red cells should establish

the diagnosis of ABO HDFN. Cord blood or

peripheral blood serum should be tested by

an indirect antiglobulin technique against

A1, B, and O red cells. The presence of anti-A,

anti-B, or anti-A,B confirms the potential

for ABO HDFN. It is often possible to elute

anti-A and/or anti-B from the infant’s red

cells despite a negative DAT, but this step is

not necessary for the presumptive diagno-

sis.IntherarecasesofABOHDFNthatre

quire transfusion, D-compatible group O

blood should be transfused, whether or not

the diagnosis has been serologically con-

firmed. Nonimmune causes of hyperbiliru-

binemia and hemolysis should still be con-

sideredinaninfantwithanegativeDAT

before concluding that it is due to ABO

HDFN because other hematologic disor-

ders might be present.50

Chapter 23: Perinatal Issues in Transfusion Practice 545

Antibodies to Low-Incidence Antigens.

If ABO HDFN is ruled out, antibody against

a low-incidence red cell antigen should be

suspected. Testing an eluate or maternal

serum against the father’s red cells with an

antiglobulin technique may provide an an-

swer. Maternal serum must be ABO com-

patible, if it is used. If either or both of these

tests are positive, it indicates that the infant

has an antigen of paternal origin that the

mother lacks, causing her to make an IgG

antibody directed against this antigen. Un-

less the mother has been exposed to red cells

from the father or his blood relations, trans-

fusion would be an unlikely immunizing

event for a low-incidence antigen. Because

there should be no difficulty in obtaining

compatible blood, diagnostic studies can

be performed after initial clinical concerns

have been resolved. If the DAT is positive

and all attempts to characterize a coating

red cell antibody are consistently negative,

causes of a false-positive DAT should be

considered (see Chapter 20).

Exchange Transfusion

Exchange transfusion for HDFN achieves

several desired effects, including:

1. Removal of antibody-coated fetal red

cells.

2. Removalofmaternalantibody.

3. Removal of bilirubin.

4. Replacement of red cells, thereby

treating anemia.

The red cells used for replacement must

be compatible with the causative antibody.

FreshFrozenPlasmaisfrequentlyusedto

reconstitute whole blood because it pro-

vides coagulation factors. Platelet values

should also be monitored and transfusion

used as necessary. Plasma frozen within 24

hours and thawed plasma can be used too,

with the understanding that there might be

decreases in the activity of the labile

clotting Factors V and VIII.

Selection of Blood

In most cases, the mother’s serum is used

for crossmatching and the red cells se-

lected for transfusion are compatible with

her ABO antibodies as well as any addi-

tional antibody(ies) responsible for the

hemolytic process. Group O red cells re-

suspended in AB plasma are commonly

used. In ABO HDFN, the red cells used for

exchange must be group O. If the anti-

body is anti-D, the red cells must be D

negative, but not every exchange transfu-

sion requires group O RBCs. If mother

and infant are ABO-identical, group-spe-

cific red cells or whole blood can be used.

If the implicated antibody is not anti-D,

D-positive red cells may be given to a

D-positive infant.

Maternal serum or plasma is the speci-

men of choice for crossmatching in ex-

change transfusion; it is available in large

quantities, decreases the volume of blood

taken from the infant, has the red cell anti-

body present in high concentration, and

can be analyzed accurately and completely

before delivery. On the other hand, use of

maternal serum may be problematic if it

contains antibodies directed against anti-

gens not present on the infant’s red cells

because of other sources of sensitization, or

if it contains IgM antibodies that have not

crossed the placenta. These additional

antibodies could complicate the serologic

picture.

If maternal blood is not available or is

unsuitable for crossmatching, the infant’s

serum and/or, preferably, an eluate from

the infant’s red cells can be used for cross-

matching. The concentration of antibody in

the infant’s serum may be low, especially if

most of the molecules are bound to the red

cells. Use of the eluate or serum, or of both

together, may be indicated if attempts to

obtain a maternal specimen would delay

therapy. Blood used for exchange transfu-

546 AABB Technical Manual

sion should be irradiated. Typically, a vol-

umeoftwicetheinfant’sbloodvolumeis

used for exchange.51

Subsequent Transfusion

Bilirubin may reaccumulate rapidly after

a successful exchange transfusion despite

appropriate phototherapy. This occurs

because most bilirubin in extravascular

fluid will reequilibrate by entering the

intravascular space and also because re-

sidual antibody-coated cells continue to

hemolyze. If rising bilirubin levels make a

second or third exchange transfusion nec-

essary, the same considerations of red cell

selection and crossmatching apply.

Infants who have undergone intraute-

rine transfusion need to be followed closely

after birth because intrauterine transfusion

suppresses fetal erythropoiesis. Weekly

hematocrit and reticulocyte counts should

be performed on the neonate for a 1- to

3-month period.18 Many of these infants

will subsequently develop anemia and

need to be supported with red cell transfu-

sion until their own production begins, as

evidenced by reticulocytosis and age-ap-

propriate hemoglobin levels.18,52

Antibody Against a High-Incidence Antigen

Rarely, the mother’s antibody reacts with

a high-incidence antigen and no compati-

ble blood is available. If this problem is

recognized and identified before delivery,

the mother’s siblings can be evaluated for

compatibility and suitability, or compati-

ble donors can be sought through a rare

donor file. Maternal red cells can also be

collected and frozen. Any products collected

from blood relatives must be irradiated. If

this very rare event is not recognized until

after delivery, three choices are available:

1. Collect blood from the mother, if the

obstetrician agrees. Remove as much

plasma as possible, preferably by sa-

line washing, and resuspend the red

cells in compatible plasma to the de-

sired hematocrit.

2. If time permits, test the mother’s sib-

lings or other close relatives for

compatibility and eligibility.

3. Use incompatible donor blood for

theexchangetransfusioniftheclini

cal situation is sufficiently urgent.

The exchange will reduce the biliru-

bin load, the most heavily anti-

body-coated cells, and the number

of unbound antibody molecules.

However, residual antibody will at-

tach to the transfused cells, and one

or more additional exchanges will

probably be needed as bilirubin ac-

cumulates.

Rh Immune Globulin

RhIG is a concentrate of predominantly

IgG anti-D derived from pools of human

plasma. A full dose of anti-D (300 µg, 1500

IU, or the actual content of a “dose” as in-

dicated by the individual manufacturer53)

is sufficient to counteract the immunizing

effects of 15 mL of D-positive red cells;

this corresponds to approximately 30 mL

of fetal whole blood. RhIG is available in a

reduced dose, approximately 50 µg, which

is protective for up to 2.5 mL of D-positive

fetal red cells. This dose can be used for

first-trimester abortion or miscarriage, when

the total blood volume of the fetus is less

than 2.5 mL. However, because of fears of

miscalculating the length of pregnancy

and concerns of inadvertently mixing up

inventory resulting in undertreatment, a

full dose is usually administered and

these low doses are frequently not stocked.

The protective effect of RhIG on D-nega-

tive individuals exposed to D-positive

cells probably results from interference

with antigen recognition in the induction

phase of primary immunization.54

Chapter 23: Perinatal Issues in Transfusion Practice 547

RhIG is available in two formulations: 1)

for intramuscular (IM) injection only and 2)

for either IM or intravenous (IV) adminis-

tration.Thedoseoftheintravenousprepa

ration is expressed in international units (5

IU is equivalent to 1 µg), with 1500 IU (300

µg) neutralizing 17 mL of D-positive red

cells, according to the package insert.

Antepartum Administration

Widespread postpartum use of Rh immuno-

prophylaxis has reduced pregnancy-asso-

ciated immunization to the D antigen to

1% to 2%.8This risk is further decreased to

0.1% if RhIG is also given antepartum at

28 weeks of gestation.7The ACOG recom-

mends antepartum RhIG prophylaxis at

28 weeks of gestation, based on the obser-

vation that, of women who develop anti-D

during pregnancy, 92% do so at or after 28

weeks.7,15

Blood obtained before injection of RhIG

should be tested for ABO and D. A D-nega-

tive woman who has antibodies other than

anti-D (eg, anti-G) is still a candidate for

anti-D immunoprophylaxis. When the

mother receives RhIG during pregnancy,

the infant may be born with a positive DAT,

but without hemolysis. The mother’s serum

will often exhibit anti-D reactivity. There

must be good communication between the

patient’s physician and the blood bank staff

at the institution where delivery takes

place, to ensure correct interpretation of

laboratory tests made at the time of deliv-

ery. The half-life of an injected dose of RhIG,

in the absence of significant FMH, is ap-

proximately 21 days. Therefore, of 300 µgof

anti-D given at 28 weeks, 20-30 µgcouldre

main at the time of delivery 12 weeks later.

Some practitioners will administer another

dose of RhIG if delivery is delayed beyond

40 weeks.18 Anti-D can be detected in the

maternal circulation for as long as 6 months.

Postpartum Administration

Cord blood from infants born to D-nega-

tive mothers should be tested for the D

antigen. A D-negative woman with a

D-positive infant should receive one full

dose of RhIG within 72 hours of delivery,

unless she is known to be alloimmunized

to D previously. The presence of residual

anti-D from antepartum RhIG does not

indicate ongoing protection.

Active vs Passive Antibody. In-vitro clues

can help distinguish passively administered

RhIG from the anti-D formed as a result of

active alloimmunization. Passively acquired

anti-D is entirely IgG; if a woman’s anti-D is

saline-reactive or can be completely or par-

tially inactivated by treating the serum with

2-mercaptoethanol or dithiothreitol, it has

an IgM component and probably repre-

sents active immunization. Passively ac-

quired anti-D rarely achieves an anti-

globulin titer above 4, so a high-titered

antibody or rising antibody titer is likely to

indicate active immunization. It is desirable

to obtain confirmation from the physician’s

records, but RhIG should always be given

when doubt cannot easily be resolved. It

should also be given if there is any problem

determining the Rh type.

Postpartum Evaluation. Asampleofthe

mother’s blood should be drawn, preferably

within 1 hour after delivery, and evaluated

for FMH of a quantity greater than that for

which 300 µg RhIG is immunosuppressive.

If the screening test demonstrates the pres-

ence of fetal cells, the extent of FMH must

be determined so that an appropriate dose

of RhIG can be administered.49(pp48,49),55

Postpartum RhIG should be given within

72hoursofdelivery.Ifprophylaxisisde

layed, the likelihood that alloimmunization

will be prevented decreases. Despite the de-

crease seen, the ACOG recommends that

treatment still be administered because

some studies have found partial protection

548 AABB Technical Manual

has occurred as late as 13 days after expo-

sure and, possibly, as late as 28 days.15

The following women are not candidates

for RhIG:

1. The D-negative woman whose infant

is D-negative.

2. Any D-positive woman. Very rare cases

of HDFN have been reported in in-

fants whose mothers had a weak/par-

tial D phenotype, but routine RhIG

prophylaxis is not routinely recom-

mended for women of the weak/

partial D phenotype.12

3. A D-negative woman known to be

immunized to D.

Other Indications for RhIG

RhIG should be given to a D-negative woman

after any obstetric event that might allow

fetal cells to enter the mother’s circula-

tion: spontaneous or therapeutic abor-

tion, ectopic pregnancy, amniocentesis,

chorionic villus sampling, molar preg-

nancy, cordocentesis, antepartum hemor-

rhage, blunt abdominal trauma, or fetal

death.55 As mentioned earlier, at 12 weeks

of gestation or earlier, a 50 µgdoseofRhIG

would be adequate to protect against the

small fetal blood volume during the first

trimester. From 13 weeks’ gestation until

term, a full dose of RhIG should be given.

At <20 weeks, the fetal blood volume is

rarely more than 30mL,56 small enough

that a single dose of 300 µgRhimmune

globulin will be sufficient for prophylaxis

for any FMH. Therefore, it is not neces-

sary to quantitate fetal red cells in the ma-

ternal circulation before 20 weeks of ges-

tation.57

Amniocentesis

Amniocentesis can cause FMH and con-

sequent Rh immunization. The D-negative

woman who has amniocentesis at 16 to 18

weeks’ gestation for genetic analysis should

receive a full dose of RhIG at that time, a

second full dose at 28 weeks of gestation,

and the usual postpartum dose if the in-

fant is D positive. If a nonimmunized

D-negative woman undergoes amniocen-

tesis for any reason in the second or third

trimester, a full dose of RhIG is indicated.

If the procedure is repeated more than 21

days later, an additional full dose should

be given.18 If amniocentesis is performed

to assess fetal maturity, and if delivery is

expected within 48 hours of the proce-

dure, RhIG can be withheld until the in-

fant is born and confirmed to be D posi-

tive. If more than 48 hours will elapse,

RhIG should be given following amnio-

centesis. If delivery occurs within 21 days

thereafter and there is no evidence of a

massive FMH,15 additional RhIG may not

be essential, but prudent management

suggests repeat RhIG administration at

delivery.

Screening for Large-Volume FMH

Postpartum administration of RhIG may

not prevent immunization if the quantity

of D-positive fetal red cells entering the

mother’s circulation exceeds the immuno-

suppressive capacity of RhIG. One 300-µg

dose protects against 15 mL of D-positive

red cells or 30 mL of fetal blood. Only 0.3%

of pregnancies are estimated to sustain

FMH greater than 30 mL, but large FMH

is an important and preventable cause of

failed immunoprophylaxis.7The ACOG

recommends postpartum testing for large

FMHonlyforhigh-riskpregnancies,

15 but

Ness and colleagues58 showed that testing

based only on the ACOG criteria would

miss 50% of mothers exposed to large-vol-

ume FMH. AABB Standards for Blood

Banks and Transfusion Services requires

examination of a postpartum specimen

from all D-negative women at risk of im-

Chapter 23: Perinatal Issues in Transfusion Practice 549

munization, to detect the presence of FMH

that requires more than one dose of RhIG.49(p49)

In rare cases, a massive FMH can cause

fetal death and infuse enough Rh-positive

cells into the maternal circulation to simu-

late a weak D phenotype in an Rh-nega-

tive patient.15,59 Unless recognized and

treated with an adequate dose of RhIG, this

will likely lead to alloimmunization.

“Microscopic Weak D.” In the past, some

workers looked for D-positive red cells in

the mother’s D-negative blood by examin-

ing the antiglobulin phase of the test for D

microscopically (“microscopic weak D

test”); mixed-field reactivity indicated a

substantial admixture with D-positive cells.

This procedure should not be used to iden-

tify large FMH, however, because of its lack

of reliability.60

The Rosette Test. The rosette test dem-

onstrates small numbers of D-positive cells

in a D-negative suspension. The suspen-

sion is incubated with an anti-D reagent of

human origin, and antibody molecules at-

tach to sites on D-positive cells in the sus-

pension. Indicator D-positive cells are

added, which react with antibody mole-

cules bound to the surface of the al-

ready-present D-positive cells and form vis-

ible agglutinates (rosettes) around them

(see Method 5.1). This method will detect

FMHs of approximately 10 mL,60 asensitiv

ity that provides a desirable margin of

safety for a screening test. Weak D-positive

cells do not react as strongly in the rosette

procedure as normal D-positive cells. If the

newborn has a weak D phenotype, FMH

can be evaluated by the Kleihauer-Betke

acid-elution test (see below), which identi-

fies fetal hemoglobin, not a surface antigen.

In all cases, the rosette test gives only quali-

tative results and a positive result must be

followed by a quantitative test, such as an

acid-elution procedure. Other tests that can

be used to detect and/or quantify FMH are

flow cytometry, gel agglutination, and the

enzyme-linked antiglobulin test (ELAT,

which is in limited use). Each of these

methods has various advantages and disad-

vantages that must be evaluated by each in-

stitution. The use of nucleic acid amplifica-

tion techniques, designed to detect very

small amounts of fetal cells, remains a re-

search endeavor.61-63

Quantifying FMH

Historically, quantification of FMH has

been achieved by the Kleihauer-Betke

acid-elution test, which relies on the dif-

ferences between fetal and adult hemo-

globin in resistance to acid elution (see

Method 5.2). Results are reported as a per-

centage of fetal cells, but the precision

and accuracy of the procedure may be

poor. Because 300 µg of RhIG will protect

against FMH of 30 mL of D-positive fetal

blood, the number of doses of RhIG re-

quired is determined by dividing the esti-

mated volume of fetal blood present by 30.

For example:

1. Kleihauer-Betke test results reported

as 1.3%

2. (1.3/100) ×5000 mL* = 65 mL of fetal

blood

3. 65/(30 mL per dose) = 2.2 doses of

RhIG required

* = mother’s arbitrarily assigned blood volume

Because quantification by this procedure

is inherently inaccurate and because the

consequences of undertreatment can be

serious, it is desirable to provide a safety

margin in calculating RhIG dosage. One

approach is as follows:

1. When the number to the right of the

decimal point is less than 5, round

down and add one dose of RhIG (ex-

ample: If the calculation comes to

2.2 doses, give 3 doses).

550 AABB Technical Manual

2. When the number to the right of the

decimal point is 5 or greater, round

up to the next number and add one

dose of RhIG (example: If the calcu-

lation comes to 2.8 doses, give 4

doses). (See Table 23-1).

Not more than five doses of RhIG should

be injected intramuscularly at one time. For

larger quantities, injections can be spaced

over a 72-hour period for the patient’s com-

fort; an optimal time sequence has not

been established. The intravenous prepara-

tion of RhIG can be used when higher

doses are required. According to the pack-

age insert, a maximum dose of 300 IU

should be given at each injection, every 8

hours, until the total calculated dose has

been administered.

Neonatal Immune

Thrombocytopenia

Maternal IgG antibodies to platelets can

cross the placenta and cause severe ante-

natal and neonatal thrombocytopenia. Two

categories of immune thrombocytopenia

are recognized, and the distinction be-

tween them is therapeutically important.

Neonatal Alloimmune Thrombocytopenia

The mechanism of NAIT is similar to that

of HDFN. Fetal platelets, expressing a pa-

ternal antigen absent from the mother’s

cells, may enter the mother’s circulation

during gestation or delivery. If she becomes

immunized, the maternal IgG antibody

crosses the placenta and causes fetal and

neonatal thrombocytopenia. The maternal

platelet count remains normal. The inci-

dence of NAIT is approximately 1 in 1500

to 2000 live births.64,65 NAIT is the cause of

the majority of cases of intracranial hem-

orrhage due to thrombocytopenia, greater

than all other etiologies of thrombocyto-

penia combined.66

Unlike HDFN, NAIT often affects firstborn

children, with about 50% to 60% of cases

occurring in a woman’s first child. The

thrombocytopenia is self-limiting, normally

resolving in 2 to 3 weeks. NAIT varies in se-

verity from mild thrombocytopenia with no

clinical signs to overt clinical bleeding. The

incidence of intracranial hemorrhage has

been reported as 10% to 30%, with approxi-

mately half occurring in utero.65,67 Recur-

rence in subsequent pregnancies is fre-

quent, with equal or increasing severity, so

Chapter 23: Perinatal Issues in Transfusion Practice 551

Table 23-1. RhIG Dosage for Massive Fetomaternal Hemorrhage, Based on the Acid

Elution Test

% Fetal Cells Vials of RhIG to Inject

Dose

In gInIU

0.3 - 0.8 2 600 3000

0.9 - 1.4 3 900 4500

1.5 - 2.0 4 1200 6000

2.1 - 2.5 5 1500 7500

Notes:

1. Based on a maternal blood volume of 5000 mL.

2. 1 vial of 300 µg (1500 IU) is needed for each 15 mL fetal red cells or 30 mL fetal whole blood.

a woman known to be alloimmunized must

receive skilled antenatal attention.

Serologic Testing

Serologic diagnosis should be sought in a

woman whose infant has had NAIT if fur-

ther pregnancies are planned. Several

platelet-specific antigen systems have

been associated with NAIT, with HPA-1a

antigen (PlA1) accounting for the vast ma-

jority of cases in Caucasians.64 Pregnancy,

rather than transfusion, is the usual im-

munizing event. Approximately 2% of the

population is HPA-1a negative; approxi-

mately 10% of HPA-1a-negative women

with HPA-1a-positive infants become im-

munized.68 Some studies have shown an

association between developing anti-HPA-

1a and possessing the HLA phenotype

DRw52a.66,68 Chapter 16 contains more in-

formation about platelet antigens. Al-

though antibodies to HLA Class I antigens

are frequently encountered in pregnancy,

and platelets express Class I antigens, this

isararecauseofNAIT,andthetrueroleof

HLA antibodies in this setting remains

controversial.69,70

Anyfamilywithahistoryofaninfant

born with a platelet count of <50,000/µL

should be evaluated. Ideally, serologic test-

ing uses maternal serum and maternal and

paternal whole blood from which platelets

are isolated. Maternal serum is screened for

both platelet-nonspecific and platelet-spe-

cific antibodies against paternal cells, as

well as panels of phenotyped platelets. Ma-

ternal and paternal platelet typing can be

performed using serologic and/or molecu-

lar typing methods. Some clinicians have

proposed screening pregnant women for

HPA-1a antigen because it is the most com-

monly implicated antigen causing incom-

patibility in Caucasians. Because only 10%

of HPA-1a-negative women are truly at risk

for forming antibody, and, of those, only

about 33% will have neonates with clini-

cally important thrombocytopenia, this has

not been widely adopted. In addition, the

cost and logistics of performing platelet an-

tigen typing are impediments to broad im-

plementation.65,71

Prenatal Considerations

With knowledge of antibody specificity

and gene frequencies, the likelihood of

subsequent offspring being affected can

be predicted (see Table 16-1). The recog-

nized platelet-specific antigens occur in

diallelic systems, so typing the father’s

platelets indicates zygosity. If the father is

homozygous for the expression of the an-

tigen, there is no need to determine the

fetal antigen status because all offspring

will be affected. If the father is heterozy-

gous for the expression of the antigen,

then there is a 50% chance that subse-

quent offspring will have the offending

antigen. In an at-risk pregnancy, the ge-

notype of the fetus (and by inference the

platelet phenotype) can be determined by

DNA typing on fetal cells obtained by am-

niocentesis.

When the risk of NAIT is high, a fetal

blood sample for platelet count determina-

tion can be obtained by cordocentesis as

early as 20 weeks’ gestation. Because

cordocentesis carries a risk of serious

bleeding in a thrombocytopenic fetus,

compatible platelets must be available at

thetimeoftheprocedureandareoftenin

fused if the platelet count is low. Some in-

stitutions will infuse platelets during the

procedure, before knowing the platelet

count, because of the risk of bleeding dur-

ing the cannulation itself. When the fetus

is found to be thrombocytopenic, the

mother is often given infusions of IGIV in

weekly doses of 1 g/kg, with or without ste-

roids, until delivery.64-67,72 Alternatively, some

would recommend empiric treatment with

552 AABB Technical Manual

IGIV in cases of a homozygous paternal ge-

notype for the specific platelet antigen or

in situations when PCR performed on

amniotic fluid reveals that the fetus carries

that platelet antigen. Many centers will pro-

ceed with elective cesarean section instead

of cordocentesis near term to determine

the fetal platelet count.

Sources of Platelets. Maternal platelets

are often prepared for use at cordocentesis

or delivery. The mother will undergo re-

quired testing for infectious disease mark-

ers. Prior administration of high-dose IGIV

may cause false-positive immunoassays;

therefore, it is desirable to test the mother

before initiating IGIV therapy. Those with

confirmed positive results (eg, hepatitis C

virus, which is transmitted more efficiently

by transfusion than perinatally) should not

be used as a source of platelets because

these results are more likely to represent

maternal infection. Of note, pregnancy it-

self can also cause false-positive results on

serologic infectious disease tests.

Platelets can be collected either from the

mother or from another donor whose

platelets lack the corresponding antigen

and whose plasma is compatible with the

fetal red cells. If maternal platelets are used,

the antibody-containing plasma should be

removed or reduced and the platelets re-

suspended in compatible plasma or saline

with reduced volume (see Method 6.15). All

components for intrauterine transfusion must

be irradiated (see Chapter 27) and should

be CMV-reduced risk.73

Scheduling Therapy. Various strategies

have been used in the management of fetal

thrombocytopenia. Although weekly plate-

let transfusions have been used in the past,

the inherent risk of repeated cordocentesis

makes the administration of IGIV to the

mother the preferred treatment in the

United States. Practice is different in Eu-

rope, where weekly platelet transfusions are

still performed. Platelet transfusion and re-

peated cordocentesis are reserved for pa-

tients when noninvasive forms of therapy

are not effective. Another approach is ad-

ministration of a single platelet transfusion

just before delivery if cordocentesis reveals

severe thrombocytopenia. This approach is

usually reserved for pregnancies at extreme

risk for intracranial hemorrhage.

Management After Delivery

Platelet counts can continue to decrease

after birth and should be monitored. For

patients at increased risk of bleeding due

to severe thrombocytopenia, compatible

platelets should be given prophylactically.

If compatible platelets are not available,

the use of high-dose IGIV should be con-

sidered, but response to this treatment is

variable. In patients who do respond, pla-

telet counts usually start increasing with-

in 24 to 48 hours, although it may take

longer in some patients.64 Because re-

sponse is slow, the neonate with an urgent

need for transfusion and no available

compatible platelets can receive platelets

from random donors, frequently resulting

in an adequate response. For patients re-

quiring platelet transfusion, giving con-

current IGIV can accelerate the recovery

of the patient’s own platelets and shorten

the period of transfusion dependency. If

the patient has mild thrombocytopenia

without bleeding, it can be managed

without specific therapeutic intervention.

Thrombocytopenia Secondary to

Maternal ITP

Infants born to mothers with active idio-

pathic (immune) thrombocytopenic purpura

(ITP) are often not profoundly thrombo-

cytopenic and have a smaller risk of hem-

orrhage than infants with NAIT.69,74 The

antibody in ITP is usually IgG, which

readily crosses the placenta. Occasionally,

delivery of a severely thrombocytopenic

Chapter 23: Perinatal Issues in Transfusion Practice 553

infant has led to the diagnosis of previ-

ously unsuspected ITP in a moderately af-

fected mother (postpartum platelet count

75,000-100,000/µL). Such cases of mild ITP

should be distinguished from gestational

thrombocytopenia, in which a mother

with no history of autoimmune thrombo-

cytopenia has a platelet count less than

150,000/µL. In cases of maternal ITP, the

risk of severe fetal thrombocytopenia

(usually defined as a platelet count less

than 50,000/µL) is 7% to 10%.74,75 The risk

of intracranial hemorrhage in infants

born to mothers with ITP is low (≤1%),

with only a few cases reported in the liter-

ature. This is lower than the rate in NAIT

because, infants born to mothers with ITP

are generally born with higher platelet

counts and their platelet function is not

impaired, as it seems to be in NAIT. Rou-

tine fetal platelet assessment is not rec-

ommended and cesarean section is re-

served for obstetric indications only.74,76

The antibody in ITP has broad reactivity

against platelets. If the infant has a high

concentration of antibody, there will be

uniformly short survival of platelets from

random donors, from the mother, or from

other family members. Responses do some-

times occur, and, in the presence of hemor-

rhage, platelet transfusions will be used as

emergency therapy.69 IGIV therapy may also

be effective for severe autoimmune throm-

bocytopenia.66,72,74,76

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556 AABB Technical Manual

Chapter 24: Neonatal and Pediatric Transfusion Practice

Chapter 24

Neonatal and Pediatric

Transfusion Practice

MANY PHYSIOLOGIC CHANGES

accompany the transitions from

fetus to neonate, neonate to

infant, and throughout childhood. Hema-

tologic values, blood volume, and physio-

logic responses to stresses such as hypo-

volemia and hypoxia vary widely. The

most rapid changes occur during early in-

fancy. Consequently, discussions of pedi-

atric transfusion are usually divided into

two periods: neonates from birth through

4 months, and older infants (>4 months)

and children. Some concerns in neonatal

transfusion practice overlap with those of

the perinatal period and are discussed in

Chapter 23.

Advances in medical care now permit

the survival of extremely premature neo-

nates. Blood providers must be capable of

furnishing blood components that are tai-

lored to satisfy the specific needs of very

low birthweight (VLBW <1500 g) and ex-

tremely low birthweight (ELBW <1000 g)

patients, whose small blood volumes and

impaired or immature organ functions pro-

vide little margin for safety. Ill neonates are

more likely than hospitalized patients of

any other age group to receive red cell

transfusions.1Advances in critical-care neo-

natology, such as surfactant therapy, nitric

oxide therapy, use of high-frequency venti-

lators, and adherence to transfusion prac-

tice guidelines, have diminished the num-

ber of blood transfusions given; most are

now given to infants with birthweights less

than 1000 g.1The doses of various compo-

nents used for simple, small-volume trans-

fusionsaregiveninTable24-1.

Fetal and Neonatal

Erythropoiesis

The predominant sites of hematopoiesis

in the developing embryo shift from the

wall of the yolk sac to the liver to the mar-

557

row in the first 24 weeks.3Hematopoiesis

is regulated by gradually increasing eryth-

ropoietin (EPO) levels stimulated by low

oxygen tensions during intrauterine life.

Fetal red cells, rich in hemoglobin F, are

well adapted to low intrauterine oxygen

tensions. The high oxygen affinity of fetal

hemoglobin enhances transfer of oxygen

from maternal erythrocytes to fetal eryth-

rocytes throughout pregnancy.

The “switch” from fetal to adult hemo-

globin begins at about 32 weeks’ gestation;

at birth, 60% to 80% of the total hemoglo-

bin is hemoglobin F. Preterm neonates,

therefore, are born with higher levels of fe-

talhemoglobinthanthosebornatterm.

The mean cord hemoglobin of healthy term

neonates is 16.9 ± 1.6 g/dL, and that of

preterm neonates is 15.9 ± 2.4 g/dL.4He-

moglobin concentration gradually falls in

the first few weeks of life. This has been

called “physiologic anemia of infancy” in

term newborns and “physiologic anemia of

prematurity” in preterm newborns. The

anemia is considered physiologic because

it is self-limited, is usually well tolerated,

and is not associated with any deleterious

effects to the infant. Erythropoietic activity

diminishes secondary to an increase in pul-

monary blood flow and a rise in arterial

pO2, as well as the increase in red cell con-

tent of 2,3-diphosphoglycerate (2,3-DPG)

and hemoglobin A, which enhance the re-

lease of oxygen to the tissues. As tissue oxy-

genation improves, levels of EPO decline

and erythropoiesis diminishes. This, along

with decreased survival of fetal red cells

and expansion of the blood volume due to

rapid growth, causes the hemoglobin con-

centration to decline. The rate of decline is

dependent on gestational age at birth; he-

moglobin may drop to as low as 8.0 g/dL at

4 to 8 weeks of age in preterm infants with

birthweights of 1000 to 1500 g, and 7.0 g/dL

in neonates with birthweights less than

1000 g.3

Despite hemoglobin levels that would

indicate anemia in older children and

adults, the normally developing infant usu-

ally maintains adequate tissue oxygenation.

Physiologic anemia requires treatment only

if the degree or timing of the anemia causes

symptoms in the patient.

Unique Aspects of Neonatal

Physiology

Infant Size and Blood Volume

Full-term newborns have a blood volume

ofapproximately85mL/kg;pretermlow

558 AABB Technical Manual

Table 24-1. Volumes for Simple, Small-Volume Transfusions of Neonates

Component Volume Estimated Change

RBC 10-15 mL/kg Hemoglobin ↑2-3 g/dL

Platelet 5-10 mL/kg Platelet ↑50,000-100,000/µL

Granulocyte ≥1×109neutrophils/kg in

volume of 10-15 mL/kg

Repeat until clinical response

FFP 10-15 mL/kg Factor activity ↑15-20%

Cryoprecipitate 1-2 units/10 kg ↑60-100 mg/dL fibrinogen (infant)

↑5-10 mg/dL fibrinogen (larger child)

Adapted with permission from Roseff.2

birthweight newborns have an average

blood volume of 100 mL/kg. As survival

rates continue to improve for infants

weighing 1000 g or less at birth, blood

banks are being asked to provide blood

components for patients whose total

blood volume is less than 100 mL on a

more frequent basis. The need for fre-

quent laboratory tests has made replace-

ment of iatrogenic blood loss the most

common indication for transfusion of low

birthweight preterm neonates. However,

the previous practice of replacing blood

mL for mL is giving way to replacement as

needed in order to maintain a target hema-

tocrit in certain clinical situations.1

Newborns do not compensate for hypo-

volemia as well as adults. After 10% volume

depletion in a newborn, left ventricular

stroke volume is diminished without in-

creasing heart rate. To maintain systemic

blood pressure, peripheral vascular resis-

tance increases, and this, combined with a

decreased cardiac output, results in poor

tissue perfusion, low tissue oxygenation,

and metabolic acidosis.5

Erythropoietin Response

Erythropoietin response in newborns dif-

fers from that in adults and older chil-

dren. In older children and adults, oxygen

sensors in the kidney recognize dimin-

ished oxygen delivery and release EPO

into the circulation. In the fetus, the oxy-

gen sensor that stimulates EPO produc-

tion is believed to be the liver, which ap-

pears to be programmed for the hypoxic

intrauterine environment.

This hyporesponsiveness to hypoxia pro-

tects the fetus from becoming poly-

cythemic in utero. Eventually, EPO produc-

tion shifts from the liver to the kidneys, a

developmental change thought to be regu-

lated based on the time of conception, not

birth, and possibly not beginning until

term. The most premature infants produce

the least amount of EPO for any degree of

anemia; this may reflect the absence of the

developmental shift of erythropoietin pro-

duction from the liver to the kidneys.6Sick

preterm neonates who receive many trans-

fusions shortly after birth have reduced cir-

culating levels of fetal hemoglobin. Circu-

lating EPO levels are lower, for a given

hematocrit, in preterm neonates with

higher proportions of hemoglobin A rela-

tive to hemoglobin F, which favors release

of oxygen to the tissues.6Erythroid progeni-

tor cells in the hypoproliferative marrow of

these preterm infants show normal intrin-

sic sensitivity to EPO. Clinical trials of re-

combinant human erythropoietin (rHuEPO)

in premature neonates show that the num-

ber of transfusions and severity of anemia

can be lessened.7Adverse effects of rHuEPO

in this age group are also different from

those seen in older children and adults and

include transient, reversible neutropenia.

Since the adherence to strict transfusion

guidelines and the decrease in phlebotomy

in VLBW infants, the ultimate role of

rHuEPO in the management of “anemia of

prematurity” has remained unclear. A re-

cent multicenter study in Europe showed

decreased need for transfusion when ad-

ministering EPO to ELBW infants within 3

to 5 days of life (early EPO administration)

and continuing for 9 weeks.8The mean

number of transfusions dropped from 2.66

to 1.86, with a reduction in donor expo-

sures from 2 to 1. Questions regarding opti-

mal dosing, route of administration, and

use of supplemental iron remain to be an-

swered.1,7-10 In any event, with the tremen-

dous strides made in decreasing donor ex-

posure in transfused newborns by using

restrictive transfusion practices alone, the

role of EPO for this purpose may no longer

be relevant.

Chapter 24: Neonatal and Pediatric Transfusion Practice 559

Cold Stress

Hypothermiainthenewborncausesex

aggerated effects, including increased

metabolic rate, hypoglycemia, metabolic

acidosis, and a tendency toward apneic

episodes that may lead to hypoxia, hypo-

tension, and cardiac arrest. Blood used for

exchange transfusion should be warmed

because blood at room temperature may

decrease a newborn’s core temperature by

0.7 to 2.5 C. The usual method is to use an

inline warmer. Blood, either large or small

volumes, should not be warmed under

a radiant heater because of the risk of

hemolysis in an unmonitored apparatus.

When transfusions are given to infants

undergoing phototherapy, the tubing

should be positioned to minimize expo-

sure to the phototherapy light in order to

prevent hemolysis.11

Immunologic Status

Infants have an immature and inexperi-

enced cellular and humoral immune system.

Antibodies present derive almost entirely

from the maternal circulation. Trans-

placental transfer of immunoglobulin and

other proteins is independent of molecu-

lar size; IgG (150 kD) is transferred much

more readily than albumin (64 kD). In hu-

mans, maternal IgM does not reach the

fetus and IgA is not readily transferred, al-

though low levels have been found in the

newborn.

All four subclasses of IgG are transported

acrosstheplacenta,buttheratevariesbe

tween individual mother-fetus pairs. Early

in pregnancy (approximately 12 weeks), IgG

probably passes from mother to fetus by

diffusion, and concentration in fetal serum

is low for all subgroups.12 Between 20 and

33 weeks of gestation, fetal IgG levels rise

markedly, apparently because of matura-

tion of a selective transport system that in-

volves, in part, specific protein receptors on

the membrane of placental cells. IgG1, the

predominant subclass in maternal blood,

crosses the placenta first and is transported

in greatest quantity. Cord blood has higher

antibody concentrations than maternal

blood. Catabolism of IgG occurs more

slowly in the fetus than in the mother, so

that transplacental maternal antibody is

conserved during the neonatal period.

A fetus exposed to an infectious process

in utero or an infant exposed shortly after

birth may produce small amounts of IgM

detectable by sensitive techniques, but un-

expected red cell alloantibodies of either

IgG or IgM class are rarely formed during

the neonatal period. The mechanisms

responsible for the lack of alloantibody pro-

duction in the neonate are not clearly un-

derstood and are most likely multifactorial,

including deficient T helper function, en-

hanced T suppressor activity, and poor an-

tigen-presenting cell function.13

The cellular immune response is critical

to the occurrence of transfusion-associated

graft-vs-host disease (TA-GVHD). In the

newborn, TA-GVHD has been reported

most often in the clinical setting of con-

firmed or suspected congenital immunode-

ficiency. It is recommended that infants

with suspected and/or documented T-cell

immunodeficiency receive irradiated blood

components. The majority of TA-GVHD cases

reported in nonimmunocompromised hosts

have occurred in infants who received intra-

uterine transfusion followed by postnatal

exchange transfusion.14 Aproposedexpla

nation is that lymphocytes given during

intrauterine transfusion could induce host

tolerance, impairing rejection of lymphocytes

given in the subsequent exchange transfu-

sions. There have also been rare cases of

TA-GVHDreportedinassociationwithex

treme prematurity, neonatal alloimmune

thrombocytopenia, and the use of extracor-

poreal membrane oxygenation (ECMO).14,15

Neonates present with TA-GVHD after a

560 AABB Technical Manual

longer latent period than adults, with fever

occurring at an average of 28 days after

exposure, rather than 10 days for immuno-

competent adults. There may be several

risk factors other than the immune status of

the recipient that predispose to TA-GVHD,

such as the number and viability of lym-

phocytes in the transfused component and

donor-recipient HLA compatibility. The

true incidence of TA-GVHD in the neonatal

setting is not known. However, on the basis

of data from Japan, it appears that the inci-

dence of reported TA-GVHD is far lower

than would be expected.15 The postulated

mechanism for this apparent decreased

susceptibility of newborns to TA-GVHD is

thought to be extrathymic and/or thymic

semitolerance of allogeneic donor T lym-

phocytes. As for all patients, directed-donor

units from biologic relatives must be irradi-

ated.15,16 There are no data to support the

practice of universal irradiation of blood

transfused to all infants or children.

Metabolic Problems

Acidosis or hypocalcemia may occur after

large-volume whole blood or plasma

transfusion because the immature liver of

the newborn metabolizes citrate ineffi-

ciently. Immature kidneys have reduced

glomerular filtration rate and concentrat-

ing ability, and newborns may have diffi-

culty excreting excess potassium, acid,

and/or calcium.

Potassium

Although potassium levels increase rapidly

in the plasma of stored red cells, small-

volume, simple transfusions administered

slowly have little effect on serum potas-

sium concentration in newborns. It has

been calculated that transfusion of 10

mL/kg of red cells (hematocrit 80%) ob-

tained from a unit of blood stored for 42

days in extended storage medium would

deliver 2 mL of plasma containing only

0.1mmol/Lofpotassium.Thisismuch

less than the daily potassium requirement

of 2 to 3 mmol/L for a 1-kg patient.17 How-

ever, serum potassium may, rise rapidly

after infusion of large volumes of red cells

in such circumstances as surgery, ex-

change transfusion, or extracorporeal cir-

culation, depending upon the plasma

potassium levels in the blood and manip-

ulation of the blood component.18,19 Of

interest, a unit of Red Blood Cells (RBCs)

preserved in AS-1 will deliver less extra-

cellular potassium compared to the

amount in RBCs stored in CPDA-1.16,20 In

stored irradiated blood, the problem of

potassium leak is potentiated; for selected

patients, it may be desirable to wash irra-

diated cells, if they have subsequently

been stored >24 hours.19 There are in-

creasing anecdotal reports of infants who

receive either older RBC units or units ir-

radiated more than 1 day before transfu-

sion having severe adverse effects (eg,

cardiac arrest, death) after transfusion of

these products into central lines or intra-

cardiac lines.21,22

The untoward consequences of washing

blood, such as reducing its shelf life and the

possible introduction of bacteria, must be

considered. It is preferable to perform irra-

diation as close to the time of administra-

tion as possible, obviating the concern for

high levels of potassium in the transfused

product.

2,3-Diphosphoglycerate

Neonates with respiratory distress syn-

drome or septic shock have decreased

levels of intracellular red cell 2,3-DPG.

Alkalosis and hypothermia may further

increase the oxygen affinity of hemoglo-

bin, shifting the dissociation curve to the

left and making oxygen even less available

Chapter 24: Neonatal and Pediatric Transfusion Practice 561

to the tissues. Arterial oxygenation may

be further compromised by respiratory

distress syndrome or other pulmonary

disease. Mechanisms that compensate for

hypoxia in adults, such as increased heart

rate, are limited in newborns. If a large

proportion of an infant’s blood volume has

come from transfusion of 2,3-DPG-de-

pleted blood, this may cause problems

that would not affect older children or

adults. Because 2,3-DPG levels decline

rapidly after the first week of storage, the

freshest blood conveniently available (up

to14days)shouldbeusedforexchange

transfusion in newborns. For small-volume

transfusions, the medical necessity for

fresh blood has never been demonstrated

and arguments have been raised to sug-

gest it is unnecessary.1,16,19

Cytomegalovirus Infection

Perinatal infection with cytomegalovirus

(CMV) may occur, acquired either in utero

or during the birth process. Neonates can

be infected during breast-feeding or by

close contact with mothers or nursery

personnel. CMV can also be transmitted

by transfusion, although the risk from the

current blood supply is small.23,24

CMV infection in newborns has ex-

tremely variable manifestations, ranging

from asymptomatic seroconversion to death.

Studies of CMV in neonatal transfusion re-

cipients reveal the following observations:

1. The overall risk of symptomatic post-

transfusion CMV infection seems to

be inversely related to the seropo-

sitivity rate in the community. Al-

though many adults are positive for

CMV antibodies, the rate of symp-

tomatic CMV infection in newborns

is low.

2. Symptomatic CMV infection during

the neonatal period is uncommon in

children born to seropositive moth-

ers.25

3. The risk of symptomatic posttrans-

fusion infection is high in multi-

transfused preterm infants weighing

less than 1200 g who are born to

seronegative mothers.17,26

4. The risk of acquiring CMV infection

is directly proportional to the cumu-

lative number of donor exposures

incurred via transfusion.

5. Cytomegalovirus in blood is associ-

ated with leukocytes. The risk of vi-

rus transmission can be reduced by

transfusing CMV-reduced-risk blood

from seronegative donors or by us-

ing leukocyte-reduced components.

Although deglycerolized and washed

red cells also have a reduced risk of

CMV infection, leukocyte reduction

by filtration is the technique of

choice.16,27-30

Red Cell Transfusions in

Infants Less than 4 Months

of Age

RBCs are the component most often

transfused during the neonatal period.

Many of the physiologic considerations

mentioned above directly affect decisions

regarding indications for transfusion, se-

lection, and administration of red cell

components, as well as the requirements

for compatibility testing.

Compatibility Testing

Because the neonate and young infant are

immunologically immature, alloimmuni-

zation to red cell antigens is rare during

the neonatal period. A study of 90 neo-

nates who received 1269 transfusions from

different donors found no instances of

antibody production even with use of

562 AABB Technical Manual

very sensitive detection techniques.31 Other

investigators confirm the relative infre-

quency of alloantibodies directed against

red cell, as well as HLA, antigens.13,28

Because alloimmunization is extremely

rare and repeated testing increases iatro-

genic blood loss, AABB Standards for Blood

Banks and Transfusion Services32(p42) requires

only limited pretransfusion serologic test-

ing for infants under 4 months old. Initial

testing must include ABO and D typing of

red cells and a screen for red cell antibod-

ies, using either serum or plasma, from the

mother or the infant.

During any one hospitalization, compat-

ibility testing and repeat ABO and D typing

may be omitted, provided that the screen

for red cell antibodies is negative; that all

red cells transfused are group O or ABO-

identical or ABO-compatible; and that red

cellsareeitherDnegativeorthesameD

type as the patient. It is unnecessary to test

the infant’s serum for anti-A and/or anti-B

as a component of blood typing. Before giv-

ing non-group-O red cells, the neonate’s se-

rum must be checked for passively ac-

quired maternal anti-A or anti-B and must

include the antiglobulin phase. If the anti-

body is present, ABO-compatible red cells

lacking the corresponding A or B antigen

must be used until the antibody is no lon-

ger detected. In this setting, it is not neces-

sary to perform crossmatches. If an unex-

pected non-ABO red cell antibody is

detected in the infant’s specimen or the

mother’s serum contains a clinically signifi-

cant red cell antibody, the infant should be

given either RBC units tested and found to

lack the corresponding antigen(s) or units

compatible by antiglobulin crossmatch.

This practice should continue for as long as

maternal antibody persists in the infant’s

blood. The institution’s policy will deter-

mine how frequently to recheck the screen

for red cell antibodies; once a negative re-

sult is obtained, subsequent crossmatches

and/or provision of blood lacking the target

antigen are unnecessary. It is important to

avoid transfusion of any component that

may transfer unexpected antibody or ABO-

incompatible antibodies to the infant.

Indications for Red Cell Transfusion

Certain events in the perinatal period cause

anemia, for which the benefits of red cell

transfusion are unquestioned. These in-

clude spontaneous fetomaternal or feto-

placental hemorrhage, twin-twin transfu-

sion, obstetric accidents, and internal

hemorrhage. A venous hemoglobin of less

than 13 g/dL in the first 24 hours of life in-

dicates significant anemia.33 For severely

anemic neonates with congestive heart

failure, it may be necessary to remove ali-

quots of their dilute blood and transfuse

concentrated red cells. This “partial ex-

change” transfusion will prevent intravas-

cular volume overload. Most red cell

transfusions in the neonatal period, how-

ever, are given either to replace iatrogenic

blood loss or to treat the physiologic de-

cline in hemoglobin (anemia of pre-

maturity) when it complicates clinical

problems.

Because tissue demand for oxygen can-

not be measured directly and because so

many variables determine oxygen availabil-

ity, no universally accepted criteria exist for

transfusion of preterm or term neonates.

Despite the widespread use of micro-

methods for laboratory tests and growing

use of bedside or noninvasive monitoring

devices, infants still sustain significant cu-

mulative blood loss from laboratory sam-

pling. In a sick neonate, red cell replacement

is usually considered when approximately

10% of the blood volume has been re-

moved. The decision to transfuse a new-

born for anemia should include evaluation

of the hemoglobin levels expected for the

patient’s age and clinical status, as well as

Chapter 24: Neonatal and Pediatric Transfusion Practice 563

the amount of blood loss over time. Trans-

fusionmaybemoreaggressiveintheinfant

in respiratory distress who is hypoxic and

more vulnerable to cerebral hemorrhage.

Considerable controversy surrounds the

correlation of the “signs of anemia” in the

preterm infant (tachycardia, tachypnea,

bradycardia, recurrent apnea, and poor

weight gain) with response to red cell trans-

fusions.1When red cells are transfused, they

are usually given in small volumes of 10 to

15 mL/kg (or less if the infant cannot toler-

ate this volume). The hematocrit of the red

cell component transfused will depend on

the anticoagulant/preservative used and

how the original unit is processed to pro-

vide small component transfusions for neo-

nates. A transfusion of 10 mL/kg of red cells

adjusted to a hematocrit greater than 80%

just before release for transfusion should

raise the hemoglobin concentration by ap-

proximately 3 g/dL. A transfusion of 10

mL/kg of red cells in additive solution,

which have a hematocrit of approximately

65%, will result in a posttransfusion hemo-

globin increment less than 3 g/dL.

Red Cell Components Used for Neonatal

Transfusion

The small-volume requirements of trans-

fusion to neonatal recipients make it pos-

sible to prepare several aliquots from a

single donor unit, thus limiting donor ex-

posure and decreasing donor-related risks.

Several technical approaches are avail-

able to realize this advantage and to mini-

mize wastage.34

Aliquoting for Small-Volume Transfusion

A multiple-pack system is a common tech-

nique for providing small-volume red cell

transfusions.34,35 Quad packs, where a sin-

gle unit of Whole Blood is collected into a

bag with four integrally attached contain-

ers, can be used to increase the number of

transfusions an infant can receive from

one donor. Because the original seal re-

mains intact, each container has the expi-

ration date of the original unit. With use

of a sterile connecting device, multiple

bags called pedi-packs or specially de-

signed syringe systems can be integrally

attached to a unit of RBCs after compo-

nent preparation. This maintains a closed

system and further increases the number

of small-volume transfusions obtained

from a single donor. Sterile connecting

devices can be used to prepare small

aliquots for transfusion in the blood bank.

If aliquots are prepared by entering the

bag through a port, the unit and the

aliquot are assigned a 24-hour shelf life, if

refrigerated.

Each aliquot must be fully labeled as it is

prepared, including the time it outdates.

The origin and disposition of each aliquot

must be recorded. Using these techniques,

a recipient can receive multiple small-vol-

ume transfusions from a single donation

until the expiration of the original unit,

thereby reducing donor exposure.34,36 The

disadvantage of any method that creates

aliquots from a single donation is that any

undetected, transmissible pathogen that

might be present in the primary unit can be

disseminated to multiple recipients.

In order to prevent waste, many transfu-

sion services assign a unit of RBCs to one or

more infants based on their weight. As an

example, 1 or 2 lower birthweight infants

may be assigned to one unit because they

will most likely require the greatest number

of transfusions. On the other hand, four

larger infants may be assigned to one unit

because their transfusion needs will not be

as great.37,38

Red Cells with Additive Solution

RBCs used for pediatric transfusions were

traditionally stored in CPDA-1.35 Additive

564 AABB Technical Manual

solutions (AS) used as anticoagulants/

preservatives contain additional adenine

and dextrose and some contain mannitol.

There has been concern about the poten-

tial side effects of these additives because

large amounts of adenine and mannitol

have been associated with renal toxicity.

Mannitol is also a potent diuretic and, be-

cause of its effect on the fluid dynamics in

preterm infants, may cause unacceptable

fluctuations in cerebral blood flow. The

different constituents of these solutions

are found in Chapter 8. However, when

the dose of transfused red cells is small (5

to 15 mL/kg), the recipient is exposed to

relatively small amounts of the preserva-

tive solutions. Clinical studies comparing

red cells stored in AS-1 and AS-3 solutions

have shown no apparent detrimental ef-

fects in neonates receiving simple trans-

fusions, and, after adjustment for the

lower hematocrit of the component, they

are as effective as CPDA-1 cells in increas-

ing hemoglobin. Furthermore, the addi-

tional sugars present have been shown to

benefit glucose homeostasis in compari-

son with CPDA-1.39 Studies on the safety

of AS-3-preserved red cells in neonates have

been published.38,40 Because the compo-

nents of AS-5 are the same as those found

in the other additive solutions, it is con-

sidered acceptable for use in neonates.

Using theoretical calculations in a vari-

ety of clinical situations, Luban et al41 dem-

onstrated that red cells preserved in

extended-storage media present no sub-

stantive risks when used for small-volume

transfusions. For preterm infants with se-

vere hepatic or renal insufficiency, however,

removing the additive-containing plasma

may be beneficial. This is particularly im-

portant if there will be multiple transfu-

sions that could have a cumulative effect.

The safety of red cells stored in additive so-

lutions and used for massive transfusions,

such as cardiac surgery or exchange trans-

fusion, has not been studied. Concern still

remains regarding the use of blood stored

in additive solutions being used outside the

setting of simple, small-volume transfu-

sion.40,41 However, with extensive anecdotal

use in large transfusion services, there have

not been reports of deleterious effects from

the infusion of additive solutions.42,43

Whether or not placental/umbilical cord

blood will become an acceptable form of

neonatal transfusion remains to be seen.

Althoughuseofthistypeof“autologous”

blood would eliminate some infectious dis-

ease risks, questions regarding quantity,

quality, and sterility raise concerns about

its safety and efficacy.18

Transfusion Administration

Vascular access is often difficult in the tiny

newborn and in any infant requiring

long-term or repeated intravenous infu-

sions. Within a short time after birth, the

umbilical artery may be cannulated. Trans-

fusion through a needle as small as 25-

gauge or a vascular catheter as small as

24-gauge has been shown to cause little

hemolysis and to be safe when constant

flow rates are used. Transfusion through

smaller gauge catheters has not been

thoroughly evaluated.

It is not usually necessary to warm small-

volume transfusions that are given slowly,

but it is important to be able to control the

volume and rate of infusion. Constant-rate

electromechanical syringe delivery pumps

provide this control and cause minimal

hemolysis, even when used with inline leu-

kocyte reduction filters.44,45

The length of the plastic tubing used can

add significantly to the volume required for

transfusion. Infusion sets identified as suit-

able for platelets or components have less

dead space than standard sets because they

have short tubing and a small 170-micron

filter. Pediatric microaggregate filters (20-

Chapter 24: Neonatal and Pediatric Transfusion Practice 565

or 40-micron) are often used for their small

priming volume, not for the removal of

microaggregates. Hemolysis may occur

when stored blood is given by negative

pressure filtration through these filters.46

Administration rates for RBCs have not

been extensively studied, nor are there

standard practices; rates of transfusion as

well as devices used vary with institutions.

The rate of administration of blood and

blood components in neonates and infants

should be individualized on the basis of the

patient’s clinical needs. Theoretical con-

cerns regarding rapid changes in intra-

vascular volume and electrolyte changes in

these small, labile patients have focused on

an increased risk for intracranial hemor-

rhage. This has not been clearly demon-

strated. For simple RBC transfusions, trans-

fusing products over 2 to 4 hours is usually

adequate. When there is an urgent need be-

cause of shock or severe bleeding, infusion

should be as rapid as possible. Products

can be transfused safely using a variety of

devices. It is important that mechanical

systems be tested and validated for use

with blood and blood components.

Exchange Transfusion for

Hyperbilirubinemia

The fetal liver has limited capacity to con-

jugate bilirubin. In utero, unconjugated

bilirubin crosses the placenta for excre-

tion through the mother’s hepatobiliary

system. After birth, transient mild hyper-

bilirubinemia normally occurs during the

first week of life and is referred to as “phy-

siologic jaundice.” Liver function is less ma-

ture, and jaundice worsens in premature

neonates. When the level of unconjugated

bilirubin is excessive, bilirubin may cross

the blood-brain barrier and concentrate

in the basal ganglia and cerebellum; the

resulting damage to the central nervous

system (CNS) is called kernicterus. Photo-

therapy with fluorescent blue lights is the

most common treatment for hyperbiliru-

binemia; exchange transfusion is reserved

for phototherapy failures. The most com-

mon reason, however, for an exchange to

be performed in a neonate is to correct

hyperbilirubinemia.

Pathologic processes that may result in

excessively high unconjugated bilirubin

levels in neonates include immune-medi-

ated hemolysis, nonimmune hemolysis,

bile excretion defects and impaired albu-

min binding. Exchange transfusion re-

moves unconjugated bilirubin and provides

additional albumin to bind residual biliru-

bin. If hyperbilirubinemia is due to anti-

body-mediated hemolysis, exchange trans-

fusion is of additional benefit by removing

free antibody and antibody-coated red cells

while providing antigen-negative red cells

that will survive normally.

Exchange transfusion should be per-

formed before bilirubin rises to levels at

which CNS damage occurs. Several factors

affect the threshold for toxicity. CNS dam-

age occurs at lower levels if there is pre-

maturity, decreased albumin binding capac-

ity,orthepresenceofsuchcomplicating

conditions as sepsis, hypoxia, acidosis, hy-

pothermia, or hypoglycemia. In full-term

infants, kernicterus rarely develops at indi-

rect bilirubin levels less than 25 mg/dL, but,

in sick VLBW infants, kernicterus has oc-

curred at levels as low as 8 to 12 mg/dL.47

Therateatwhichbilirubinrisesismore

predictive of imminent need for exchange

transfusion than the absolute level attained.

Neonates with severe anemia and a rapid

rise in bilibrubin, despite phototherapy, re-

quire exchange transfusion. A two-volume

exchange transfusion removes approxi-

mately 70% to 90% of circulating erythro-

cytes and about 25% of the total bilirubin.

Because of reequilibration between the

extravascular tissue and plasma bilirubin,

levels may again rise, resulting in the need

566 AABB Technical Manual

for a second exchange transfusion.48 Indica-

tions for repeat exchange are similar to

those for the initial exchange.

The American Academy of Pediatrics re-

cently released new guidelines for the man-

agement of newborn infants born ≥35

weeks of gestation with hyperbilirubine-

mia. It is hoped that raising awareness

about the potential for hyperbilirubinemia

in this patient group will reduce its fre-

quency and provide a framework for opti-

mal treatment, to include the use of photo-

therapy, exchange transfusion, and Immune

Globulin, Intravenous (IGIV).49

Exchange Transfusion for Other Causes

The safety and efficacy of exchange trans-

fusion in the neonatal period for other in-

dications should be evaluated on a case-

by-case basis, using guidelines in pub-

lished literature. Treatment categories for

disorders based on proven vs theoretical

benefits have been recommended (see

Table6-1).Thetreatmentofdisseminated

intravascular coagulation (DIC) using ex-

change transfusion has yielded variable

results, perhaps because only the sickest

infants have been selected to receive this

therapy. The most important aspect of

therapy for neonatal DIC is to treat the

underlying disease.

Exchange transfusion is occasionally

used to remove other toxins, such as drugs

or chemicals given to the mother near the

time of delivery, drugs given in toxic doses

to the neonate/infant, or substances such

as ammonia that accumulate in the new-

born because of prematurity or inherited

metabolic diseases.50,51

Technique of Exchange Transfusion

Choice of Components

Red cells are resuspended in compatible

thawed Fresh Frozen Plasma (FFP) for ex-

change transfusion. If AS-RBC units are

used, depending on the clinical situation,

some institutions would choose to remove

the additive-containing plasma, to reduce

the volume transfused. As discussed ear-

lier, washing components may not be

necessary or desirable. Many transfusion

services use red cells that have been

screened and found to lack hemoglobin S

for exchange transfusion, to avoid the

possibility of intravascular sickling.

The glucose load administered during

exchange transfusion can be extremely

high. This stimulates the infant to secrete

insulin, which may lead to rebound hypo-

glycemia. It is important to monitor blood

glucose levels for the first few hours after

the procedure.

Because unconjugated bilirubin binds to

albumin, albumin is frequently used to in-

crease intravascular binding. With addi-

tional albumin in the circulation, bilirubin

from the extravascular space diffuses out to

the intravascular space. This, in turn, in-

creases the total quantity of bilirubin re-

moved during the exchange. There have

been conflicting results, however, about the

efficacy of administering albumin either

before or during exchange to enhance bili-

rubin removal. A study that compared 15

hyperbilirubinemic neonates given albu-

min with 27 who received none found simi-

lar efficiency of bilirubin removal in both

groups.52 Infusing albumin raises the colloid

osmotic pressure and increases intravas-

cular volume. Therefore, it should be given

cautiously, if at all, to neonates or infants

who are severely anemic, have increased

central venous pressure, or are in renal or

congestive heart failure.

Exchange transfusion may cause dilu-

tional thrombocytopenia and/or coagulo-

pathy that require transfusion of platelets

and/or other components containing coag-

ulation factors. Platelet counts and coagu-

lation parameters should be monitored af-

ter exchange transfusion.

Chapter 24: Neonatal and Pediatric Transfusion Practice 567

Volume and Hematocrit

An exchange transfusion equal to twice the

patient’s blood volume is typically recom-

mended for newborns; rarely is more than

one full unit of donor blood required. In

practice, the volume calculated for ex-

change is an estimate. The final hemato-

crit of transfused blood should be approx-

imately 40% to 50%, with sufficient plasma

to provide clotting factors, if needed. In

the unusual event that the infant’s condi-

tion demands a high postexchange hemato-

crit, a small-volume transfusion of red cells

can be given after the exchange, or units

with a higher hematocrit used for ex-

change. It is important to keep the blood

mixed during the exchange; if it settles in

the container, the final aliquots will not

have the intended hematocrit. The in-

fant’s hematocrit and bilirubin level

should be measured using the last aliquot

removed in the exchange.

Vascular Access

Exchange transfusions in the newborn

period are usually accomplished via cath-

eters in the umbilical vessels. Catheteriza-

tion is easiest within hours of birth, but it

may be possible to achieve vascular ac-

cess at this site for several days. The cath-

eters should be radio-opaque to facilitate

radiographic monitoring during and after

placement. If umbilical catheters are not

available for exchange transfusion, small

central venous or saphenous catheters

may be used.

Methods Used

Two methods of exchange transfusion are

incommonuse.Intheisovolumetric

method, there is vascular access through

two catheters of identical size. Withdrawal

and infusion occur simultaneously, regu-

lated by a single peristaltic pump. The

umbilical artery is usually used for with-

drawal and the umbilical vein for infu-

sion.

The manual push-pull technique can be

accomplished through a single vascular ac-

cess. A three-way stopcock joins the unit of

blood, the patient, and an extension tube

that leads to the graduated discard con-

tainer. An inline blood warmer and a stan-

dard blood filter should be incorporated in

the administration set. The maximum vol-

ume of each withdrawal and infusion will

depend on the infant’s size and hemo-

dynamic status. The rate at which exchange

transfusion occurs may alter the infant’s

hemodynamic status. It is important to

maintain careful records during an ex-

change transfusion. The procedure should

take place over 1 to 1.5 hours.

Transfusion of Other

Components

Although the percentage of VLBW and

ELBW infants being transfused has de-

creased significantly since the 1980s, be-

tween 61% and 94% of these neonatal pa-

tients can be expected to receive multiple

red cell transfusions. The smallest pa-

tients will receive the greatest number of

transfusions. It is estimated that a much

lower percentage of infants receive other

components.1,9,53,54

Platelet Transfusion

The normal platelet count of newborns is

similar to that of adults. A platelet count

less than 150,000/µL in a full-term or pre-

mature infant is abnormal. Approximately

20% of infants in neonatal intensive care

units have mild-to-moderate thrombo-

cytopenia, which is the most common

hemostatic abnormality in the sick in

fant.55 Neonatal thrombocytopenia may

result from impaired production or in-

568 AABB Technical Manual

creased destruction of platelets, abnormal

distribution, or a dilutional effect second-

ary to massive transfusion such as ex-

change transfusion. Increased destruction

is the most common cause; it may be as-

sociated with a multitude of conditions and

is usually transient. Neonatal alloimmune

thrombocytopenia is discussed in Chap-

ter 23.

Indications

Platelet transfusion is indicated in neo-

nates and young infants with platelet

counts below 50,000/µLwhoareexperi

encing bleeding.55 The use of prophylactic

platelet transfusions in the newborn re-

mains controversial. In thrombocyto-

penic adults, the risk of severe bleeding is

rare unless the platelet count is less than

10,000/µL.56 Conversely, preterm neonates

and infants with other complicating ill-

nesses may bleed at higher platelet counts.

Factors contributing to this increased risk

of bleeding include a quantitatively lower

concentration of plasma coagulation fac-

tors; circulation of an anticoagulant that

enhances inhibition of thrombin; intrin-

sic or extrinsic platelet dysfunction; and

increased vascular fragility.57 Of major

concern is intraventricular hemorrhage,

which occurs in up to 40% of preterm ne-

onates in the first 72 hours. Although pro-

phylactic platelet transfusions increase

platelet counts and shorten the bleeding

time in these infants, the incidence or ex-

tent of intraventricular hemorrhage is not

reduced.57 Because of the apparent lack of

clinical benefit, controversy exists about

the use of platelet transfusion in this set-

ting, as well as the selection of an optimal

dose. After a platelet transfusion, a post-

transfusion platelet count soon after

transfusion can be used to evaluate sur-

vivalinthecirculationbutmaynotpre

dict hemostatic efficacy. Repeated platelet

transfusions, without an appropriate rise,

may not be beneficial.

Platelet Components

A platelet dose of 5 to 10 mL/kg body

weight should raise the platelet count of

an average full-term newborn by 50,000

to 100,000/µL, depending on the platelet

concentration in the component used.17,57

The platelet component should be group

specific, if possible, and should not con-

tain clinically significant unexpected red

cell antibodies. Transfusion of ABO-in-

compatible plasma is more dangerous in

infants than in adults because of their

very small blood volumes. If it is neces-

sarytogiveaplateletunitthatcontains

incompatible plasma (due to antibodies

in the ABO or other blood groups), plasma

can be removed (see Method 6.15) and

the platelets resuspended in saline. FFP

can be substituted as the resuspending

medium if the patient also requires clot-

ting factors, but it carries the risk of infec-

tious disease transmission. Routine cen-

trifugation of platelets to reduce the

volume of transfusion is not necessary.53,55

If platelets have been volume reduced

and placed in a syringe, the pH declines

rapidly, a potential problem for an already

ill, acidotic patient.58 Therefore, if there is

a need to reduce the volume of platelets,

it should be done just before transfusion

and the component must be infused

within4hours,ifdoneinanopensystem.

Granulocyte Transfusion

Neonates are more susceptible to severe

bacterial infection than older children be-

cause of both the quantitative and quali-

tative defects of neutrophil (polymorpho-

nuclear cell or PMN) function and, in the

Chapter 24: Neonatal and Pediatric Transfusion Practice 569

absence of pathogen-specific maternal

antibody, to deficiency of humoral immu-

nity. Group B streptococcus is the most

frequent cause of early-onset neonatal

sepsis, and, despite improvement in

antimicrobial therapy and intensive care,

it is still associated with a high mortality

rate. Controversy surrounds several issues

in granulocyte transfusions for neonates,

including dose, neutrophil level at which

to transfuse, and efficacy as compared to

other forms of therapy.59 Because it ap-

pears that efficacy is related to dose, the

smaller blood volume of infants and

young children may result in these pa-

tients having a better response to this

form of therapy. A meta-analysis pub-

lished in 1996 concluded that granulocyte

doses greater than 1 ×109PMN/kg re-

sulted in a better clinical response.60

Granulocyte concentrates prepared by

apheresis are more desirable than those

prepared from buffy coats because they

produce a higher yield.17,60 Products col-

lected from donors on a regimen of

granulocyte colony-stimulating factor

(G-CSF) and steroid mobilization can

yield higher numbers of granulocytes

than those collected from an unstimulat-

ed donor. There have been some encour-

aging observations on the use of IGIV in

the treatment of early neonatal sepsis, al-

though conflicting data result in a lack of

consensus on its use.59,61-64 Preliminary stud-

ies of hematopoietic growth factors (eg,

G-CSF) show promise in the treatment of

overwhelming bacterial infection in the

newborn.59,61,65,66 For adults and larger chil-

dren, a minimum dose of 1 ×1010 PMN/kg

is recommended.60,67,68

Indications

Although the precise role of granulocyte

transfusion for neonatal sepsis is unclear,

certain clinical situations exist in which

granulocyte transfusion may be consid-

ered as an adjunct to antibiotic therapy.

Candidates for possible granulocyte

transfusion are infants with strong evi-

dence of bacterial septicemia, an absolute

neutrophil count below 3000/µL, and a

diminished marrow storage pool, such

that less than 7% of their nucleated cells

in the marrow are granulocytes at the

stage of metamyelocytes or more mature

forms.18,69

Granulocyte Components

Granulocytes are harvested by standard

apheresis techniques. For infants, a dose

of 10 to 15 mL/kg is recommended, which

is about 1 ×109to 2 ×109PMN/kg.17,60 Be-

cause neonatal neutrophil function is of-

ten abnormal, the use of granulocyte

transfusions in this age group may be

beneficial.17,60,67,70-72 Administration should

be continued daily until an adequate

white cell mass is achieved or the patient

has clinically improved. Based on the fact

that granulocyte concentrates contain

large numbers of lymphocytes, there is

general consensus that all granulocyte

transfusions should be irradiated to pre-

vent graft-vs-host disease. For granulo-

cyte transfusions to neonates, donors are

usually selected to be CMV seronegative

and must be ABO compatible with the in-

fant, in accordance with Standards,be

cause there is a significant volume of red

cells in these products.32(p40) Many institu-

tions also provide products that are

D-compatible (see Chapter 21).

Transfusion to Enhance Hemostasis

The elements of the hemostatic system of

the newborn are similar to those of older

children and adults, but the concentration

of many plasma proteins is decreased.

Coagulation factors do not cross the pla-

centa, but are independently synthesized

570 AABB Technical Manual

by the fetus. Plasma levels of coagulation

proteins increase progressively with ges-

tational age. At birth, the infant’s pro-

thrombin time and partial thrombo-

plastin time are prolonged, compared to

older children and adults, primarily the

result of physiologically low levels of the

vitamin-K-dependent factors (II, VII, IX,

and X) and contact factors (XI, XII, pre-

kallikrein, and high-molecular-weight

kininogen).73 Proteins C and S and anti-

thrombin inhibitors of coagulation are

also at low levels. These two systems usu-

ally balance each other, so that spontane-

ous bleeding and thrombosis in the healthy

newborn are rare, but very little reserve

capacity exists for response to pathologic

insults. Therefore, serious bleeding may

occur in the first week of life in the sick

premature infant as a result of hemostatic

immaturity coupled with an acquired dis-

order of hemostasis.

In addition to having physiologically low

levels of the vitamin-K-dependent factors,

neonates may also become vitamin-K-defi-

cient during the first 2 to 5 days of life, plac-

ing them at risk of bleeding. This “hemor-

rhagic disease of the newborn” is rare in

developed countries because intramuscular

vitamin K is routinely given at birth. If vita-

min K therapy is omitted, especially if the

neonate is breast fed, life-threatening hem-

orrhage may occur. This should be treated

with FFP.2,74

Although hereditary deficiencies of coag-

ulation factors may be apparent in the

newborn, significant bleeding is rare.

Coagulopathy more often results from an

acquired defect such as liver disease or

DIC.74 Although component therapy re-

placement may temporarily correct the

hemostatic problem, treatment of the un-

derlying disease will ultimately reduce the

need to treat the acquired defect.

Newborns who are heterozygous for de-

ficiencies of inhibitory proteins rarely expe-

rience complications in the absence of an-

other pathologic insult. However, the ho-

mozygous form of protein C deficiency has

caused life-threatening thrombotic compli-

cations in the newborn period. In countries

where it is available, protein C concentrates

prepared from human plasma should be

used as the initial treatment for neonates

with homozygous protein C deficiency pre-

senting with purpura fulminans. Protein C

concentrates may be available on a com-

passionate use basis in the United States.

Otherwise, plasma infusion is used as the

initial treatment during the acute event,

with subsequent anticoagulant therapy for

long-term management.74,75

Fresh Frozen Plasma

Freshfrozenplasmamaybeusedtore-

place coagulation factors in newborns,

particularly if multiple factors are in-

volved, such as in vitamin K deficiency.

The usual dose is 10 to 15 mL/kg, which

should increase factor activity by 15% to

20% unless there is marked consumptive

coagulopathy.74 As with red cell transfu-

sions, there are several methods to pro-

vide small-volume FFP infusions while

limiting donor exposure and wastage of

components. Blood can be collected into

a system with multiple integrally attached

bags, creating aliquots that can be pre-

pared for freezing.34 Once thawed, these

aliquots can be further divided and used

for several patients within a 24-hour pe-

riod. If not used within 24 hours as aliquots,

the thawed plasma (stored at 1 to 6 C) can

still be used as a means to decrease donor

exposure. As with all patients, newborns

must receive FFP that is ABO compatible

and free of clinically significant unex-

pected antibodies. Group AB FFP is often

used because a single unit provides com-

patible small aliquots for several neonates

requiring FFP simultaneously.

Chapter 24: Neonatal and Pediatric Transfusion Practice 571

Cryoprecipitate

Cryoprecipitate is rich in fibrinogen, co-

agulation Factors VIII and XIII, and von

Willebrand factor. This component is of-

ten used in conjunction with platelet

transfusions to treat DIC in the newborn.

In DIC, fibrinogen and platelets are the el-

ements most often severely depleted. The

plasma in which the platelets are sus-

pended is a source of stable coagulation

factors. Cryoprecipitate provides concen-

trated levels of additional fibrinogen and

storage-labile Factor VIII. For an infant, one

bag is sufficient to achieve hemostatic

levels. As with FFP and platelets, the

cryoprecipitate should be ABO compati-

ble with the neonatal recipient. Directed

donor cryoprecipitate is not recommended

as first-line therapy in the newborn with

hemophilia A because safer alternatives are

available. Recombinant Factor VIII prod-

ucts or virus-inactivated, monoclonal-an-

tibody-purified, plasma-derived products

are the standard treatment.76 Cryoprecipi-

tate should be used to treat von Wille-

brand disease only as a last resort, when

safer products are not readily available.

More information on the use of cryopreci-

pitate can be found in Chapter 21.

Neonatal Polycythemia

A venous hematocrit greater than 65% or

hemoglobin in excess of 22 g/dL any time

in the first week of life defines polycythemia,

a condition that occurs in approximately

5% of all newborns. Small-for-gestational-

age infants and infants of diabetic moth-

ers are at increased risk for developing

polycythemia. As the hematocrit rises

above 65%, the viscosity of blood in-

creases exponentially and oxygen trans-

port decreases. For neonates, the expo-

nential rise may occur at a hematocrit

closer to 40%.77 Infants have a limited

ability to increase cardiac output to com-

pensate for hyperviscosity and may

develop congestive heart failure. Impair-

ment of blood flow can cause CNS abnor-

malities, pulmonary and renal failure, and

necrotizing enterocolitis. Phlebotomy can

be used to normalize the hematocrit to

55% to 60% and improve tissue perfusion,

while maintaining the blood volume.

To treat polycythemia, whole blood is re-

moved and the volume replaced with

crystalloid (such as normal saline), the

choice being based on the quantity needed

and on the infant’s clinical condition.

Plasma is not recommended because the

volume administered will be insufficient to

correct any coagulopathy and because

necrotizing enterocolitis has been reported

when it is used in this procedure.78 Afor-

mula to approximate the volume of colloid

replacement required (and the volume of

blood to be drawn) for the exchange is47:

Volume of replacement fluid =

blood volume ×

(observed hematocrit – desired hematocrit)

observed hematocrit

Extracorporeal Membrane

Oxygenation

ECMO is a modified cardiopulmonary by-

pass technique that has been used for

short-term support for cardiac or respira-

tory failure. It is performed in specialized

centers and only for patients in whom

conventional medical therapy has failed

and anticipated survival with such ther-

apy is limited (see Table 24-2). The use of

ECMO in patients other than neonates is

not widespread. The therapy is more suc-

cessful in infants, whose small blood vol-

ume allows for total cardiorespiratory

support and whose primary respiratory

problem often resolves after 1 to 2 weeks

572 AABB Technical Manual

of support. ECMO provides gas exchange

independent of the patient’s lungs, allow-

ing them time to improve or heal without

exposure to aggressive ventilator support

and the secondary lung damage this may

cause.79

Individual ECMO centers establish their

own specific criteria for transfusion and

blood component selection, and standard

transfusion practices are lacking. Because

of the combination of factors present (in-

cluding systemic heparinization, platelet

dysfunction, thrombocytopenia, and other

coagulation defects) as well as the ECMO

circuitry itself, bleeding complications are

frequent. The ECMO team should be in

close communication with the blood bank

or transfusion service staff, and there

should be mutual agreement on protocols

to ensure consistency of care. Many infants

requiring ECMO have been transferred

from other hospitals, where they may al-

ready have received numerous transfu-

sions. The amount of red cell, platelet, and

FFP support required to maintain hemato-

logic and hemodynamic equilibrium will

vary depending on the clinical situation

and in accordance with the institutions’

practices.68 When platelet transfusion is re-

quired, some practitioners think it is im-

portant to transfuse through peripheral ac-

cess to avoid platelet damage, whereas

others will transfuse directly into the ECMO

circuitry because all blood will eventually

flow through the equipment. It is also im-

portant to monitor ionized calcium levels

and supplement them as needed.47,79 (See

Tables 24-3 and 24-4.)

Leukocyte Reduction

The benefit of leukocyte reduction of

components transfused to infants re-

mains controversial. Difficulty in identify-

ing transfusion reactions in this patient

population makes this question hard to

study. In addition, infants are rarely alloi-

mmunized because of the immaturity of

their immune system during this period

of development. The reduction of risk of

CMVtransmissiontoinfantsistheonly

benefit of leukocyte reduction that has

been well documented, as discussed ear-

lier.16,30,80

Of interest, a recent study in Canada

compared the clinical outcomes of prema-

ture infants weighing <1250 g before and

after implementation of universal leukocyte

reduction. Although neither mortality nor

bacteremia were reduced in the setting of

universal leukocyte reduction, other sec-

ondary clinical outcomes, such as retino-

pathy of prematurity and bronchopul-

monary dysplasia, were improved. Length

Chapter 24: Neonatal and Pediatric Transfusion Practice 573

Table 24-2. Disorders Treated by ECMO

■Meconium aspiration

■Diaphragmatic hernia

■Persistent pulmonary hypertension of the

newborn

■Severe group B streptococcal sepsis

Table 24-3. Risks of ECMO

■Bleeding

■Thrombosis

■Thrombocytopenia

■Neutropenia

■Platelet dysfunction

■Stroke

■Seizure

■Air embolism

■Hemolysis

■Systemic hypertension

■Cannulization of carotid artery

■Infectious complications of blood

transfusion

of stay was decreased with universal leuko-

cyte reduction.81

Transfusion Practices in

Older Infants and Children

The indications for transfusion of red cells

and other components in older infants

(>4 months) and children are similar to

those for adults but must take into ac-

count differences in blood volume, ability

to tolerate blood loss, and age-appropri-

ate hemoglobin and hematocrit levels.

The most common indication for red cell

transfusioninchildrenistoreverseor

prevent tissue hypoxia resulting from de-

creased red cell mass associated with sur-

gical procedures or in response to anemia

of chronic diseases or hematologic malig-

nancies. It is important to remember that

normal hemoglobin and hematocrit levels

are lower in children than adults. Pediat-

ric patients may remain asymptomatic

despite extremely low levels of hemoglo-

bin, particularly if the anemia has devel-

oped slowly.

The decision to transfuse should be

based not only on the hemoglobin level but

also on the presence or absence of symp-

toms, the functional capacity of the child,

the etiology of the anemia, the possibility of

using alternative therapies, and the pres-

ence or absence of additional clinical con-

ditions that increase the risk for developing

hypoxia. If small-volume transfusions are

required, many of the methods described

to provide small-volume transfusions to ne-

onates could be applied. All pediatric pa-

tients over 4 months of age, however, must

be tested for ABO and D type as well as for

the presence of clinically significant antibod-

ies before red cell transfusions. Compatibil-

ity testing must be done in accordance with

Standards.32(pp37-41) Of note, a published ab-

stract reported that leukocyte reduction

does decrease the occurrence of febrile non-

hemolytic transfusion reactions in pediatric

hematology/oncology patients.82

Red Cell Support for Children with

Hemoglobinopathies

In certain childhood conditions, chronic

red cell transfusions are given not only to

treat tissue hypoxia but also to suppress

endogenous hemoglobin production. Ap-

proximately 6% to 10% of children with

sickle cell disease suffer a stroke, with

two-thirds experiencing a recurrence.83

The goal of transfusion for these patients

is to reduce the risk of stroke by decreas-

ing the percentage of circulating red cells

capable of sickling, while simultaneously

avoiding an increase in blood viscosity. It

is important to remember that raising the

hematocrit, without significantly reduc-

ing the percent of sickle cells, could in-

crease viscosity and negate any beneficial

effects of the transfusion.83,84 Therateof

recurrent stroke can be reduced to less

than 10% by maintaining a hemoglobin

level of 8 to 9 g/dL, with a hemoglobin S

level less than 30%, in children who have

had a cerebrovascular accident. This can

usually be achieved with a simple or par-

tial exchange transfusion every 3 to 4 weeks.

Therapy is continued indefinitely, as ces-

sation can lead to subsequent stroke.83,84

Because of concern about iron overload,

some workers follow several uneventful

years of transfusions to keep hemoglobin

574 AABB Technical Manual

Table 24-4. Contraindications for ECMO

■High risk for intraventricular hemorrhage

■Irreversible lung disease

■Systemic bleeding

■Severe asphyxia

■Presence of lethal malformations

S below 30% with a less aggressive proto-

col that maintains hemoglobin S between

40% and 50%.84 Erythrocytapheresis has

been shown to improve iron balance in a

small cohort of patients.85 Adams et al

showed that transfusion to maintain a he-

moglobin S level less than 30% in children

who have abnormal results on trans-

cranial Doppler ultrasound reduced the

risk of a first stroke.86 The benefits of this

transfusion therapy, however, must be

weighed against the complications of

transfusion, such as iron overload and

alloimmunization, as well as the risks of

increased donor exposure during erythro-

cytapheresis. Although simple transfusion

increases blood viscosity, exchange trans-

fusion does not. Blood for transfusion to a

patient with sickle cell disease should ide-

ally be screened for hemoglobin S. In ad-

dition, most centers now provide leuko-

cyte-reduced blood for patients with sickle

cell disease to prevent alloimmunization

to platelets, which could complicate trans-

plantation.87

Red cell transfusions are also used to

treat acute complications associated with

sickle cell disease, such as splenic seques-

tration and aplastic crisis.88 Acute chest syn-

drome, a new pulmonary infiltrate in a pa-

tient with sickle cell disease, other than

atelectasis, with additional respiratory

symptoms and fever, carries a poor progno-

sis if untreated. Simple transfusion can be

used as a first-line therapy to improve oxy-

genation. For those patients who continue

to deteriorate or who do not improve, red

cell exchange transfusion should be per-

formed. There are no randomized con-

trolled trials comparing exchange and sim-

ple transfusion in this setting.89 Astudy

evaluating preoperative transfusion proto-

cols found that a conservative protocol, in

which the hemoglobin was raised to 10 g/

dL, was as effective in preventing perio-

perative complications as an aggressive ap-

proach to decrease hemoglobin S levels to

below 30%.90

Patients with sickle cell disease might

also be at risk for severe delayed hemolytic

transfusion reactions that could be life-

threatening because of the coincident sup-

pression of erythropoiesis. When a patient’s

hemoglobin level decreases after transfu-

sion, this “hyperhemolytic” syndrome—

wherein it appears that autologous red cells

are destroyed through an innocent by-

stander mechanism—should be suspected.

In these circumstances, transfusion should

be stopped and corticosteroid therapy, or a

combination of corticosteroid therapy

and IGIV, considered, based on reports of

efficacy in case studies.91,92 Autoantibody

formation also occurs in these patients af-

ter transfusion.93 In the hope of decreasing

the need for transfusion, medical interven-

tions are being explored. One therapy uses

hydroxyurea to increase the percentage of

hemoglobin F. By having a higher percent-

age of cells that are hemoglobin F, the for-

mation of hemoglobin S polymers is re-

duced. In addition, the concomitant decrease

in neutrophil counts that accompanies

hydroxyurea administration was found to

be independently associated with a reduc-

tion in the rate of crisis.94 Marrow trans-

plantation has also been used in some pa-

tients and may have a role in the future

treatment of patients with sickle cell dis-

ease.95,96

For children with thalassemia and severe

anemia, transfusion not only improves tis-

sue oxygenation but also suppresses

erythropoiesis. By suppressing ineffective

erythropoiesis, many of the complications

associated with the disease are amelio-

rated. So-called hypertransfusion, in which

the pretransfusion hemoglobin is kept be-

tween 8 and 9 g/dL, allows normal growth

and development, as well as normal levels

of activity for the child’s age. Supertransfu-

sion programs aim to maintain a pre-

Chapter 24: Neonatal and Pediatric Transfusion Practice 575

transfusionhemoglobinconcentrationbe

tween 11 and 12 g/dL, in order to decrease

iron absorption from the gastrointestinal

tract. The results of maintaining near-nor-

mal hemoglobin levels are still controver-

sial. Iron overload is a complication of

treatment, requiring chelation therapy be-

ginning early in childhood.97

Antibody Production in Sickle Cell and

Thalassemia Patients

The frequency of red cell alloimmuni-

zation in chronically transfused children

varies with the disease, the age of first

transfusion, the number of transfusions

given, and the ethnic background of do-

nors and recipients, although patients

with sickle cell disease have the highest

rates of alloimmunization of any patient

group.98-100 Antibodies to the common an-

tigens of the Rh, Kell, Duffy, and Kidd

systems are often identified. It may be

prudent, therefore, to phenotype the pa-

tient’s red cell antigens as completely as

possible before beginning transfusion

therapy and to maintain a permanent re-

cord of the results. This can be helpful in

selecting compatible blood if alloim-

munization occurs. There is evidence that

transfusing K, C, and E antigen-negative

red cells can significantly reduce the rate

of alloimmunization.101 However, the

practice of transfusing only phenotypi-

cally matched units is controversial, espe-

cially in patients who have not yet devel-

oped the corresponding antibody, because

many of these units are difficult to ob

tain.83,102 A recent survey of 50 academic

medical centers in the United States and

Canada found that the most common

practice is to perform pretransfusion

phenotypicmatchingforC,E,andK.

103 In

patients who have already become immu-

nized and who are at high risk of develop-

ing additional antibodies, use of pheno-

typically matched units may be a reason-

able approach to prevent further allo-

immunization.102 Method 2.16 can be used

to perform red cell phenotyping on auto-

logous red cells of recently transfused pa-

tients.

African Americans with sickle cell dis-

ease frequently become immunized be-

cause the majority of red cell transfusions

are obtained from Caucasian donors, with

major differences in antigen exposure. In

some areas, blood collectors have devel-

oped programs to specifically recruit Afri-

can American donors to supply blood for

patients with sickle cell disease, in order to

reduce rates of alloimmunization.101 Leuko-

cyte-reduced blood components may be of

particular value for these chronically trans-

fused patients. One benefit would be to di-

minish the development of alloimmuniza-

tion to HLA antigens, in light of the prospect

of future marrow transplantation.87 There

are conflicting data whether leukocyte re-

duction can prevent alloimmunization to

red cell transfusion.104,105 Preventing febrile

transfusion reactions is also important for

patients who may be receiving a unit of

phenotypically matched blood. Discarding

such a unit would be both wasteful and

may have an impact on the ability to ade-

quately transfuse the patient.106

Platelets and Plasma

The indications for FFP and platelet

transfusions in older infants and children

parallel those for adults. Platelet transfu-

sions are most often given as prophylaxis

to children receiving chemotherapy. Pro-

phylactic platelet transfusions are seldom

givenwhenplateletcountsareabove

10,000 to 20,000/µL, but, as with red cell

transfusion and hemoglobin, the indica-

tion for platelet transfusion should not be

based solely on the platelet count. When

additive risk factors such as fever, sepsis,

576 AABB Technical Manual

DIC, or clotting abnormalities are present,

the platelet count may need to be higher

to prevent spontaneous hemorrhage. In the

absence of such factors, a much lower level

may be safe.56 In recent studies, the use of

ABO-compatible platelets has been asso-

ciated with better clinical outcomes.107-109

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54. Maier RF, Sonntag J, Walka MW, et al. Chang-

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57. Andrew M, Vegh P, Caco C, et al. A random-

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58. Pisciotto P, Snyder EL, Snyder JA, et al. In vi-

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59. Sweetman RW, Cairo MS. Blood component

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67. Price TH. The current prospects for neutro-

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reduction of red cell transfusions is associ-

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alloimmunization. Transfusion 2003;43:945-

52.

105. Van de Watering L, Jermans J, Witvliet M, et

al. HLA and RBC immunization after filtered

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106. Lane TA, Anderson KC, Goodnough LT, et al.

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580 AABB Technical Manual

Chapter 25: Cell Therapy and Cellular Product Transplantation

Chapter 25

Cell Therapy and Cellular

Product Transplantation

THERAPEUTIC CELLS INCLUDE cell

populations collected and proces-

sed to provide a special therapeu-

tic effect. The classic example of cell

therapy is the use of pluripotent stem

cells, which are capable of self-renewal

and differentiation into all blood-cell lin-

eages. These stem cells—transplanted in

preparations of marrow, stimulated pe-

ripheral blood mononuclear preparations,

or cord blood—also give rise to other re-

generative tissues such as hepatocytes,

endothelial cells, and other tissue under

the proper microenvironmental circum-

stances.1Other cell populations may serve

therapeutic purposes such as immune

modulation in posttransplant donor lym-

phocyte infusion (DLI). The hemato-

poietic progenitor cells (HPCs), which are

committed to a blood-cell lineage, were

first studied in the laboratory for their

power to give rise to complete, sustained

hematopoietic engraftment when given in

sufficient numbers. This chapter primarily

discusses the hematopoietic repopulation

cells. Cell preparations for HPC transplan-

tation are thought to contain both hemato-

poietic stem cells (HSCs) capable of

self-renewal and HPCs committed to a

blood-cell lineage. However, committed

progenitor cells lack capacity for sus-

tained self-renewal or the ability to differ-

entiate into other blood-cell lineages. The

committed cells are important for the

speed of engraftment.2Current measure-

ment methods cannot easily separate the

earlier and more committed cell popula-

tions. Both cell populations are referred to

as HPCs in this chapter. HPCs collected

from peripheral blood via apheresis are

referred to as HPC-A. Those collected by

harvesting marrow are termed HPC-M and

those from cord blood are called HPC-C.

HPC transplantation has advanced from

a research procedure performed in a few

centers to a common medical procedure

581

performed in many tertiary care centers.

HPC transplantation can be classified ac-

cording to the source of the HPCs used for

engraftment as autologous, allogeneic,

syngeneic, and xenogeneic.

Autologous HPC transplantation is tech-

nically not a transplant but, rather, the “res-

cue” of a patient with the patient’s own

HPCs, which are removed and stored frozen

or in nonfrozen conditions for later infusion

as protection from the lethal effects of ther-

apeutic or ablative irradiation or chemo-

therapy. The autologous graft may be ma-

nipulated to retain HPCs and leave behind

or exclude tumor cells or damaging im-

mune cells. Autologous HPCs are reinfused

to repopulate the patient’s marrow after an

otherwise lethal or near-lethal dose of radi-

ation or chemotherapy given to treat malig-

nancies of the marrow and metastatic or re-

current solid tumors. In the special case of

an identical twin donor and recipient, such

transplants are referred to as syngeneic.

Allogeneic HPC transplantation involves

the infusion of HPCs from another human

(an HLA-matched related or unrelated

donor) in order to establish donor cell

chimerism to rescue the patient from dose-

intense therapy and/or as an active immuno-

therapy against a disease after a potentially

lethal dose of radiation or chemotherapy.

Such transplants are preferred in patients

who have acute myelogenous leukemia

(AML), acute lymphocytic leukemia (ALL),

severe immunodeficiency, aplastic anemia,

marrow involvement with their malignancy,

or those who are incapable of supplying

their own autologous “normal” HPCs, as

with hemoglobinopathies (thalassemias or

sickle cell disease). As new indications for

transplantation are developed, some others

are drastically changed by new discoveries.

For instance, patients with chronic myelo-

genous leukemia (CML) used to receive the

largest number of adult hematopoietic

transplants; however, imatinib mesylate

has been such a successful treatment for

this disease that the number of CML trans-

plants has dropped sharply since licensure

of this drug.3

Another advance in marrow transplanta-

tion has been the recognition and exploita-

tion of the effects of transplanted allogeneic

immune cells on the malignancy of the pa-

tient. Because the transplanted cells are the

treatment modality for the patient, the

preparation for transplantation needs only

to modify the patient’s immune system to

allow the new cells to engraft. This non-

myeloablative transplantation expands the

number of patients eligible for transplant

by accepting patients of more advanced age

and with more health problems. These pa-

tients would not be candidates to enter

more toxic myeloablative regimens because

the risk of the treatment would be so great.

In this setting, chemotherapy and/or low-

dose total body irradiation targets the re-

cipient’s T lymphocytes to allow tolerance

for the healthy, allogeneic graft.

In successful marrow transplants, the

cells gradually produce full chimerism in

the patient’s marrow and attack and elimi-

nate the tumor cells immunologically.4-6 In

patients where healthy cell replacement is

the intended result of treatment, such as in

children with immune deficiencies, this

method allows replacement cells to engraft

without putting the child through the dan-

ger of full myeloablation. The patient does

not have to undergo extensive periods of

cytopenia with exposure to infection and

bleeding risk. The engrafted cells become

the treatment agent, allowing the chemo-

therapy and/or radiation to be low-dose

and relatively low toxicity. This approach

uses the immune reconstitution as the tool

to control disease, but it is not without risk

because the immune cells can also attack

the healthy tissues of the patient, causing

graft-vs-host disease (GVHD).7The number

of nonmyeloablative transplants has in-

582 AABB Technical Manual

creased from 20 in 1997 to 870 in 2001, ac-

cording to the Center for International

Blood and Marrow Transplant Research

(CIBMTR) reporting system.3

Xenogeneic HPC transplantation would

involve HPC transplants derived from a

nonhuman species. However, because of

currently insurmountable immunologic

barriers and disease concerns, these trans-

plants are not now clinically feasible.

Sources of progenitor cells include mar-

row, peripheral blood, umbilical cord

blood, and fetal liver (although this source

is experimental and not in routine clinical

use).1Once collected, the HPCs may be

subjected to ex-vivo processing, eg, re-

moval of incompatible red cells or plasma

and/or cell selection (purging) before the

transplantation procedure. In some cases,

certain cell populations are “positively” se-

lected (selectively isolated) for their special

therapeutic effects. T lymphocytes may be

isolated and later infused in measured

doses for antitumor effects, thus minimiz-

ing GVHD. Separation of cell populations

can be accomplished using a cell separa-

tion device approved by the Food and Drug

Administration (FDA), such as the Isolex

system (Baxter Healthcare, Deerfield, IL).

Alternatively, some cell populations may be

“negatively selected” (culled out or de-

stroyed), as by antibody-mediated lysis of

malignant cells or by flow cytometry sepa-

ration of cell populations in clinical trial sit-

uations.

Diseases Treated with

Hematopoietic Cell

Transplantation

Many diseases have been treated with

HPC transplantation.8Malignant diseases

are the most frequent indications for HPC

transplantation. Although many nonma-

lignant diseases have also been treated with

life-saving transplantation, as a group,

they represent less than 15% of all trans-

plants.3,9 The success rate of HPC transplan-

tation depends on the condition and age

of the patient, type and stage of the dis-

ease being treated, the cell dose, and de-

gree of HLA matching between the donor

and the patient. Overall, long-term sur-

vival rates are generally 30% to 60% for

otherwise fatal diseases. Table 25-1 de-

scribes general outcomes.10

Sources of Hematopoietic

Progenitor Cells

Historically, marrow was the primary source

of hematopoietic cells for transplantation.

However, HPC-A transplants constituted

approximately 90% of adult autologous

and about half of allogeneic transplanta-

tion procedures in the 1998-2000 Interna-

tional Bone Marrow Transplant Registry

report.3Data on umbilical cord blood

transplants have shown promising results

in pediatric patients for whom a matched

unrelated allogeneic HPC-M or HPC-A do-

nor is unavailable. Clinical studies are in

progress to determine 1) the safety and ef-

ficacy of cord blood transplantation, 2)

whether adults can be successfully trans-

planted, and 3) the extent of HLA mis-

match that can safely and effectively pro-

vide durable engraftment.

HPCs from Marrow (Autologous)

Currently, only about 5% of autologous

transplants are performed with HPC-M.

This source has been largely replaced by

mobilized autologous HPC-A.

HPCs from Marrow (Allogeneic)

Allogeneic transplants address the prob-

lem of the inability to obtain a tumor-free

Chapter 25: Cell Therapy and Cellular Product Transplantation 583

584 AABB Technical Manual

Table 25-1. Examples of Diseases Responsive to Hematopoietic Stem Cell

Transplantation10

Disease Type of Transplant Timing Clinical Results

AML Allogeneic First CR OS 40%-50%

ALL (children) Allogeneic Second CR OS 40%-65%

ALL (high risk) Allogeneic First CR OS 50%

Chronic phase CML Allogeneic Chronic phase (CP) OS 50%-80%

Accelerated phase CML Allogeneic Individualized OS 30%-40%

Blast phase CML Allogeneic Second CP OS 15%-25%

Myelodysplastic

syndrome

Allogeneic Age <60 OS 40%

Aplastic anemia Allogeneic Individualized OS 70%-90%

CLL Allogeneic or

autologous

Participation in

clinical trial

Small series of patients

with durable CR;

nonablative transplants

under investigation

Intermediate-grade NHL Autologous Chemosensitive

relapse

OS 40%-50%

High-risk NHL Autologous First CR OS 50%-60%

Low-grade NHL Allogeneic or autologous Chemosensitive

relapse

DFS 25%-50% at 5 years

Mantle cell lymphoma Allogeneic or autologous

clinical trial

First CR Small series with durable

CR rates of 25%-50%

Lymphoblastic lymphoma Allogeneic or autologous

clinical trial

Chemosensitive

relapse or first CR

Small series with durable

NHL or Hodgkin's disease Allogeneic clinical trial Advanced refractory

disease

DFS 15%-25%

Multiple myeloma Autologous Chemosensitive re-

lapse or first CR

OS 50% at 5 years; DFS

20%

High-risk breast,

testicular, or ovarian

cancer

Autologous clinical trial Chemosensitive

disease

Improved survival over

historical controls not

confirmed in

randomized trials

Renal cell carcinoma Nonablative allogeneic Clinical trial Small series with durable

Thalassemia Allogeneic Clinical trial OS 75% for patients

without cirrhosis

Sickle cell anemia Allogeneic Clinical trial OS 75%

Autoimmune disorders Allogeneic Clinical trial Small series of remissions

CR = complete response; DFS = disease-free survival; OS = overall survival; AML = acute myelogenous leukemia; CML =

chronic myelogenous leukemia; ALL = acute lymphocytic leukemia; CP = chronic phase; NHL = non-Hodgkin's lymphoma.

graft with an autologous transplant for a

patient with malignant disease and pro-

vide possible immune help after trans-

plantation from graft-vs-tumor response.

For other patients, such as those with

marrow failure, immunodeficiency, in-

born errors of metabolism, or hemoglob-

inopathy, an allogeneic transplant is the

only appropriate type of graft. Sources of

allogeneic HPC-M may be from matched

or partially matched related or unrelated

donors.

Matched, Unrelated Donor Transplantation

Matched, unrelated donor searches can

be initiated for the 60% to 70% of candi-

dates without an HLA-identical related

(usually sibling) donor. Several marrow

donor databases are available worldwide.

The largest is the National Marrow Donor

Program (NMDP) database. Since its

founding in 1986, the NMDP has facili-

tated approximately 12,000 transplant

procedures with a total of over 2000 trans-

plants per year. A directory of transplant

centers, outcome results, and charges is

available from the NMDP. (See Appendix

11 at the end of the book).

Upon initial search, 80% of transplanta-

tion candidates usually find an HLA pheno-

typic match. However, patients from racial

or ethnic minorities have a lower chance of

success in such a donor search (Caucasian,

81%; Hispanic, 64%; Asian/Pacific Islander,

55%; African American, 47%; and American

Indian/Alaska Native, 50%; according to the

NMDP). The median time from initiating a

search to receiving a transplant has been

120 days, but a new expedited search and

donor preparation program is being imple-

mented to shorten this time.

The NMDP is conducting a clinical trial

of HPC-A collections from unrelated, HLA-

matched volunteers stimulated with granu-

locyte colony-stimulating factor (G-CSF or

filgrastim).11 The donors’ physical symp-

toms as well as their attitudes and feelings

about the process are being monitored for

thetrial.TheuseofHPC-Aoffersthepoten

tial advantage of improved engraftment ki-

netics and enhanced graft-vs-leukemia

(GVL) effect. In allogeneic, related trans-

plant settings clinical trials comparing

HPC-M to HPC-A, the latter showed im-

proved survival. Although chronic GVHD

was a problem in the HPC-A study arm, it

was manageable in a sufficient number of

cases to give those patients a survival ad-

vantage.12

Initially, the most common diagnosis in

patients undergoing matched, unrelated

donor transplantation was CML. The dis-

covery of imatinib mesylate has revolution-

ized the treatment of CML such that the

most common indication for allogeneic

transplantation is acute leukemia followed

by non-Hodgkin’s lymphomas, multiple

myeloma, and other diseases of marrow

failure such as aplastic anemia.13

Traditionally, HLA typing for Class I

(HLA-A and HLA-B) has been dependent

on serologic techniques. However, it is

likely that the posttransplant complications

of GVHD and failure to engraft result from

use of phenotypically matched, unrelated

donors with significant disparities in allo-

antigens that were not identified through

serologic matching techniques.14 HLA-A

and HLA-B molecular Class I typing, inter-

mediate resolution, and high-resolution

typing of Class II alleles are the current

standard of care, particularly in unrelated

transplants.

The risk of GVHD is greater with HLA

Class II disparity than with Class I dispar-

ity.15 HLA typing for HLA-DR and HLA-DQ

is routinely performed by DNA-based tech-

niques. Molecular technology provides

greater resolution, including subtypes of al-

leles identified as cross-reactive groups us-

ing conventional serologic techniques. Mis-

Chapter 25: Cell Therapy and Cellular Product Transplantation 585

matching for a single Class I or Class II

antigen has no effect on survival, but mor-

tality increases with more than one Class I

mismatch or simultaneous mismatches in

Class I and Class II antigens.15 Recent stud-

ies demonstrated the importance of recipi-

ent HLA-DRB1 and HLA-DQB1 allele dis-

parity in the development of GVHD.16,17

With further experience in molecular typ-

ing and transplant outcomes, the extent to

which successfully transplanted cells can

tolerate disparities in specific alleles will be

elucidated. Although the importance of

HLA-A, -B, and -DR disparities is well

known, the significance of HLA-C disparity

is being investigated. HLA-C typing has been

hampered by poor serologic identification,

and its significance has been thought, until

recently, to play a minor role in the T-cell

immune response because of its reduced

polymorphism and low level of cell surface

expression. Early studies showed that

HLA-C antigens can be recognized by

alloreactive cytotoxic T lymphocytes and

natural killer cells, which may be associated

with an increased risk of graft failure.18

Graft-vs-Host Disease

The negative outcomes of HLA mismatch-

ing are graft rejection, host-vs-graft reac-

tions, and graft-vs-host reactions. In acute

GVHD, the transplanted cells may attack

the tissues of the recipient early in the

engraftment—within 100 days after the

initial engraftment-associated events. The

skin, the gastrointestinal tract, and the

liver are most commonly involved, al-

though usually not concurrently. The site

and severity determine the clinical grade

ofacuteGVHD.TheriskofGVHDis

greater with HLA-mismatched, unrelated

and related transplants than with HLA-

identical transplants.19

Chronic GVHD characteristically occurs

spontaneously months after transplanta-

tion or after acute GVHD (generally after

posttransplant day 50) and may severely af-

fect the patient’s quality of life. In addition

to the symptoms found in the acute form,

chronic autoimmune-type disorders such

as biliary cirrhosis, Sjogren’s syndrome, and

systemic sclerosis may develop as the

transplanted immune cells attack the secre-

tory epithelial cells in the saliva glands, the

biliary tree, or the patient’s connective tis-

sue. Chronic GVHD was reported in 55% to

65% of allogeneic transplant patients who

survived beyond day 100 in a large study.2

Both forms of GVHD impair the patient’s

immune response and predispose the pa-

tient to infections. To decrease or eliminate

GVHD in these transplants, HPCs can

undergo procedures for T-cell reduction

(depletion) and the patient can be treated

prophylactically with a variety of immuno-

suppressive drug therapies.

GVHD has been associated with both a

decreased disease relapse and an improved

overallsurvivalinleukemiapatientsifthe

GVHD is relatively mild. Such a GVL effect

is believed to be secondary to the graft at-

tacking residual malignant cells. A major

clinical challenge is to maximize the GVL

effect while minimizing the adverse se-

quelae of GVHD. The mechanism of the

GVL effect is incompletely understood, but

donor-derived cytotoxic T lymphocytes

specific for the patient’s minor histocom-

patibility antigens may contribute to the ef-

fect. Donor lymphocyte infusion of thera-

peutic T cells in patients with leukemic

relapse after allogeneic transplantation has

been attempted to induce a GVL effect.20

Conflicting data exist about whether a GVL

effect can occur independently of GVHD. A

strategy to maximize the GVL effect and

minimize GVHD has been to titrate the

number of donor lymphocytes until GVHD

Grade II-III occurs in order to create a GVL

effect without severe GVHD. However, the

optimal number of donor lymphocytes ca-

586 AABB Technical Manual

pableofinducingaGVLeffectwithoutsig

nificant GVHD is not known. Immunosup-

pression, number of T cells transfused,

T-cell phenotype, myelosuppression, and

thetimingoftheDLIoftherapeuticTcells

all play a role in the balance between the

GVL effect and GVHD. The GVL effect is best

documented in CML and less well shown in

AML, ALL, and other lymphoid malignancies.

However, because it is difficult to identify

a good HLA match, allogeneic transplanta-

tion is associated with a major risk that

immunocompetent donor T cells reacting

against recipient tissues will cause GVHD.

Even in HLA-identical (six-antigen match)

donor/recipient pairs, up to 6% of the grafts

will fail and GVHD will occur in 20% to 60%

of cases as a result of nucleated cells exhib-

iting minor histocompatibility antigens that

are not linked to the major histocompati-

bility complex antigens.21 Improved HLA

typing techniques now employ molecular

techniques that give a fuller picture of the

antigen mapping and match/mismatch

picture, but serologic terms of estimation of

number of matches are still frequently used

in clinical descriptions for reference. This

occurs despite immunosuppressive therapy

administered for several months after the

procedure.22 New and emerging immune

cell manipulations are being explored to

exploit the cancer-controlling effects of

these cells while seeking to avoid the un-

wanted consequences of GVHD. Extracor-

poreal photopheresis is being used to treat

patients with GVHD and to reduce the dose

of immunosuppressive medications re-

quired.23

HPCs by Apheresis (Autologous)

Autologous HPC-A collection involves

mobilizing the hematopoietic cells from

the patient’s marrow compartment into

the peripheral blood with hematopoietic

growth factors, most commonly filgrastim,

with or without treatment with chemo-

therapy before collection. Once in the

circulation, the HPCs are collected by

leukapheresis. HPC-A collection carries

no anesthesia risk, is less invasive, and

contains fewer tumor cells than marrow

harvests.

HPCs by Apheresis (Allogeneic)

Related Transplantation

For adult allogeneic transplantation, the

best clinical results are obtained with a

completely HLA-matched, related donor.

The best chance of finding a six-antigen

HLA match is among the patient’s genetic

sisters and brothers. Parents and children

will be at least a haplotype match.

Genetically, there is a 25% chance of a

sibling being a complete match, a 50%

chance of a haplotype match, and a 25%

chance of a complete mismatch. Pediatric

patients are more tolerant of partially mis-

matched grafts and, therefore, have a larger

available donor pool.24 Intherareinstancea

recipient has an identical twin, a syngeneic

transplant may be optimal because the do-

nor and recipient cells are genotypically

identical and the risk of GVHD is reduced.

However, syngeneic grafts do not provide

the graft-vs-tumor effect found in allo-

geneic transplants.

HPC-A has largely replaced HPC-M for

related transplantation.25 The use of G-CSF

in healthy donors has been shown to mobi-

lize sufficient HSCs for allogeneic trans-

plantation,26 thus avoiding the need for an-

esthesia and a marrow harvest. Clinical

data comparing allogeneic HPC-A vs allo-

geneic HPC-M from HLA-identical siblings

have shown that HPC-A recipients have

faster engraftment, improved immune re-

constitution, lower transplant-related mor-

bidity, and a similar incidence of acute

GVHD.2,27 Retrospective analyses reported a

higher incidence of chronic GVHD in re-

Chapter 25: Cell Therapy and Cellular Product Transplantation 587

lated allogeneic HPC-A recipients.28 How-

ever, a recent prospective randomized

study found no difference in the risk of

chronic GVHD.12

HPCs from Cord Blood

Despite the fact that over 3 million indi-

viduals are registered with the NMDP, pa-

tients in need of an allogeneic HPC trans-

plant have ≤85% chance of finding a

matched donor. Finding and qualifying a

willing donor for HPC-A or HPC-M collec-

tion typically takes weeks. Because of the

time and availability constraints, atten-

tion has turned to the third available

source—HPC-C. Umbilical cord blood,

which in clinical practice is routinely dis-

carded, is being banked as an alternative

source of HPCs, especially for children,

for whom smaller collection numbers of

HPCs are adequate for successful engraft-

ment. The advantages of cord blood as a

source of HPCs for transplantation in-

clude: no risk to the donor, potential

availability of cord blood from donor pop-

ulations underrepresented in the NMDP,

more rapid availability, and possibly

lower risk of viral infection and GVHD. Ar-

eas of concern regarding HPC-C include

ethical and informed consent issues, speed

of engraftment, higher mortality in the

posttransplant period, and ability to

achieve engraftment in adults as a result

of the limited number of nucleated cells

in cord blood.

Cord blood obtained from a delivered

placenta is known to be rich in early and

committed progenitor cells.29 Since the first

cord blood transplant was reported in 1989

for Fanconi anemia,30 more than 2500 pa-

tients have received cord blood for a variety

of hematologic malignant and nonmalig-

nant conditions.31

Cord blood is collected from the placenta

at the time of delivery using a variety of

techniques, but the preferred system uses a

small collection bag (150 to 250 mL) with

appropriate anticoagulant. In-vitro data

have suggested that placental blood has an

increased capacity for proliferation.32 The

volume of units retained for processing is

typically 80 to 100 mL (range, 40-240 mL),

with a median nucleated cell count of 1.2 ×

109in the units chosen for transplantation

and a median CD34+ cell count of 2.7 ×

105/kg.33 Clinical studies have reported suc-

cessful engraftment in children.34,35 The me-

dian time to neutrophil engraftment (500/

µL) is 30 days; median time to platelet

engraftment (50,000/µL) is 56 days.36 Com-

pared with engraftment observed after allo-

geneic marrow transplantation, neutrophil

and platelet engraftment appear to be de-

layed. Clinical studies have also suggested

that unrelated HPC-C transplants are asso-

ciated with a lower risk of GVHD compared

with unrelated HPC-M transplants in chil-

dren, even considering the lower risk sus-

ceptibility of pediatric patients to GVHD.31,34,36,37

Two studies have compared engraftment

and outcomes of transplantation of adults

with one- or two-antigen mismatched

HPC-C, matched HPC-M, and one-antigen

mismatched HPC-M. The findings in the

two studies were very similar: HPC-M

transplant recipients recovered neutrophils

on day 18 to 19, and HPC-C recipients re-

covered neutrophils on day 26 to 27.38,39

Platelet engraftment in these studies re-

peated the finding that HPC-M transplan-

tation provided more rapid recovery at 29

days compared with 60 days for HPC-C.

Both of these studies found that the HPC-C

transplantation compares favorably with

one-antigen mismatched HPC-M trans-

plantation, with equal advantage for sur-

vival for each group. A matched HPC-M

transplant was superior to HPC-C or mis-

matched HPC-M, but the advantage was

small. This is encouraging news for adults

who need an HPC-M transplant but have

588 AABB Technical Manual

difficulty finding a suitably matched donor.

Current clinical trials are testing whether

multiple HPC-C units received by adults

will facilitate faster engraftment. A study of

23 such recipients with advanced-stage dis-

ease showed a mean neutrophil engraft-

ment of 23 days and a 57% chance of dis-

ease-free survival after 1 year. In these

two-unit transplants, the cells of one of the

units prevailed and provided the lasting

engraftment population.40

Related Cord Blood Donors

Sibling-derived cord blood has been used

as a source of hematopoietic engraftment

in more than 250 allogeneic transplants in

Europe and North America, representing

15% of the allogeneic cord blood trans-

plants.31,35 The kinetics of hematopoietic

recovery are similar to those observed with

unrelated cord blood recipients.31 Times to

both platelet and neutrophil engraftment

are slower than those observed in HLA-

matched sibling marrow transplants. How-

ever, recipients of cord blood from HLA-

identical siblings have a lower incidence

of acute and chronic GVHD compared with

marrow from HLA-identical siblings.31

Autologous Cord Blood

Companies are marketing the freezing and

long-term storage of an infant’s cord blood

cells to parents, in case the child may need

them.41,42 The chance of an individual

needing a cord blood transplant by age 18

is estimated to be 1 in 200,000.42 The first

successful autologous cord blood transplan-

tation procedure was reported in a 14-

month-old patient with neuroblastoma.43

Cord Blood Banks

The first large-scale community cord

blood program was the Placental Blood

Program at the New York Blood Center,

which began collecting placental prod-

ucts in 1992. The program has stored in

excess of 14,000 cord blood units and has

provided cells for more than 1500 trans-

plant procedures. The outcomes of the

first 562 procedures have been reported

in detail.34 The NMDP has developed a

registry of participating cord blood banks.

The National Cord Blood Bank of the New

York Blood Center, NetCord, Bone Marrow

Donors Worldwide, and the Caitlin Ray-

mond Registry are additional search sites

available today.

Donor Eligibility

Donor Evaluation: Autologous Setting

In the autologous setting, the major con-

cern regarding eligibility for transplanta-

tion arises from the condition of the pa-

tient. For HPC-M, the patient’s marrow

first should be assessed for residual ma-

lignancy and marrow cellularity. Patients

scheduled for an autologous transplant

should undergo an extensive history and

physical examination to identify any risks

from the marrow harvest and/or aphere-

sis procedures. For HPC-A, the patient/

donor is evaluated for the likelihood that

he or she can undergo successful mobili-

zation and collection.

Donor Evaluation: Allogeneic Setting

Allogeneic donors must be selected ac-

cording to their degree of HLA match;

qualifications according to the FDA regu-

lations; standards of the AABB, NMDP,

and Foundation for the Accreditation of

Cellular Therapy (FACT); and physical

ability and willingness of the donor to un-

dergo the collection procedure. Ideally, a

full six-antigen match should be found;

however, transplant procedures have been

performed successfully using one-antigen

mismatched and haploidentical donors.

Chapter 25: Cell Therapy and Cellular Product Transplantation 589

Cytomegalovirus (CMV) status of the do-

nor is also a deciding factor in the selec-

tion process in the case of CMV-negative

recipients. The use of parous females or

gender-mismatched individuals as donors

is associated with an increased risk of

GVHD,asisahistoryoftransfusioninthe

donor.44-46 Therefore, the preferred HLA-

identical donor would be CMV negative

(in the case of CMV-negative recipients);

of the same gender as the recipient; if fe-

male, nonparous; and untransfused.

Infectious Disease Testing

Autologous and tested allogeneic donors

must be screened and tested for certain

infectious diseases.47 In the Code of Fed-

eral Regulations (CFR) Title 21, sections

of the regulations relevant to cell therapy

donors are Donor Screening (1271.75),

Donor Testing: General (1271.80), and Do-

nor Testing: Specific Requirements

(1271.85). The rules are described by the

FDA as “comprehensive, but flexible.” The

donor screening rules are consistent with

the 1994 Centers for Disease Control and

Prevention (CDC) guidelines to prevent

transmission of human immunodeficiency

virus (HIV), and 1993 FDA guidelines for

prevention of transmission of hepatitis B

virus (HBV) and hepatitis C virus (HCV).

Testing for syphilis is required, and tissue

rich in leukocytes must be tested for hu-

man T-cell lymphotropic virus (HTLV)

and CMV. They also include requirements

for questioning donors about risk factors

for variant Creutzfeldt-Jakob disease and

new or emerging pathogens such as West

Nile virus and severe acute respiratory

syndrome, and requirements for tests for

carriers of these illnesses as soon as they

are practically available. The regulations

and guidance are extensive and fit the

FDA’s stated “layered approach” that in-

tensifies the amount of screening and test-

ing of the donor to the level of risk to

which the recipient may be subjected.

Thus, unrelated allogeneic donors repre-

sent the highest level of risk, whereas

family members or regular sexual partners

are considered a lower risk source of new

exposure to patients.

The donor must be screened in order to

minimize the risk of disease transmission

to an already immunocompromised recipi-

ent. FDA regulations specify that the HPC

donor sample should normally be tested up

to7daysbeforecollection,oratthetimeof

collection but before release of the product.

HPC-A donor samples may be obtained up

to 30 days before collection in order to have

infectious disease testing completed before

the patient begins myeloablative chemo-

therapy [21 CFR 1271.80(b)]. For purposes

of optimal donor selection, it may be advis-

able to test the donor earlier in the trans-

plantation workup period as well.48,49

The use of approved nucleic acid tests

for HIV and HCV has considerably short-

ened the possible “window period” in do-

nors who may have contracted either of

these infections but who do not yet test

positive on antibody tests. Donors being

stimulated with filgrastim develop higher

sample-to-cutoff ratios. This makes false-

positive reactions more likely in the

immunoassays currently used for infectious

disease. False-positive test results for hepa-

titis B surface antigen and HCV antibody

have been associated with G-CSF adminis-

tration in normal donors.50,51

Donors who are confirmed positive for

HIV should not be used as a source for the

transplant. Other positive disease markers

do not necessarily prohibit use of collec-

tions from a particular donor (eg, anti-HBc

positive), and special evaluation of donors

with this marker is under way by the NMDP

at this time. The allograft material from

such donors may be used when the pa-

tient’s transplantation physician informs

590 AABB Technical Manual

him or her of the status of the donor and

documents consent of the patient. Segre-

gated methods of storage must be used for

the biohazardous material collected until it

is infused so that cross-contamination of

otherstoredproductsisprevented.

48(p78),49,52

In cases of allogeneic HPC-C transplants,

a sample for infectious disease studies

should be obtained from the donor’s

mother within 48 hours of collection of the

cord blood and tested. Any positive results

should be reported to the mother and the

mother’s physician. The HPC-C donation

involving a mother or a child who has a

positive infectious disease history or results

should either be discarded or the patient

must go through a special notification and

consent process.52 In cases of allogeneic

transplantation, CMV status of the donor

and the recipient should be carefully con-

sidered.

A recipient may develop primary CMV

infection if he or she is CMV negative and

receives a CMV-positive graft but may have

some protection from the immunity in the

graft. CMV-positive individuals receiving a

CMV-negative graft may have a severe pri-

mary infection of the graft from virus resi-

dent in the recipient’s tissue.46

Collection of Products

HPC-M Collection

A marrow harvest is the same for an allo-

geneic donor as for an autologous patient.

The procedure is performed under sterile

conditions in the operating room. The

posterior iliac crest provides the richest

site of marrow. In the autologous patient,

prior radiation therapy to an aspiration

site may result in hypocellular yields. In

general,thesesitesareunsuitableforhar

vestandshouldbeavoided.Onceaspi

rated, the marrow should be mixed and

diluted with an anticoagulant (usually

preservative-free heparin and/or ACD).

The marrow aliquots are pooled into a

sterile vessel or a harvest collection bag

equipped with filters of graduated pore

size. The marrow is then transferred to a

sterile blood bag and transported to the

processing laboratory, where samples are

removed for graft evaluation, quality as-

surance testing, possible manipulation of

the product, final labeling, and/or cryo-

preservation.

Collection Targets

The recipient’s body weight and type of

manipulation of the collection, if any, will

determine the volume of marrow to be

collected. A frequently used minimum

target (after processing), for both autologous

and allogeneic transplants, is 2.0 ×108nu-

cleated cells per kilogram of recipient

body weight. Marrow harvests in autolo-

gous patients who have received alkylat-

ing agents as therapy may yield fewer pro-

genitor cells relative to the total nucleated

cells collected. In these cases, extra mar-

row should be obtained if possible.53 If the

harvested product is to be processed (ie,

T-cell depletion, ex-vivo tumor purging),

additional marrow (up to double the re-

infusion target) should be collected. The

NMDP limits the volume harvested from

its donors to a maximum of 1500 mL. Many

centers use the cell number requested for

transplant and the cell count on the prod-

uct during collection to determine the fi-

nal collection volume.

Clinical Considerations

Theageofthedonorand,intheautolo

gous setting, the underlying disease and

previous treatment regimen influence the

HPC yield (Table 25-2).54 Autologous or allo-

geneic donors may require RBC transfu-

sions to replace blood taken with marrow

Chapter 25: Cell Therapy and Cellular Product Transplantation 591

collection. Frequently, allogeneic marrow

donors will have an autologous RBC unit

collected 2 to 3 weeks before the harvest.

Most RBC transfusions occur after the har-

vest to avoid marrow dilution. Allogeneic

blood components must be irradiated if

given before or during the procedure, to

incapacitate donor lymphocytes from re-

plication and attack of the transplant re-

cipient, possibly causing GVHD. In trans-

plants that require marrow manipulation,

such as red cell depletion, the recovered

red cells may be returned to the donor.

HPC-A Collection

HPCs can be mobilized into the periph-

eral blood with the use of recombinant

colony-stimulating factors and collected

in sufficient numbers to achieve long-

term hematopoietic engraftment in a trans-

plant recipient.

CD34

CD34 is the cluster designation given to a

transmembrane glycoprotein present on

immature hematopoietic cells, some ma-

ture endothelial cells, and stromal cells.55

The antigen has an approximate molecu-

lar weight of 110 kD and carries a negative

charge. Cells expressing the CD34 antigen

encompass the lineage-committed cells as

well as the pluripotent stem cells. CD34+

cells purified from marrow are capable of

trilineage reconstitution in humans.55 One

to three percent of normal marrow cells

express this antigen. In normal peripheral

blood, CD34+ cells circulate in low num-

bers (0.01-0.1%).56 Monoclonal antibodies

(MoAbs) to CD34 have been developed

and are used in flow cytometric assays to

measure the stem/progenitor content of

HPC collections and CD34+ cell concen-

tration in donor peripheral blood.57

HPC-A Collection (Autologous)

In the autologous setting, HPC-A donors

are routinely mobilized with hematopo-

ietic growth factors (with or without che-

motherapy), which include G-CSF and

granulocyte-macrophage colony-stimu-

lating factor (GM-CSF) or a combination

of the two. Mobilization with chemother-

apy or growth factors alone results in a

10- to 30-fold increase in the concentra-

tion of HPCs over the steady state.8Com-

bined mobilization with growth factors

and chemotherapy enriches HPC concen-

trations 50- to 200-fold.58 Transplantation

of autologous HPC-A results in a reduced

time to hematopoietic recovery compared

with autologous HPC-M.59 Platelet recon-

stitution is most striking and patient hos-

592 AABB Technical Manual

Table 25-2. Factors Reported to Affect the Mobilization of Hematopoietic Cells

1. Mobilization technique

a. Chemotherapy—degree of transient myelosuppression

b. Growth factors—type, schedule, dose

c. Use of combined chemotherapy and growth factors

2. Extent of prior chemotherapy/radiation

3. Type of underlying disease

4. Age of patient

5. Presence of marrow metastases

Adapted with permission from Lane.54

pital stays are reduced by 7 to 10 days.

Schwella and colleagues60 demonstrated

that the median time to an unsupported

platelet count of ≥20,000/µLwas10days

in the group receiving chemotherapy plus

G-CSF-mobilized HPCs vs 17 days in the

group receiving autologous HPC-M. Ef-

fective mobilization in the autologous pa-

tient is influenced by previous chemo-

therapy or radiation.

There is a linear relation between the

precollection peripheral blood CD34 count

and the volume of blood that needs to be

processed to achieve a target dose. Graphs

and predictive models would be expected

to vary based on the efficiency of CD34+

collection related to the specific equipment

and software employed. It would also be

expected to be least predictive at low pe-

ripheral blood CD34+ concentrations, where

the enumeration is least accurate. White

cell counts, the number of mononuclear

cells, and the number of circulating CD34+

cells have all been used as surrogate mark-

ers for the timing of the HPC-A collec-

tion.59,61-63 It has been suggested that the op-

timal time to begin HPC collection in pa-

tients who have been primed with chemo-

therapy is when the white cell count first

exceeds 5 ×109/L.59 However, the white cell

concentration does not correlate with the

number of HPCs in the peripheral blood.

Phenotypic analysis of CD34+ cells by flow

cytometry provides a more real-time mea-

surement of CD34+ cell content in an HPC

collection or hematopoietic graft. The tech-

nique can be used to indicate the timing of

the first collection and the total number of

CD34+ cells/kg finally collected (as a func-

tion of the volume of blood processed).56,57,64-66

The length of time for engraftment corre-

lates with the number of CD34+ cells in the

collection.65,67-69 In general, a peripheral blood

CD34+ cell concentration of 10/µLcanbe

expected to result in a yield of at least

1.0 ×106CD34+ cells/kg.60,61,64

In most autologous patients, venous ac-

cess is obtained through a dual- or triple-

lumen central venous apheresis catheter.

Operators of blood cell separators generally

process two to three blood volumes per

procedure. In the pediatric setting, it may

be necessary to prime the cell separator

with compatible, irradiated red cells. The

donor will undergo daily procedures of ap-

proximately 2 to 5 hours each. Large-vol-

ume leukapheresis procedures (processing

at least three blood volumes or 15-20 liters)

are performed at many centers to reduce

the overall number of collections. Investiga-

tional studies have suggested that there is

equilibration of noncirculating HPCs into the

peripheral circulation with large-volume

collections.70-72 Hillyer73 reported a 2.5-fold

increase in colony-forming units–granulo-

cyte-macrophage (CFU-GM) per mL pro-

cessed in collection when the collection

volumewas15literscomparedtotheyield

in the first blood volume of the collection.

Collection Targets. The adequacy of

autologous HPC collection is gauged by the

CD34+ dose (the number of CD34+ cells per

kilogram of recipient body weight). The re-

ported minimum threshold of CD34+ cells

necessary for neutrophil and platelet engraft-

ment in the autologous patient has ranged

from 0.75 to 1.0 ×106/kg.8,64 This minimum

target is a broad guideline and higher doses

have been associated with accelerated

platelet engraftment,74 reduced febrile com-

plications, and use of antibiotic therapy af-

ter transplantation.75 Recent data support

an economic benefit associated with greater

CD34+ cell collections (greater or equal to

5.0 ×106/kg) compared to the minimum ac-

ceptable collections required for engraft-

ment (1.0 ×106/kg).76 Thetypeofprocessing

will also influence the volume necessary for

collection.

Poor Autologous HPC Mobilization. Pa-

tients who have been heavily pretreated

with chemotherapy and/or radiation ther-

Chapter 25: Cell Therapy and Cellular Product Transplantation 593

apy may fail to mobilize enough HPCs after

stimulation with growth factors/chemo-

therapy. The best way to obtain an ade-

quate collection from poorly mobilized pa-

tients is unknown. Collection of HPC-M in

combination with, or in place of, HPC-A is

frequently ineffective in improving engraft-

ment.77 Second attempts at mobilization

with G-CSF alone have been successful in

obtaining adequate CD34+ cell counts and

can be as effective as G-CSF and chemo-

therapy.78 Increasing the dose of G-CSF or

combining with GM-CSF has also success-

fully mobilized some autologous donors af-

ter prior failed attempts.79

Mobilization can be very difficult in some

patients, particularly if they have been

heavily pretreated or are older. Another ap-

proach to increase production of stem/pro-

genitor cells by growth factors is manipula-

tion of the chemokines that attach the cells

to their microenvironment. The interaction

of stromal-derived growth factor-1 (SDF-1)

and CXCR4, its ligand, controls mobiliza-

tion. The molecule AMD 3100 was devel-

oped originally as a potent and selective

inhibitor of CXCR4 in order to control repli-

cation of HIV-containing cells. An observed

side effect of the drug was a rapid increase

in white cells. Broxmeyer and colleagues

measured a 40-fold increase in HPCs after

injection of AMD 3100 into human volun-

teers.80 Phase I and II clinical trials are un-

der way in conjunction with G-CSF to en-

hance mobilization and HPC-A yields in

difficult-to-mobilize patients.81

Clinical Considerations for Autologous

HPC-A Collection. The frequency and length

of HPC-A collections may result in donor

discomfort and side effects. Complete blood

counts are performed before and after each

apheresis procedure to monitor the hemato-

crit and platelet values. Red cell and/or

platelet transfusions may be required. Red

cell loss should be minimal in HPC-A col-

lection, but platelet counts typically de-

crease from 25% to 40% because the HPCs

lie close to the platelet layer in the buffy

coat. Collection can be performed via pe-

ripheral or central venous access. Patients

who have received prior chemotherapy are

more likely to need a central venous cathe-

ter because of poor peripheral access. Cath-

eter-associated thrombosis (either in the

catheter or in the vein surrounding it) is the

most common complication associated

with HPC-A collection.82

HPC-A Collection (Allogeneic)

Collection of allogeneic HPCs from HLA-

matched relatives is primarily performed

using G-CSF mobilization. Typically, do-

nors are given 10 to 16 µg/kg as a subcu-

taneous injection once or twice daily. The

concentration of CD34+ cells in the pe-

ripheral blood begins to rise after 3 days

of G-CSF administration and peaks after 5

to 6 days. Standard volume leukapheresis

(processing 8-9 L) results in a component

with the following median values: white

cells, 32.4 ×109; mononuclear cell count,

31.4 ×109; CD34+ cells, 330 ×106;plate-

lets, 470 ×109;andRBCs,7.6mL.

83 Clinical

trials have suggested a minimum dose of

2.0 ×106/kg of CD34+ cells to be adequate

for allogeneic HPC-A transplants.84

Allogeneic donors with poor venous ac-

cess may require central venous catheters.

In the NMDP report on normal donor col-

lections of HPCs, 5% of men and 20% of

women require central venous access for

collection.85 There is a 1% risk of complica-

tions associated with these catheters includ-

ing infection, hemorrhage, and pneumo-

thorax. Thrombocytopenia has been reported

as a complication of G-CSF administration

and HPC-A collection in healthy donors.86,87

There is also a risk in exposing normal

donors to G-CSF. Ninety percent of donors

experience side effects.88 The most com-

mon complaint is bone pain followed by

594 AABB Technical Manual

headaches, body aches, fatigue, nausea,

and/or vomiting. Bone pain, headaches,

and body aches can be successfully man-

aged with nonsteroidal analgesics such as

acetaminophen. As mentioned earlier,

false-positive infectious disease serologies

have been reported.51 Serious adverse ef-

fects are rare but include reports of splenic

rupture,89,90 neutrophilic infiltration of the

skin (Sweet’s syndrome),91 arterial thrombo-

sis,92 and an anaphylactoid reaction.93 Do-

nors with complex hemoglobinopathies

(eg, sickle trait and β+ thalassemia) have

been observed to have complications with

G-CSF stimulation for the purpose of be-

coming an HPC donor.94,95

HPC-C Collection

Umbilical cord blood can be collected from

either a delivered or an undelivered pla-

centa.96 Cord blood collection should not

interfere with obstetric care of the mother

or the infant. If the birth provider intends

to collect the cord blood, plans should be

made in advance to abandon the collec-

tion if care of the mother or the infant re-

quires the care provider’s attention in-

stead. Cord blood collected after delivery

of the placenta is preferably initiated

within 15 minutes of parturition, if ade-

quatevolumesaretobeobtained.

97 To

minimize the risk of bacterial contamina-

tion, the surface of the cord should be

cleaned with alcohol, then disinfected, in

a similar manner to the preparation of skin

for a blood donation. A large-bore needle

connected to a blood collection bag con-

taining CPDA, CPD, or ACD anticoagulant

is inserted into the umbilical vein so that

the placental blood drains by gravity into

the bag. Alternatively, a delivered pla-

centa can be suspended above the collec-

tion bag. CPDA or CPD are the preferred

anticoagulants because they are isotonic

and have a neutral pH. However, other

closed system arrangements using ACD or

heparinareinuseinsomecenters.In

formed consent from the biologic mother

or legal representative of the child must

be obtained, preferably before delivery.

A personal and family medical history of

the biologic mother (and, if available, of the

infant donor) must be obtained and docu-

mented before, or within 48 hours of, the

collection. If available, his medical history

should be obtained from the biologic fa-

ther. HPC-C collections are not acceptable

for allogeneic use if there is a family history

(biologic mother, father, or sibling) of ge-

netic disorders that may affect the graft’s ef-

fectiveness in the recipient or otherwise ex-

pose the recipient to a genetic disorder

through the transplant.

Red cells from the HPC-C collection or

from the infant donor must be typed for

ABO/Rh and a screen for unexpected red

cell antibodies must be performed using ei-

ther the mother’s serum or plasma or a

sample from either the infant donor or the

cord blood collection.52,56 White cells from the

cord blood must be typed for HLA (HLA-A

and -B antigen testing, and DNA-based

Class II typing) by a laboratory accredited

by the American Society of Histocompati-

bility and Immunogenetics (ASHI) or an

equivalent accrediting organization.49,52 In

order for HPC-C to be a feasible alternative

to allogeneic HPC-M or HPC-A transplants,

it is essential to have a frozen inventory of

ready-to-use HLA-typed products.

Samples, preferably from an attached

segment, should be frozen for pretrans-

plant confirmation of HLA type by the

transplant facility. Many centers freeze a

sample of the mother’s serum to allow new

infectious disease tests to be done if they

become available after the HPC-C has been

collected and are thought to be important

at the time the transplant is planned. The

emergence of West Nile virus as a threat to

Chapter 25: Cell Therapy and Cellular Product Transplantation 595

mothers, infants, and transplant recipients

is an example. A sample of the mother’s

blood containing cells or DNA (a dried filter

paper sample is adequate) may be useful if

questions concerning the child’s HLA type

arise. These samples from the mother were

more useful when HLA typing was done by

serologic methods to investigate ambigu-

ous types in the infant. CD34+ cell enumer-

ation at the time of thawing may also be

performed to estimate the stem cell con-

tent of the HPC-C graft but is useful only if

a viability marker is employed so that only

viable cells are counted.

Thedesiretoincreasethedoseofstem

cells in HPC-C has fueled interest in ex-vivo

expansion of the cells in a laboratory set-

ting.98 Early clinical trials with expanded

cord blood progenitors are promising, hav-

ing shown successful engraftment; how-

ever, no improvement in patient survival

has been reported.99,100 Ex-vivo expansion of

cord blood is an area of active experimental

investigation.

Processing of Hematopoietic

Progenitor Cells

Techniques for Cell Selection and/or

Purging of Hematopoietic Progenitor Cells

Selection of the CD34+ Cells

As an HPC undergoes differentiation and

maturation, CD34 antigen levels decrease.

This property, coupled with the use of MoAbs

specific for the different epitopes of the

CD34 molecule, permits physical separa-

tion procedures. There are several meth-

ods of immunoselection available, such as

fluorescence-activated cell sorting (FACS)

and immunomagnetic beads.101 Selection

of CD34+ HPCs may be associated with a

reduction of tumor cells (autologous grafts)

or T cells (allogeneic grafts). The clinical

utility of CD34 and AC133 selection for tu-

mor or T-cell reduction is under investiga-

tion.102

Immunomagnetic Separation. Various

immunomagnetic separation techniques,

direct or indirect, are available. Typically, a

CD34 antibody is coupled to a magnetic

bead. This complex is incubated with mono-

nuclear cells, and the cells expressing the

CD34 antigen bind to the antibody-coated

beads, forming rosettes. A magnet is ap-

plied to separate the rosetting CD34+ cells

from the nonrosetting cells. Bead detach-

ment, which varies among methods, may

be accomplished through anti-Fab frag-

ments or enzymatic treatment (eg, chymo-

papain).103 The Isolex 300 system (Baxter

Healthcare Immunotherapy Division, Irvine,

CA), a magnetic cell separator, is a semi-

automated instrument for clinical-scale

CD34+ selection applications. This method

uses antibody-coated Dynal paramagnetic

beads to rosette the CD34+ cells. A fully au-

tomated device (Isolex 300i, Baxter) and a

peptide release agent are available for clini-

cal use.104 Other techniques of magnetic cell

separation employ superparamagnetic micro-

beads that remain attached to the HPC sur-

face.105 One such method, magnetic cell

sorting (MACS, Miltenyi Biotec, Bergisch

Gladbach, Germany), was first introduced

on a small scale by Miltenyi.106 The

CliniMACS (Miltenyi Biotec) is a clinical-

scale version of the MACS system and is

available in the United States for investiga-

tional use only. This system employs anti-

body-conjugated iron-dextran microbeads.

The magnetically stained cells are sepa-

rated over a high gradient magnetic column

and a microprocessor controls the elution

of the CD34+ cells.

Counterflow Centrifugal Elutriation.

Counterflow centrifugal elutriation (CCE) is

a method of separating cells based on their

size and density. A continuous-flow centri-

fuge (Beckman Instruments, Palo Alto, CA,

and Gambro, Golden, CO) and unique

596 AABB Technical Manual

chamber design allow for the basic separa-

tion principle: two opposing forces (centrif-

ugal force and counter media flow) acting

upon cells at the same time. As cells are

pumped into the chamber (centripetal di-

rection), they align according to their sedi-

mentation properties. With adjustment of

the counterflow rate, the centrifugal and

counterflow forces are balanced, and a gra-

dient of flow rates exists across the cham-

ber. By gradually increasing the flow rate of

the medium or decreasing the speed of the

rotor, cells can be eluted out of the cham-

ber and collected. Smaller, slower sedi-

menting cells elute first. Although this tech-

nique is used primarily for T-cell depletion,

CCE has been applied to CD34+ cell selec-

tion. CD34+ cells are heterogeneous and

will elute in subset fractions that are useful

for repopulation experiments and ex-vivo

expansion trials.107

Autologous Tumor Purging

Purging or negative selection refers to the

removal of tumor cells that may contami-

nate the autologous graft. In patients with

hematopoietic disease or malignancies

that frequently involve the marrow (eg,

lymphomas), minimal residual disease

contributes to relapse.108 Although autolo-

gous HPC-A collections have a lower prob-

ability than HPC-M of tumor contamina-

tion and fewer tumor cells/mL, they still

may contain large numbers of viable tu-

mor cells. Studies have demonstrated that

tumor cells may be mobilized from the

marrow into the peripheral circula

tion.109,110 Whether tumor purging de-

creases the likelihood of relapse is contro-

versial. Because some purging methods

may damage HPCs, the probability of re-

sidual disease or relapse should be care-

fully balanced against the higher graft

failure rate or increased mortality from

prolonged aplasia that may be associated

with tumor purging.111 Numerous tech-

niques for autologous purging are

available. The goal of all purging meth-

ods— whether physical, immunologic, or

pharmacologic—is the destruction or re-

moval of the malignant clone while main-

taining the efficacy of the HPCs necessary

for engraftment.112

Pharmacologic Techniques. In-vivo

antineoplastic therapy produces greater tu-

mor cell kill because of the differential sen-

sitivity of malignant cells over normal cells.

In-vitro pharmacologic purging is an effort

to expand this therapeutic ratio. In-vitro

purgingallowsfordoseandexposurein

tensification without concern for organ tox-

icity because higher drug concentrations

can be used on isolated hematopoietic

grafts ex vivo, which then can be adminis-

tered in vivo. However, the drug concentra-

tion must be at nontoxic levels before re-

infusion. Activated oxazaphosphorines

(4-hydroperoxycyclophosphamide, mafos-

famide) were the most frequently used

compounds but are generally no longer in

use in the United States. Other chemo-

therapeutic agents have been investigated

in preclinical models but are not in clinical

use. Results from purged autologous trans-

plants for AML with either of these drugs

showed prolonged aplasia with less than

1% CFU-GM survival.112

Physical Techniques. Separation meth-

ods based on cell size and density through

gradient-generating reagents or CCE do not

achieve adequate tumor cell depletion.111

However, the combined use of physical and

immunologic techniques requires further

study.

Immunologic Techniques. The develop-

ment of MoAbs coupled with the discovery

of tumor-associated antigens opened the

field for immunologic purging.113 MoAbs

may be directed at tumor-specific antigens

or cell-differentiation antigens.111 The im-

munologic techniques differ primarily by

Chapter 25: Cell Therapy and Cellular Product Transplantation 597

the method of target cell removal. MoAbs

are used in conjunction with complement,

bound to toxins, or coupled to magnetic

beads.ThechoiceofMoAbandthehetero

geneity of antigen expression on the target

cell affect the success of the purge or the

level of depletion. Many investigators em-

ploy a cocktail of MoAbs in an effort to en-

hance the purging efficiency.114,115 Comple-

ment-mediated cytotoxicity is frequently used

if the antibody is of IgM isotype or if the ex-

pected level of antigen density is high.111

Immunomagnetic cell separation, either

by direct or indirect method, employs an

antibody-coated magnetic bead to target

the antigen or antigens of interest. A recent

study described a 5-log tumor cell deple-

tion with two cycles using the indirect

method for B-cell lymphoma. Colony assays

showed only a 20% reduction in CFU-GM

and multipotential CFU (CFU-GEMM).114

T-Cell Depletion

GVHD is a significant complication in

allogeneic HPC transplantation. Because

the disease process is mediated by donor

T cells that are reactive against the host,

depletion strategies are used to target and

remove these cells in an effort to decrease

the incidence or at least lessen the sever-

ity of GVHD. Many of the techniques out-

lined for positive selection and tumor purg-

ing are applicable to T-cell depletion (Table

25-3).17

In T-cell-depleted grafts, two major areas

of concern are graft failure and recurrent

leukemia. Most instances of graft failure

(initial or late) are caused by immunologic

rejection. Early transplants involving de-

pleted grafts in patients with CML showed

a 50% or greater relapse rate. In contrast,

patients who developed GVHD had a lower

relapse rate. Subsequent clinical and inves-

tigational data provided evidence of an im-

mune-mediated GVL effect.116

Research and developmental efforts have

focused on determining the optimal level of

T-cell depletion. Dreger et al115 reported a

comparison of T-cell depletion methods

(CAMPATH-1 plus complement, immuno-

magnetic CD34+ selection, and biotin-

avidin-mediated CD34+ selection). The

immunomagnetic method provided a 4-log

reduction in T cells vs a 3.1-log reduction

with the biotin-avidin method. MoAb treat-

ment with autologous complement yielded

a 2.1-log reduction. The challenge of mini-

mizing the severity of GVHD and maintain-

ing the GVL effect is ongoing. Additional

studies are examining the role of sub-

populations of T cells and/or cytokines in

potentiating the GVL effect.117

ABO Incompatibility

Although HLA compatibility is crucial in

the successful engraftment of myelosup-

pressed or ablated patients, ABO compat-

ibility is not. Pluripotent and very early

committed HPCs do not possess ABH anti-

gens, allowing engraftment to occur suc-

cessfully regardless of the ABO compati-

bility between the recipient and donor.

ABO incompatibility does not affect neutro-

phil or platelet engraftment, graft failure,

or rejection. However, delayed red cell

engraftment may occur after a major

ABO-incompatible transplant and delayed

hemolysis may occur after a minor ABO-

incompatible transplant where the donor

598 AABB Technical Manual

Table 25-3. Methods of T-Cell Depletion

Nonimmunologic

Counterflow centrifugal elutriation

Pharmacologic/cytotoxic drugs

Immunologic

Monoclonal antibodies with or without

complement

Immunotoxins

Immunomagnetic beads

has antibodies to the recipient’s blood

cells (Table 25-4).118

Major ABO Incompatibility

Major incompatibility occurs when the

recipient has ABO antibodies or other red

cell antibodies, such as anti-D or anti-

Kell, against the donor red cell antigens.

The HPC preparation can be processed to

remove mature red cells. The group O re-

cipient who receives a group A graft may

continue to produce anti-A and anti-B for

3 to 4 months or longer in rare instances,

and the presence of anti-A will delay

erythropoiesis by the group A graft; group

A red cells appear in the circulation when

the recipient’s anti-A disappears. Granulo-

cyte and platelet production are not

affected.119 Red cell engraftment may be

delayed to ≥40 days after the transplanta-

tion.120 Red cells used for transfusion of the

recipient must be compatible with both the

donor and the recipient. In some centers,

group O red cells are given to all major

ABO-incompatible transplant recipients

in order to avoid confusion. Recommen-

dations for optimal blood component se-

lectionareshowninTable25-5.

Minor ABO Incompatibility

Minor ABO incompatibility occurs when

the donor has antibodies against the re-

cipient red cell antigens (such as a group

O donor to a non-group O recipient). Be-

Chapter 25: Cell Therapy and Cellular Product Transplantation 599

Table 25-4. Potential Problems with ABO- and Rh-Incompatible HPC

Transplantation

Example

Incompatibility Donor Patient Potential Problems

ABO (major) Group A Group O Hemolysis of infused donor RBCs, delay

of RBC engraftment, hemolysis at the

time of donor RBC engraftment

ABO (minor) Group O Group A Hemolysis of patient RBCs from infused

donor plasma; in pediatric cases,

hemolysis of patient RBCs 7-10 days

after transplant caused by passenger

lymphocyte-derived

isohemagglutinins

Rh Negative Positive Hemolysis of patient RBCs by donor

anti-D produced after engraftment

Positive Negative

(with

anti-D)

Hemolysis of donor RBCs from newly

engrafted HPCs (rare), or delayed

RBC recovery

Other RBC

antigens

Negative Positive Hemolysis of patient RBCs by donor an-

tibodies in plasma produced after

engraftment

Positive Negative

(with

antibody)

Hemolysis of donor RBCs from newly

engrafted HPCs (rare), or delayed

RBC recovery

600 AABB Technical Manual

Table 25-5. Transfusion Support for Patients Undergoing ABO-Mismatched Allogeneic HPC Transplantation

Phase I Phase II Phase III

Recipient Donor

Mismatch

Type

All

Components RBCs

First

Choice

Platelets

Next Choice

Platelets* FFP

All

Components

A O Minor Recipient O A, AB AB; B; O A, AB Donor

B O Minor Recipient O B, AB AB; A; O B, AB Donor

AB O Minor Recipient O AB A; B; O AB Donor

AB A Minor Recipient A, O AB A; B; O AB Donor

AB B Minor Recipient B, O AB B; A; O AB Donor

O A Major Recipient O A AB; B; O A, AB Donor

O B Major Recipient O B AB; A; O B, AB Donor

O AB Major Recipient O AB A; B; O AB Donor

A AB Major Recipient A, O AB A; B; O AB Donor

B AB Major Recipient B, O AB B; A; O AB Donor

A B Minor & major Recipient O AB A; B; O AB Donor

B A Minor & major Recipient O AB B; A; O AB Donor

*Platelet concentrates should be selected in the order presented. Modified from Friedberg et al.121

Phase I = the time when the patient/recipient is prepared for HPC transplantation.

Phase II = from the initiation of myeloablative therapy until:

For RBC—DAT is negative and antidonor isohemagglutinins are no longer detectable (ie, the reverse typing is donor type).

For FFPs—recipient’s erythrocytes are no longer detectable (ie, the forward typing is consistent with donor’s ABO group).

Phase III = after the forward and reverse type of the patient are consistent with donor’s ABO group.

Beginning from Phase I, all cellular components should be irradiated and leukocyte reduced.

fore infusion of a graft from a plasma-in-

compatible donor, the plasma may be re-

moved to avoid infusion of preformed

anti-A and/or anti-B. Minor ABO-incom-

patible transplants can be characterized by

rather abrupt onset of immune hemolysis,

which begins about 7 to 10 days after trans-

plantation and may last for 2 weeks. The

direct antiglobulin test (DAT) is positive;

anti-A and/or anti-B can be recovered in

theeluateandhemoglobinemiaand/or

hemoglobinuria may occur. An additional

30% of such transplant recipients develop

a positive DAT without experiencing gross

hemolysis. This phenomenon is the result

of red cell antibodies produced by pas-

senger B lymphocytes in the stem cell

graft.121 Non-ABO antibodies from passen-

ger lymphocytes in HPC transplants have

also been reported.122 Although transient,

the hemolysis may persist for up to 2 weeks

and may require transfusion with group O

red cells. Minor ABO-mismatched HPC

transplants may be associated with a

higher risk of hemolysis, possibly caused

by greater B-cell content.123 In all cases,

plasma used for transfusion should be

compatible with both the donor and the

recipient. In some centers, group AB plasma

products are given to all minor ABO-in-

compatible transplant recipients in order

to avoid confusion. RBC transfusions, be-

ginning with the conditioning regimen,

should be of donor type. Recommenda-

tions for optimal blood component selec-

tion are shown in Table 25-5.

Chimerism

In spite of intensive pretransplant chemo-

therapy and irradiation, some of the host’s

hematopoietic cells may survive and sub-

sequently coexist with cells produced by

the transplanted HPCs. This dual cell pop-

ulation, called partial hematopoietic

chimerism, may have an effect on immu-

nologic tolerance.124 This tolerance devel-

ops in successful transplants and allows

normal immune reconstitution. Full chim-

erism (replacement of host marrow cells

entirely by donor cells) usually occurs in

successful HPC-M transplantation.

Processing in the Presence of ABO

Incompatibilities

Major ABO Incompatibility. Two ap-

proaches have been employed with major

ABO incompatibilities: 1) removal of or

decrease in the isoagglutinin level in the

recipient or 2) removal of the red cells in

the HPC-M collection. Processing is not

generally required with HPC-A because

such a small volume of red cells is usually

included.

Attempts to remove or decrease the iso-

agglutinin titer in recipients involve the use

of one or more large-volume plasma ex-

changes with or without the subsequent in-

fusion of donor-type red cells as a secondary

effort to absorb any additional isoaggluti-

nins.125,126

Other approaches to ABO-incompatible

products include red cell depletion. The

most prevalent method in use is red cell

sedimentation.

Some institutions accomplish red cell

depletion using density gradient separation

and cell washers (Gambro BCT, Lakewood,

CO); others use continuous-flow apheresis

machines such as the Spectra (Gambro) and

the Fenwal CS-3000 (Baxter Healthcare),

with mononuclear cell recoveries of 94%

and 87% and red cell depletion of 99% and

98%, respectively.

Minor ABO Incompatibility. Theoreti-

cally, the level of hemolysis is dependent on

thevolumeofHPC-Minfusedrelativeto

the recipient’s plasma volume and the IgM

titer of the donor. Incompatible plasma can

be easily removed by centrifugation.

Plasma removal using this method removes

Chapter 25: Cell Therapy and Cellular Product Transplantation 601

approximately 75% of the plasma volume

while recovering >70% of the initial nucle-

ated cell count. Plasma removal is not gen-

erally required in HPC-A because the vol-

ume of incompatible plasma is usually small.

Umbilical Cord Blood Processing

Advances in the processing of umbilical

cord blood cells have solved the early

problems of unacceptably high progeni-

tor cell losses with manipulation.127 Unlike

cryopreservation laboratories that store

relatively few autologous or allogeneic

HPC-M or HPC-A products (typically, less

than a few hundred products at any one

time), a successful cord bank would be

expected to store thousands of products

for prolonged periods. Therefore, attempts

to reduce umbilical cord blood bulk (and,

therefore, storage space, liquid nitrogen

requirements, and cost) have been ac-

tively pursued. Currently, many centers

arefollowingtheapproachusedbythe

New York Blood Center, which involves

sedimentation and volume reduction be-

fore cryopreservation.128

Cultures for Microbial Contamination

Sterility is essential in blood products for

infusion to immunosuppressed patients.

This is particularly important with HPC-M,

which is frequently collected in an open

system, and with products that require

multiple manipulations (Table 25-6).129-134

Culture growth can result from contami-

nation during collection or product pro-

cessing, or as a result of an infected cathe-

ter or the patient’s sepsis. Skin commensals

are the predominant isolates from these

cultures. In all cases it is important to

identify the source, the degree of contam-

ination, and the causative organism,

given the fact that this product is in-

tended for transplantation in an immuno-

compromised recipient. Each product

must be tested for microbial contamina-

tion at least once during the course of

processing, usually just before freezing or

infusion with allogeneic transplants.48 A

positive culture does not necessitate im-

mediate discard of the product because

these are frequently irreplaceable cells.135

Thus, positive results need to be reviewed

by the appropriate physician on a case-

by-case basis and sensitivity tests ordered

to better define the organism and to allow

for appropriate clinical management at

the time of transplant. This should also

serve as the impetus to review all proce-

dures and techniques to ensure that they

602 AABB Technical Manual

Table 25-6. Percent Microbial Contamination of Cell Therapy Products

Source of HPCs No. of Products Tested % Contaminated Reference

Marrow 291 0 129

227 1.3 130

317 6.0 131

194 <0.1 132

Peripheral blood 1380 0.65 129

560 0.7 130

576 0.5 131

1040 0.2 133

Cord blood 1000 <1.0* 134

*Initial rates were as high as 28%; however, with improved donor selection and technique, the rate fell to <1%.

provide safeguards to prevent contamina-

tion.

Stem Cell Enumeration: CD34 Analysis,

Aldehyde Dehydrogenase Activity, and Others

As stem cells differentiate into hemato-

poietic cells, CD34 surface markers ap-

pear. As the cells continue to differentiate

and acquire other surface markers, CD34

expression diminishes. Stages of differen-

tiation may be studied by the expression

or co-expression of other antigens or

properties. There are currently two assays

for CD34 that are FDA-cleared for in-vitro

diagnostic use. These CD34+ measure-

ment methods are the most widely used

for the evaluation of grafts, have the lon-

gest history of use, and show the most

clinical correlations in the marrow trans-

plantation literature.

The principle of gain and later loss of

CD34 expression has been applied to evalu-

ate the quality of HPC grafts. The current

approach is to use a method based on

multiparameter flow cytometry to deter-

mine the number of CD34+ cells and to cal-

culate a subsequent CD34+ dose based on

the recipient’s weight. Numerous flow

cytometric analysis methods for CD34 enu-

meration exist.56,136,137 Because these meth-

ods differ, correlation of CD34 dose values

from site to site is often unreliable. Multiple

studies have documented the variability of

CD34 analysis methods and results and

have examined possible causes of the vari-

ability.56,57,138,139 Becauseofthe“rareevent”

nature of CD34 enumeration, several pro-

cedural components play a critical role in

the assay. Selection of the CD34 antibody

clone, fluorescent conjugate, lysing solu-

tion, lyse-wash format, gating strategy, and

the number of events analyzed are some of

the factors that influence the end result.140

Growing awareness of, and concern

about, the need for a standardized ap-

proach to CD34 analysis have prompted

several collaborative groups such as the In-

ternational Society for Cellular Therapy

(ISCT) to propose guidelines for CD34+ cell

determination by flow cytometry.56,141 Alter-

nate approaches are available and will con-

tinue to be developed and investigated as

more is discovered about the CD34 antigen

and other markers for the pluripotent stem

cell.142

A second method for enumeration of

early cells has gained FDA clearance. The

cytosolic expression of aldehyde dehydro-

genase (ALDH) in stem cells was found to

betheprotectivepropertyofHSCsincyclo

phosphamide treatment of patients and in

preservation of stem cells in graft purging

with 4HC. Flow cytometric analysis of

ALDH-positive cells with the enzymatic ex-

citation and trapping inside the stem cells

of a fluorescent substrate (because the

fluorophore becomes charged and does not

diffuse freely) allows measurement of a via-

ble population enriched in colony-forming

progenitor cells. Both long-term repopulat-

ing and committed progenitor cells are

enriched and the ALDHbr contains the pop-

ulation responsible for hematopoietic engraft-

ment of NOD-SCID mice.143,144 This method

to enumerate ALDHbr SSclo cells identifies

only cells with intact membranes and is

well correlated with engraftment in post-

thaw HPC-A grafts.145 Another measure-

ment technique involving the use of Flk-2

and Thy 1.1lo showed that Flk-2–, Thy 1.1lo

cells represented long-term repopulating

cells, whereas Flk-2+, Thy 1.1lo cells were

short-term engraftment-enhancing cells.

These tools may be developed for use in fu-

ture graft engineering approaches to finely

separate early cell populations.146

Colony-Forming Cell Assays

Culture systems are available that can

demonstrate in-vitro proliferative capacity

Chapter 25: Cell Therapy and Cellular Product Transplantation 603

of a hematopoietic sample. It is thought

that short-term (generally within 2 weeks)

repopulating potential is produced from

the committed HPCs. Long-term repopu-

lating ability is thought to be a result of

the pluripotential HPCs that are princi-

pally necessary for a complete and sus-

tained engraftment. Cultures may be use-

ful to assess engraftment potential; however,

because media, culture techniques, and

colony identification are quite variable,

interinstitutional comparisons are diffi-

cult. Reported CFU-GM doses below

which engraftment may be delayed range

from 1.5 ×105to 5.0 ×105/kg.147

Long-term cultures are not routinely used

clinically because of the 5- to 8-week incu-

bation requirements. However, they may be

useful in evaluating developmental proce-

dures involving product manipulation.

Freezing and Storage

Although easily performed by many trans-

plant centers, HPC cryopreservation and

reinfusion are not without risk of loss of

HPCs. The infusion of cryopreserved cells

also results in risks to the recipient, many

of which are life-threatening, and some of

which are not well understood. The loss of

HPCs from cryopreservation and the ef-

fect of this loss on engraftment speed have

not been quantified. There are multiple

aspects to successful cryopreservation of

HPCs. Each of these variables affects the

recovery of HPCs during the cryopreser-

vationstepsand,aswithanymanufacturing

process, must be rigidly controlled for re-

producible results. The question that still

faces cryopreservation facilities is whether

other solutions or processing techniques

will provide better cryosurvival of cells, at

less cost, with greater simplicity, or with

less toxicity to either the recipient or to

the cell type being frozen.

HPC-A, HPC-M, and HPC-C are frozen

and stored using the same techniques. The

general parameters include cryopreservation

in dimethylsulfoxide (DMSO) and a source

of plasma protein with or without hydrox-

yethyl starch (HES), cooling at 1 to 3 C/

minute, and storage at –80 C or colder. Vari-

ations on this technique include the con-

centration at which the cells are frozen, the

amount and source of the plasma protein,

and the cooling techniques used.148 Most of

these variations probably have little effect

on the survival of the HPCs as shown by the

consistent engraftment of cryopreserved

components. However, cryopreservation

results in the loss of an undefined but po-

tentially substantial proportion of HPCs, and

delay in engraftment can occur if the com-

ponent being frozen has borderline quanti-

ties of HPCs. There is also considerable but

generally minor toxicity associated with the

infusion of cryopreserved cells that must be

considered when developing cryopreserva-

tion techniques.

Allogeneic Products

The collection of allogeneic marrow is

generally timed to coincide with the com-

pletion of the recipient’s preparative regi-

men. Because of the brief storage period

required, the product is maintained in the

liquid state. Unseparated HPC-M can be

stored in the liquid state for up to 3 days

at either 4 C or 22 C without any signifi-

cant loss in viability of either uncommit-

ted or committed progenitors. The ability

to store unmanipulated marrow under

these conditions is vital in the context of

the NMDP or other transplant registries

where unrelated HPC-M units are col-

lected and then transported great dis-

tances for transplantation. Similarly,

HPC-A units may be stored in the liquid

stateunderthesameconditionsasHPC-M

for up to 3 days at 4 C.149,150 The cell con-

604 AABB Technical Manual

centration is important in determining

the storage time and temperature that the

cells will tolerate without damage. High

concentrations of white cells in liquid

storage at room temperature quickly ex-

pend the supplies of oxygen and glucose

and cause a decrease in pH in the storage

medium that is toxic to the cells. A lower

concentration of cells (higher volume of

suspending medium, often autologous

plasma) and refrigerated storage are pro-

tective.

Autologous Products

Because of the length of time required for

treatment and/or HPC product collection,

autologous products are usually cryopre-

servedandstoreduntilthetimeofinfu-

sion. Cryopreservation also allows HPCs to

be collected while the patient is in remis-

sion and stored for use in case a relapse

occurs (prophylactic storage). At present,

no expiration date has been defined for

these products; however, HPC-M stored

for 11 years has been used for transplanta-

tion, with sustained engraftment.151

Computer-Controlled Cryopreservation

The purpose of cryopreservation is to freeze

cells in such a way as to allow for their

long-term storage with a minimal loss of

cell viability or reconstitutive ability upon

thaw. The main obstacle to maintaining

viability during cryopreservation and

storage is the formation of intracellular

ice crystals along with an increase in ex-

ternal osmolarity, causing exit of water from

the cell, both resulting in cell lysis. Ther-

mal shock, the time required for phase

change (liquid state to solid state), and

the posttransition freezing rate also pres-

ent specific conditions to be managed for

cryopreservation.152

The adverse effects caused by the forma-

tion of intracellular ice crystals or by cell

dehydration can be minimized by a slow

cooling rate and the addition of a cryo-

protective agent such as DMSO. Cryo-

protectants such as DMSO prevent the for-

mation of large ice crystals within the cells

by penetrating the cell and providing some

balance of the external hyperosmolar sol-

ute conditions in the freezing medium sur-

rounding the cell.153 A commonly used

cryoprotectant consists of 20% DMSO and

20% plasma or albumin prepared in a buf-

fered electrolyte solution or tissue culture

media. The plasma or albumin provides a

proteinsource,whichaidsinpreventingcell

damage during freezing and thawing. The

cryoprotectant is combined with an equal

volume of product immediately before

freezing, resulting in a final cell suspension

containing 10% DMSO and 10% plasma.

Phase transition time is a physical phe-

nomenon of freezing water where forming

ice crystals release heat energy into the cell

solution that needs to be offset by contin-

ued cooling to prevent crystallization of ice,

thawing, and recrystallization during this

energy exchange, with damage to the cell

membrane.152 This method of freezing re-

quires physical conditions that support this

process. This may be accomplished by a

computerized programmable freezing

chamber that adds liquid nitrogen in suffi-

cient amounts to push the freezing process

through the phase change smoothly. The

freezer is programmed to cool cells at an

optimal rate of 1 to 3 C/minute until a tem-

perature of –90 to –100 C is reached.154-156 With

the combination of a cryoprotectant and a

programmed rate of freezing, cryopreserva-

tion and long-term storage of HPCs are

possible, with minimal damage to the cells.

Passive Controlled-Rate Freezing

Mechanical, methanol bath immersion,

or “passive controlled-rate” freezing is ad-

vocated by some investigators as a simple,

Chapter 25: Cell Therapy and Cellular Product Transplantation 605

reliable, cost-effective, and validated

method of managing the physical condi-

tions of cooling as an alternative to a con-

trolled-rate device.157-159 The principle is

that products can be cooled without the

aid of a programmable freezer or liquid

nitrogen if the conditions of freezing are

predetermined, measured, and performed

according to standard operating proce-

dures. The products are stored in a –80 C

mechanical freezer or colder after the ini-

tial freeze. HPCs stored in this fashion

have successfully engrafted after as long

as 2 years of storage.159 In some cases, the

combination of the metal canisters the

bags are placed in, the bag itself, and the

volume of product frozen produces a

freezing rate of approximately 3 C/min-

ute, which falls within the optimal range

previously discussed.160 The major deter-

rent to use of mechanical freezers is fear

of mechanical failure and the lack of data

on long-term storage and engraftment.

Frozen Storage

Although products frozen in a mechanical

freezer are stored at –80 to –150 C, prod-

ucts cryopreserved using a programmable

freezer generally are stored in a liquid ni-

trogen freezer. The storage temperature

achieved with vapor phase, although not

as cold as liquid phase, averages –140 C, a

temperature that has been shown to allow

for viable long-term marrow storage.151

The major drawbacks to vapor phase stor-

age include the potential for large fluctua-

tion in temperature when the freezer is

entered as well as the variation in storage

temperature throughout the freezer itself,

dependent on the design of the storage

chamber and the procedure for entering

and retrieving units. The time to rising

temperatures of the stored products in

cases of electrical or liquid nitrogen sup-

ply emergencies is significantly shorter

than with liquid phase. The advantage of

vapor phase storage is the possibly de-

creased risk of cross-contamination that

has become an issue with liquid phase

storage.161

Storage of Untested or Infectious Products

Regulations [21 CFR 1271.60(a) and

1271.65(a)] and standards48(p35),49 require al-

ternative storage for products that are un-

tested or have a positive disease marker

test result. Some institutions comply by

storing all products in vapor phase and

physical separation by category of prod-

uct. Other institutions use an overwrap,

placing the component in an outer plastic

bag that is sealed before storage. A third

alternative is a separate storage compart-

ment for units that are untested or posi-

tive for infectious disease markers.

Final Suitability Criteria

Before each autologous or allogeneic unit

is released, it must undergo review and

meet the predetermined release criteria

described in the facility’s quality plan.

The pertinent facts describing donor se-

lection, product collection, processing,

and storage must be reviewed. The con-

tainer label and associated information

must accurately reflect the product classi-

fication, storage/preservative medium in

the final container, the product content

(usually cell content), and results of re-

lease testing including infectious disease

tests. If exceptions to standard practice

were made, they must be explained either

on the label or in the accompanying re-

lease material.

Transportation and Shipping

In some cases, a hematopoietic component

must be transported from one center to

another. The product must be positively

606 AABB Technical Manual

identified upon its removal from inven-

tory in preparation for shipping. In all

cases, precautions should be taken to pro-

tect the component from rough handling,

out-of-range temperatures, X-ray exami-

nation, breakage, and spillage. The ship-

ping container must undergo quality con-

trol to ensure that it is capable of holding

the expected temperature during ship-

ping. In the case of cryopreserved compo-

nents, the use of a liquid nitrogen “dry

shipper” is desirable. Such “dry shippers”

have liquid nitrogen absorbent material

between the walls of the container that al-

lows the inside of the container to main-

tain temperatures in the range of –180 C

forupto10to14daysiftheyareproperly

filled with liquid nitrogen and shipped in

the upright position. Tipping or inversion

of the container during shipping permits

the liquid nitrogen to drain out, allowing

the container to warm toward ambient

temperature.

Thawing and Infusion

For all components, final identification is

done by the nurse or physician perform-

ing the infusion. Flow through the central

venous catheter is confirmed and the cells

are infused by gravity drip, calibrated

pump, or manual push with or without an

in-line filter (a standard 170-micron red

cell infusion filter is acceptable). Although

DMSO was thought to be toxic to HPCs, it

is now known to be nontoxic after short-

term exposure (up to 1 hour) at the con-

centration used for cryopreservation of

HPCs.162 However, prolonged exposure to

DMSO ex vivo at 22 to 37 C may be harm-

ful to HPCs. To minimize the exposure of

thawed cells to DMSO, many centers rap-

idly thaw one bag at a time near the bed-

side. Some centers place product bags in

secondary containment bags before thaw-

ing; others immerse the bag (all but the

access ports) directly into sterile water or

salineat37to40C.

152 The bag is kneaded

gently until all solid clumps have thawed.

The cells are then infused (usually 10-15

mL per minute). Products may also be

washed and resuspended in the laboratory

before infusion to prevent cell aggregation.152

Side effects associated with infusions in-

clude nausea, diarrhea, flushing, brady-

cardia, hypertension, and abdominal pain.

In general, such side effects may justify

slowing, but not halting, the infusion until

the symptoms pass. A limit of 1 gram of

DMSO per kilogram of body weight of the

patient at one infusion is recommended to

allow the patient to tolerate both the DMSO

and the volume infusion effects of the ad-

ministration. Sudden and severe hypoten-

sion can occur in the absence of adequate

antihistamine premedication. The patient

should receive fluids and treatment to en-

sure that the urine is “alkalinized.” This fa-

cilitates the clearance of hemoglobin caused

by red cell lysis, which occurs during freez-

ing and reduces the risk of renal complica-

tions. If the total infusion volume exceeds

10 mL/kg of recipient body weight, many

centers divide the volume over a morning

and an afternoon infusion or over 2 consec-

utive days.

It is best to collect postthaw laboratory

samples directly from the patient’s infusion

bags instead of freezing separate individual

specimens because these samples are iden-

tical to the infused product.163

Evaluation and Quality Control

of Hematopoietic Products

Cell Counts

Each hematopoietic product is analyzed

to determine the total cell concentration

and the mononuclear cell concentration,

Chapter 25: Cell Therapy and Cellular Product Transplantation 607

which are used to calculate the number of

cells per kilogram (of the recipient) or cell

dose for each product.48(p55),49 These doses,

in combination with other assays, deter-

mine the number of collections necessary

to achieve engraftment.135 In addition,

they are used to calculate the percent re-

covery, providing a quality control mea-

sure for processing procedures and

equipment.164

In general, automated cell counts provide

the most rapid and accurate value. How-

ever, platelet/cellular aggregates in HPC-A

or fat globules in HPC-M specimens can

falsely decrease or increase cell counts.135,165

In such cases, manual cell counts may be

preferable. It is important that cell counts

are not overestimated because this may re-

sult in inaccurate estimation of time to

engraftment or graft failure.

Engraftment Data

Ultimately, engraftment of neutrophils,

platelets, and red cells is the primary de-

terminant of graft quality. Monitoring and

documenting days to engraftment for

neutrophil and platelet lineage are re-

quired by FACT49 and AABB48(p41) for ac-

creditation.

Tumor Cell Detection

Tumor cell detection techniques have been

developed to screen products suspected

of tumor cell contamination and to evalu-

ate purged products. The majority of these

assays use MoAbs that specifically bind

tumor antigens. Detection and quantita-

tioncanbedonebyflowcytometry,immuno

fluorescence, or immunohistochemical

staining. Sensitivity varies with technique

from 0.1% to 0.0004% of cells examined.166

Preliminary studies indicate that the pres-

ence of tumor cells may be associated

with a reduced disease-free survival.167

Regulations

In 1997, the FDA announced a new com-

prehensive approach to the regulation of

cellular and tissue-based products.168

HPCs from placental/umbilical cord

blood and peripheral blood were covered

by this proposal. Many of the policies,

proposed regulations, and guidance doc-

uments needed to implement this ap-

proach have been published.47,169-173

These regulations require establishment

registration and listing of facilities collect-

ing, processing, or distributing tissue or cell

therapy products. Although the regulations

are similar to those for blood donor qualifi-

cation, there are differences appropriate to

the specific tissue source under consider-

ation. The cGTP regulations (found in Title

21 CFR 1271.145 to 320) are specific in-

structions intended to ensure that facilities

establish and maintain a quality program

that documents that personnel, proce-

dures, facilities, equipment, supplies, and

reagents are set up and maintained in an

acceptable and a standard manner. Process

control is required: there must be written

procedures and documented validation.

Products are required to be collected,

tested, labeled, and stored in ways that pre-

serve their identity and prevent contamina-

tion and cross-contamination. It may be

necessary to demonstrate regulatory com-

pliance during FDA inspection visits. Re-

porting of adverse events (when applicable)

is also required within 15 days of receipt of

the information if the event is serious as de-

fined in 21 CFR 1271.350. This body of reg-

ulations represents a major effort on the part

of the FDA to ensure that tissue and cell

therapy products are safe for the recipient.

608 AABB Technical Manual

Standards

The AABB and FACT have published sepa-

rate, but substantially similar, standards

for HPCs.48,49 The AABB Standards for Cel-

lular Therapy Product Services addresses

the collection, processing, storage, and

distribution of HPCs. Other organizations

such as the NMDP also publish voluntary

standards. FACT standards address clini-

cal issues provided by HPC clinical trans-

plant programs as well as laboratory ser-

vices performing the collection, processing,

storage, and distribution of HPCs. Both

AABB and FACT provide an inspection

and certification program for requesting

members. These program reviews are an

important, external, and impartial look at

program organization and how the pro-

gram’s organization affects patient out-

come. As an outgrowth of the standards

writing process, representatives from the

constituent organizations of AABB and

FACT cooperated to produce a uniformly

endorsed Circular of Information for the

Use of Cellular Therapy Products.174 In this

circular, the organizations agreed upon a

number of principles, including a com-

mon set of names for cell therapy prod-

ucts. This agreement on product names

allowed the International Committee on

Commonality of Blood Bank Automation’s

(ICCBBA) North American Task Force to

approve progenitor cell label designs, in-

cluding ICCBBA’s quality design require-

ments.

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Marrow Transplant 1998;21:511-7.

158. Galmes A, Besalduch J, Bargay J, et al. Cryo-

preservation of hematopoietic progenitor

cells with 5-percent dimethyl sulfoxide at

–80 degrees C without rate-controlled freez-

ing. Transfusion 1996;36:794-7.

159. Stiff PJ, Murgo AJ, Zaroules CG, et al. A sim-

plified bone marrow cryopreservation method.

Blood 1988;71:1102-3.

160. Stiff PJ, Murgo AJ, Zaroules CG, et al. Un-

fractionated marrow cell cryopreservation

using dimethylsulfoxide and hydroxyethyl

starch. Cryobiology 1983;21:17-21.

161. Tedder RS, Zuckerman MA, Goldstone AH, et

al. Hepatitis B transmission from contami-

Chapter 25: Cell Therapy and Cellular Product Transplantation 615

nated cryopreservation tank. Lancet 1995;

346:137-40.

162. Rowley SD, Anderson GL. Effect of DMSO ex-

posure without cryopreservation on hemato-

poietic progenitor cells. Bone Marrow Trans-

plant 1993;11:389-93.

163. Gee AP. Quality control in bone marrow pro-

cessing. In: Gee AP, ed. Bone marrow process-

ing and purging: A practical guide. Boca

Raton, FL: CRC Press, 1991:19-27.

164. Lasky LC, Johnson NL. Quality assurance in

marrow processing. In: Areman EM, Deeg HJ,

SacherRA,eds.Bonemarrowandstemcell

processing: A manual of current techniques.

Philadelphia: FA Davis, 1992:386-443.

165. Bentley SA, Taylor MA, Killian DE, et al. Cor-

rection of bone marrow nucleated cell counts

for the presence of fat particles. Am J Clin

Pathol 1995;140:60-4.

166. Moss TJ. Detection of metastatic tumor cells

in bone marrow. In: Gee AP, ed. Bone marrow

processing and purging: A practical guide.

Boca Raton, FL: CRC Press, 1991:121-35.

167. Pecora AL, Lazarus EM, Cooper B, et al.

Breast cancer contamination in peripheral

blood cell (PBPC) collections association

with bone marrow disease and type of mobi-

lization (abstract). Blood 1997;89(Suppl):99a.

168. Food and Drug Administration. A proposed

approach to the regulation of cellular and tis-

sue-based products (March 4, 1997). Fed

Regist 1997;62:9721-2.

169. Food and Drug Administration. Human tissue

intended for transplantation; final rule, 21

CFR 16 and 1270 (July 29, 1997). Fed Regist

1997;62:40429-47.

170. Food and Drug Administration. Guidance for

industry: Screening and testing of donors of

human tissue intended for transplantation.

(July 29, 1997) Rockville, MD: CBER Office of

Communication, Training, and Manufactur-

ers Assistance, 1997.

171. Food and Drug Administration. Draft guid-

ance for industry: Preventive measures to re-

duce possible risk of transmission of Creutz-

feldt-Jakob disease (CJD) and variant

Creutzfeldt-Jakob disease (vCJD) by human

cells, tissues, and cellular and tissue-based

products (HCT/Ps) (June 25, 2002). Fed

Regist 2002;67:42789-90.

172. Food and Drug Administration. Request for

proposed standards for unrelated allogeneic

peripheral and placental/umbilical cord

blood hematopoietic stem/progenitor cell

products; request for comments (January 20,

1998). Fed Regist 1998;63:2985-8.

173. Food and Drug Administration. Current good

tissue practice for human cell, tissue, and

cellular and tissue-based product establish-

ments; inspection and enforcement (Novem-

ber 24, 2004). Fed Regist 2004;69:68612-88.

174. AABB, American Red Cross, America’s Blood

Centers, American Society for Blood and

Marrow Transplantation, Foundation for the

Accreditation of Cellular Therapy, Interna-

tional Society for Cellular Therapy, National

MarrowDonorProgram.Circularofinforma-

tion for the use of cellular therapy products.

Bethesda, MD: AABB, 2003.

616 AABB Technical Manual

Chapter 26: Tissue and Organ Transplantation

Chapter 26

Tissue and Organ

Transplantation

IN RECENT YEARS, the numbers of

cornea, bone, skin, heart valve, and other

tissue donations and transplants1-3 have

exceeded those of solid organs,4such as

kidneys, livers, hearts, and lungs. The

hospital blood bank, transfusion service,

community blood center, and regional

blood center are uniquely qualified to

provide essential support for organ and

tissue transplantation and to serve as tis-

sue banks (Table 26-1).5It is common for

hospital blood banks to provide transfu-

sion support for organ and tissue recipi-

ents and, in some cases, to store, keep records

of, and dispense tissue for allografts. AABB

Standards for Blood Banks and Transfu-

sion Services6(pp8,14,15,60,79) addresses the re-

ceipt, storage, transportation, and records

of tissue allografts. Additional guidance for

the collection and preparation of tissue

and organ allografts is available from fed-

eral and state regulations, Public Health

Service Guidelines, and the standards,

guidelines, and technical manuals of other

national or local organizations.7-14

Transplant-Transmitted

Diseases and Preventive

Measures

The widened availability of tissue and or-

gan grafts has encouraged new clinical

uses and highlighted not only their effec-

tiveness and advantages but also their

drawbacks, side effects, and complications.

Organs and tissues can transmit bacterial,

fungal, viral, and prion diseases from the

donor to the recipient3,15-26 (Table 26-2),

but careful donor screening and testing,

along with disinfection and sterilization

steps for specific tissues, can markedly re-

duce the risk.

617

Risk Reduction for Tissues

Crucial to the safety of transplanted tissue

is an evaluation of the potential donor’s

eligibility. Listed below are the questions,

examinations, and tests undertaken to en-

sure that material from the potential donor

poses a low threat of disease transmission.

Additional measures apply to reproductive

tissue donors.10,11

1. Review of health history, through in-

terviews with next of kin and/or sig-

nificant other, and possibly health-

care provider, and review of medical

records. Review includes an evalua-

tion (although not necessarily rejec-

tion for asterisked items*) of the po-

tential donor for:

a. History of infection,* malignant

disease,* or neurodegenerative

disease

b. History of autoimmune processes*

c. History of exposure to hormone

derived from human pituitary

gland or dura mater transplant

618 AABB Technical Manual

Table 26-1. Skills and Experience Appropriate for Institutions Undertaking

Tissue Banking

■Community support

■Public accountability

■Public education with a broad-based public information system

■Donor recruitment

■Counseling

■Medical overview

■Donor selection

■Donor testing including automated virology testing to avoid transcription errors

■Cellular cryopreservation

■Temperature-controlled and monitored storage

■Regulatory compliance

■Transportation infrastructure

■Financial relations with hospitals

■Computerized inventory control

■Record-keeping

■Logistics management

■Investigation of adverse reactions

■Peer review of medical, scientific, and operational practice

■Recipient matching

■Concern over the balance of the adequacy and safety of supply

■Reputation for dependable service

■Commitment to research and development

■24-hours-per-day, 7-days-per-week operation

Reproduced with permission from Warwick et al.5

Chapter 26: Tissue and Organ Transplantation 619

Table 26-2. Infectious Diseases Reported to Have Been Transmitted by Organ and

Tissue Allografts15-26

Allograft Infectious Disease/Disease Agent

Bone HIV-1

Hepatitis C

Hepatitis, unspecified type

Bacteria

Tuberculosis

Cornea Hepatitis B

Creutzfeldt-Jakob disease

Rabies

Cytomegalovirus (?)

Bacteria

Fungus

Dura mater Creutzfeldt-Jakob disease

Heart valve Hepatitis B

Tuberculosis

Skin Bacteria

Cytomegalovirus (?)

HIV-1 (?)

Pericardium Creutzfeldt-Jakob disease

Bacteria

Solid organ (eg, kidney, liver,

heart)

HIV-1

Hepatitis B

Hepatitis C

Cytomegalovirus

Epstein-Barr virus

Parvovirus

Toxoplasmosis

Chagas’ disease

Malaria

Bacteria

Tuberculosis

HHV-8

Strongyloidiasis

Sarcoidosis

West Nile virus

Rabies

Lymphocytic choriomeningitis

(cont’d)

2. Review for evidence of high-risk be-

havior (exclusion for any of the do-

nor-deferral criteria included in items

9c, 9e, and 10 of Standard 5.4.1A of

Standards for Blood Banks and Trans-

fusion Services.6(pp62-65)

3. Serologic testing on suitable blood

specimens10 (see below for more de-

tail).

a. Hepatitis B surface antigen

(HBsAg)

b. Antibodies to human immuno-

deficiency viruses 1 and 2 (anti-

HIV-1 and -2)

c. Antibody to hepatitis C virus

(anti-HCV)

d. Antibody to human T-cell lym-

photropic virus types I and II

(anti-HTLV-I and -II)

e. Serologic test for syphilis

4. Physical examination to detect:

a. Evidence of intravenous drug use

b. Jaundice

c. External signs of infection

d. Signs of AIDS

5. Review of results of autopsy exami-

nation, if performed.

Consent and Donor Eligibility

Written consent for clinical use of any tis-

sue or organ must be obtained from a liv-

ing donor or from the next of kin of a de-

ceased donor (formerly referred to as a

cadaveric donor), except when corneas are

procured under statutory consent. State

and federal referral statutes and regula-

tions exist, mandating that hospitals 1)

maintain policies for notifying organ pro-

curement organizations and appropriate

620 AABB Technical Manual

Table 26-2. Infectious Diseases Reported to Have Been Transmitted by Organ and

Tissue Allografts15-26 (cont'd)

Allograft Infectious Disease/Disease Agent

Pancreatic islet Bacteria

Semen Hepatitis B

Hepatitis C (?)

Gonorrhea

Syphilis (?)

HIV-1

HTLV-I (?)

Human papilloma virus (?)

Trichomonas vaginalis

Chlamydia trachomatis

Cytomegalovirus (?)

Ureaplasma urealyticum

HSV-2

Mycoplasma hominis

Group B streptococcus

tissue banks, including eye banks, when

death of a patient has occurred or is im-

minent and 2) designate requestors to ap-

proach families about organ and tissue

donation. All applicable federal, state, and

local laws concerning the consent of next

of kin must be obeyed. When the next of

kin signs consent for tissue donation, he

or she should specify which tissues may

be donated and whether the permission

includes tissue to be used for research or

other specific uses.

Living donors and the families of de-

ceased donors are not responsible for ex-

penses involved in recovering and process-

ing donated tissues and organs. Donors do

not receive compensation for the donation,

although donors of reproductive tissue are

often compensated for their time, risk, and

inconvenience, and donor families may re-

ceive incentives, such as a contribution to-

ward funeral expenses, for donation from

deceased donors. Tissues can be recovered

up to 24 hours after death if the body is re-

frigerated within 12 hours of death. Tissues

can be recovered up to 15 hours after death

if not refrigerated. Organs must be recov-

ered from a donor whose neurologic death

has been declared and whose circulation

has been maintained (see Table 26-3). Eyes

as a source of corneal allografts should be

removed as soon as possible after death to

ensure viability of endothelial cells.

Each prospective deceased donor must

be evaluated against eligibility criteria for

the specific tissue(s) and organ(s) to be col-

lected; eg, deceased newborns are not suit-

able for bone donation because of their

cartilaginous skeletal structure but may be

candidates for heart valve donation. Al-

though exceptions may be made in specific

cases, the medical eligibility of a donor is

determined on the basis of absence of in-

fection and malignancy as revealed by the

medical history, physical examination, lab-

oratory tests, and autopsy, if performed.

Donation of organs and tissues does not or-

dinarily cause delays in funerals or prevent

family viewing of the body.

Serologic Testing

Federal regulations require that donors of

tissues be tested for HBsAg, anti-HIV-1/2,

and anti-HCV, and given a syphilis screen-

ing test, with tests licensed by the Food

and Drug Administration (FDA). Testing

must be performed by a laboratory that is

certified under the provisions of the Clini-

cal Laboratory Improvement Amendments

of 19887,8 or that has met equivalent re-

quirements as determined by the Centers

for Medicare and Medicaid Services. A

screening test approved for cadaveric

specimens must be used when available.27

National standard-setting organizations,

such as the American Association of Tis-

sue Banks (AATB),10 Eye Bank Association

of America (EBAA),12 and the United Net-

work for Organ Sharing (UNOS),13 may re-

quire additional tests for infectious dis-

ease markers. The American Society for

Reproductive Medicine also has issued

recommendations for gamete and embryo

donation.11 For anonymous semen do-

nors, tests for anti-HIV-1/2 and anti-HCV

must be repeated on a sample obtained 6

months after donation and the results

found negative before semen can be re-

leased for use.9,10 Testing for anti-HBc

must also be performed on this sample to

rule out hepatitis B infection at the time

of collection. ABO typing is required for

organ grafts. HLA typing is essential for

organ grafts and hematopoietic progeni-

tor cells13 but not for tissues.28 Other tests,

such as antibody to cytomegalovirus (anti-

CMV), are also generally performed on or-

gan donors. For deceased donors, tests are

optimally performed on a blood sample

obtained before administration of transfu-

sions or fluids; these patients may have re-

Chapter 26: Tissue and Organ Transplantation 621

ceived large volumes of replacement

fluids shortly before death, and the conse-

quent plasma dilution may cause false-

negative results.19,20

If donor serum collected before blood

transfusion or intravenous fluid adminis-

tration is not available from other sources, a

pretransfusion sample is often available

from the blood bank because blood banks

hold specimens collected for compatibility

for at least 7 days. For samples obtained af-

ter transfusion or infusion of intravenous

fluids, algorithms for determining the suit-

ability of a donor sample are available.7,8 For

example, the sample is not suitable for in-

fectious disease marker testing if either one

of the following situations exists:

1. The total volume of colloid (plasma,

dextran, platelets, or hetastarch) trans-

fused in 48 hours plus the total vol-

ume of crystalloid infused in the

hour before the sample is obtained

exceed the patient’s plasma volume.

2. The sum of the volume of blood

transfused (RBCs, whole blood) and

colloid transfused in 48 hours plus

the total volume of crystalloid infused

in the hour before the sample is ob-

tained exceed the patient’s blood

volume.

Testing of cadaveric blood specimens can

be complicated by postmortem hemolysis,

which can cause misleading test results (eg,

false-positive HBsAg).15,29 Many tests li-

622 AABB Technical Manual

Table 26-3. Kinds of Donors Providing Organs and Tissue for Transplantation

Living Donor

Deceased Donor

(Neurologic Death)

Deceased Donor

(Cardiorespiratory and

Neurologic Death)

Amniotic membrane

Bone

Fetal tissues (mother is the

donor)

Foreskin

Kidney

Liver

Lung

Marrow

Milk

Pancreas

Parathyroid (frequently

autologous)

Peripheral blood progenitor

cells

Reproductive tissue

Umbilical cord blood

Umbilical vein

Heart

Kidney

Liver

Lung

Pancreas (with or without

small intestine)

Bone

Cartilage

Cornea

Dura mater

Fascia lata

Heart valve

Marrow

Pericardium

Skin

Tendon

Vascular tissue

censed for use on blood donor specimens

are not licensed for use with postmortem

specimens, but licensed assays for post-

mortem specimens are available for anti-

HIV-1/2, HBsAg, and HIV-1/HCV nucleic

acid.27

Bone Banking

Except for blood cells, bone is the most

commonly transplanted tissue1or organ.

When bone grafting is needed, fresh auto-

logous bone, usually removed from the

iliac crest during surgery, is generally con-

sidered the most effective graft material.

As with blood, the use of autologous bone

for graft material is not risk free and there

may be morbidity and infectious compli-

cations. The quantity of bone graft needed

for some surgical procedures may make

the use of autologous bone impractical.

Allografts are used for these patients and

for patients in whom the prolongation of

surgery, extra bleeding, and potential

complications of autograft collection are

considered undesirable. Bone allografts

have achieved widespread clinical appli-

cation for acetabular and proximal femur

support in revisions of failed hip prosthe-

ses; packing of benign bone cysts; spinal

fusion to treat disc disease or scoliosis; re-

construction of maxillo-facial defects; and

correction of healed fractures. Demineral-

ized bone powder is commonly used by

periodontal surgeons to restore alveolar

bone in periodontal pockets.

The surgeon today has access to a wide

choice of processed bone allografts: freeze-

dried or frozen, cancellous or cortical, with

or without treatment with sterilants. Com-

mon preparations include frozen or freeze-

dried cancellous cubes or chips, cortical

struts, and cortical-cancellous blocks and

dowels. Bone can be stored frozen or, if

freeze-dried to a low residual moisture con-

tent (6% or less by gravimetric analysis or

8% by nuclear magnetic resonance spec-

trometry), at room temperature for 5 years

(Table 26-4). Frozen bone is processed

aseptically, some without a microbial inac-

tivation step. Such tissue carries a greater

risk of bacterial contamination. A series of

infection cases, including one death, linked

to aseptically processed musculoskeletal allo-

grafts underscores the importance of recov-

ery cultures of donated tissue.24 Freeze-

dried tissue has undergone extensive pro-

cessing to remove blood and marrow and

may have been exposed to alcohol. Thus,

the risk of disease transmission is reduced.

Most bone allografts used in the United

States are freeze-dried to simplify storage.

Some grafts, whether frozen or freeze-dried,

may be treated with gamma irradiation or

ethylene oxide to reduce the risk of infec-

tious disease transmission. Demineraliza-

tion of bone is believed to make its proteins

and growth factors more readily available,

thereby enhancing its capacity to promote

healing and bone formation.

The implantation of frozen bone allo-

grafts has stimulated blood group antibod-

ies that have been implicated in hemolytic

disease of the newborn. Frozen, unpro-

cessed bone allografts contain sufficient red

cells to stimulate production of Rh and

other red cell antibodies. When indicated,

the risk of blood group sensitization can be

avoided by using processed frozen or freeze-

dried bone allograft, both of which are de-

void of blood cells and marrow. With these

grafts, matching blood groups of donors

and recipients is not necessary. When using

unprocessedfrozenboneinRh-negative

females with childbearing potential, it is

advisable to use bone from Rh-negative do-

nors to prevent alloimmunization or ad-

minister Rh immune globulin prophylacti-

cally. There is no evidence to date that ABO

or Rh incompatibility between the bone

Chapter 26: Tissue and Organ Transplantation 623

624 AABB Technical Manual

Table 26-4. Recommended Preservation Conditions and Dating Periods for

Human Tissue and Organs

Storage Condition Dating Period

Tissue

Bone –40 C 5 years*

–20 C 6 months

1-10 C 5 days

Liquid nitrogen Not defined

Lyophilized, room tempera-

ture

5 years*

Tendon –40 C 5 years*

Fascia lata Lyophilized, room tempera-

ture

5 years*

–40 C 5 years*

Articular cartilage –40 C 5 years*

1-10 C 5 days

Liquid nitrogen, immersed Not defined

Skin 1-10 C 14 days

–40 C Not defined

Lyophilized, room tempera-

ture

Not defined

Cornea 2-6 C 14 days

Hematopoietic progenitor

cells

Liquid nitrogen, immersed Not defined

Liquid nitrogen, vapor phase Not defined

Semen Liquid nitrogen, immersed Not defined

Liquid nitrogen, vapor phase Not defined

Heart valve, vein, artery –100 C Not defined

Dura mater Lyophilized, room tempera-

ture

Not defined

Organ

Kidney Refrigerated 48-72 hours

Liver Refrigerated 8-24 hours

Heart Refrigerated 3-5 hours

Heart-lung Refrigerated 3-5 hours

Pancreas Refrigerated 12-24 hours

*Unless a longer dating period has been validated by the processor

donor and recipient has an adverse effect

on the success of the bone graft.30

Skin Banking

A human skin allograft is the dressing of

choice for deep burn wounds if sufficient

amounts of skin for autografting are un-

available. A skin allograft provides tempo-

rary coverage; speeds reepithelialization;

acts as a metabolic barrier against loss of

water, electrolytes, protein, and heat; and

provides a physical barrier to bacterial in-

fection. Skin allografts are replaced peri-

odically until sufficient autograft skin can

be obtained. A skin allograft may also be

used for donor sites for pedicle flaps and

skin autografts, and for traumatically de-

nuded areas or unhealed areas of chronic

injury, such as decubitus ulcers.

Following preparation, skin donation in-

volves removing a layer of skin approxi-

mately 0.015 inch thick. After collection, re-

frigerated skin can be stored at 1 to 10 C for

up to 14 days. For refrigerated storage, stan-

dard tissue-culture nutrient media are

used, with added antibiotics. Skin can be

frozen soon after collection, usually with

10% to 15% glycerol, although dimethyl

sulfoxide (DMSO) is an acceptable alterna-

tive.31 Skin is often cryopreserved on fine-

mesh gauze in flat cryopreservation bags.

Cryogenic damage is minimized by con-

trolled-rate freezing at about 1 C per min-

ute, or by freezing, using a validated heat

sinking method, followed by storage in liq-

uid nitrogen or in a mechanical freezer at a

temperature colder than –40 C. Alternatively,

skin placed in aluminum plates inside in-

sulated boxes can be placed directly into a

–40 C mechanical freezer. This simple pro-

cess also provides a slow, predictable freez-

ing rate and maintains cellular viability. The

optimal freezing procedure and the maxi-

mal storage period that maintain viability

and structural integrity in the frozen state

have not been determined. Skin should be

transported to the operating room on wet

ice if stored at 4 C, or on dry ice if cryo-

preserved. A variety of bioengineered skin

substitutes, such as cultured keratinocyte

allografts and autografts, are available for

treatment of acute and chronic wounds.

Keratinocytes extracted from foreskin can

be seeded onto biodegradable platforms to

create bioengineered tissue products used

in wound healing or as cadaveric skin sub-

stitutes for burns.32

Heart Valves

Human heart valve allografts provide long-

term function for valve replacement—su-

perior to that of mechanical or porcine

valves. Recipients of human heart valve

allografts do not require anticoagulation

and the incidence of thromboembolism is

low. These allografts may rarely transmit

bacterial or fungal infection. They are the

graft of choice for children, to avoid long-

term anticoagulation; for pregnant women,

to avoid teratogenic risks of anticoagulants;

and for patients with infection at the aor-

tic root. Despite their advantages, wide-

spread use of human valve allografts has

been slow because implantation is techni-

cally difficult and appropriate size valve

allografts are not always readily available.

To obtain allograft valves, hearts are

aseptically collected in an operating room,

autopsy room, or morgue. Subsequently, in

thetissuebank,thepulmonicandaortic

valves are dissected out, cryopreserved with

DMSO, and stored in liquid nitrogen. Com-

pared with valves stored at 5 C in antibiot-

ics and culture medium, cryopreserved heart

valves are associated with increased cell vi-

ability; reduced incidence of valve degener-

ation, rupture, and leaflet perforation; and

reduced occurrence of valve-related death.33

Chapter 26: Tissue and Organ Transplantation 625

Records of Stored Tissue

Allografts

In two cases in which HIV and HCV were

transmitted (through unprocessed frozen

bone or organs and tendon allografts, re-

spectively) from two deceased donors to

multiple recipients,22,23 investigations re-

vealed that several hospitals had insuffi-

cient records to identify recipients of other

tissue from the same infected donors. Vol-

untary standards of national professional

associations and government regulations

requiretissuebankstohavearecord-

keeping system that identifies the donor

and allows tracking of any tissue from the

donor (or supplier source) to the con-

signee.6(pp12,47,79),10,14,34 Records must show the

source facility, the identification number

of the donor or lot, storage temperatures,

and final disposition of each tissue. These

records must be retained at least 10 years

beyond the distribution date, transplanta-

tion date, disposition date, or expiration

date, whichever is latest. Donor eligibility

records for dura mater must be retained

indefinitely. Hospitals should also have

records that identify recipients who re-

ceived tissue from a specific donor or tis-

sue lot. Hospitals should have procedures

in place to recognize adverse outcomes of

tissue use and to report them to the tissue

bank supplying the tissue.

FDA Regulation of Tissue

The FDA regulates human tissue collected

for transplantation.7,8 There are require-

ments for infectious disease testing, do-

nor screening, and record-keeping. A tis-

sue establishment must have and follow

written, validated procedures to prevent

contamination and cross-contamination

during processing.7,35 The FDA has pub-

lished rules for determining eligibility for

donation of human cells, tissues, and cel-

lular and tissue-based products (HCT/

Ps)36 that became effective May 25, 2005.

Federal rules also require all facilities that

recover, process, store, or distribute HCT/Ps

or screen or test the donor to register with

the agency and list their products by mail-

ing or faxing Form FDA 3356.37 Informa-

tion may be submitted electronically at

the FDA website www.fda.gov/cber/tis-

sue/tisreg.htm. Products not covered by

these regulations include xenogeneic tis-

sue, vascularized organs, transfusable

blood products, products used in the

propagation of cells or tissues, and prod-

ucts that are secreted or extracted from

cells or tissues. Minimally manipulated

marrow, such as marrow that undergoes

cell separation, the relevant characteris-

tics of which are not altered by the pro-

cessing, is not covered.

Under the federal regulations, infectious

disease testing is required for allogeneic tis-

sues, except reproductive tissue from sexu-

ally intimate partners. Current good tissue

practice rules to address concerns about

proper handling, storage and processing of

tissuehavebeenfinalized.

34 Until such time

that the comprehensive regulatory frame-

work for human cells, tissues, and cellular

and tissue-based products is effective, in-

cluding donor eligibility requirements,

good tissue practice regulations, and ap-

propriate enforcement provisions, human

dura mater and human heart valves will re-

main subject to the medical devices re-

quirements under the Federal Food, Drug,

and Cosmetic Act.38 Federal regulation of

tissue banks, formerly under the purview of

the FDA’s Center for Biologics Research and

Review, Office of Blood Research and Re-

view, is now the responsibility of the Center

for Biologics Evaluation and Research, Of-

fice of Cellular, Tissue, and Gene Therapies’

Division of Human Tissues.

626 AABB Technical Manual

The Importance of ABO

Compatibility

The ABO antigens are important in trans-

plantation practice because they constitute

very strong histocompatibility antigens that

are expressed on vascular endothelium.

Major ABO mismatching can cause rapid

graft rejection due to endothelial damage

by ABO antibodies and subsequent wide-

spread thrombosis within the graft. ABO

matching is important to the success of

vascularized grafts (ie, kidney,39 heart,

liver, and pancreas), but ABO matching is

not important in tissue grafts (ie, fascia,

bone, heart valves, skin, and cornea).40

The definition of an ABO-compatible

graft is the same as that for a red cell trans-

fusion. A group O donor of tissue or organ

is a universal donor whose graft can be

transplanted into recipients of all blood

groups. Case reports document rare suc-

cessful organ transplants with major ABO

incompatibility, but these are few in num-

ber.41 In some cases, A2donor kidneys can

be successfully transplanted into group O

recipients with survival comparable to that

of group O donor kidneys.42 ABO-incompat-

ible transplants have occurred, often with

fatal results, due to errors of record-keeping

or labeling. It has been estimated that inad-

vertent ABO-incompatible heart or kidney

transplants occur with a frequency of 1 per

1000.43 This underscores the importance of

a final ABO check of donor and recipient

blood at the transplant facility to reduce

this risk.

The Role of Transfusion in

Kidney Transplants

In 1973, Opelz et al44 noted decreasing kid-

ney allograft survival when hemodialysis

staff attempted to avoid priming the dial-

ysis equipment with blood and to limit

pretransplant blood transfusions. The as-

sociation between fewer transfusions and

declining renal allograft survival led

transplant centers to initiate deliberate

pretransplant transfusion protocols in the

mid-1970s. Subsequent studies have sup-

ported the theory that pretransplant blood

transfusions enhance renal allograft sur-

vival through mechanisms of inducing

tolerance that remain imperfectly under-

stood.45,46 However, with the availability of

cyclosporine and other immunosuppres-

sive agents, interest in this approach has

waned.47 If the controversial practice of

transfusion to induce tolerance in patients

before transplantation is begun, nonleuko-

cyte-reduced Red Blood Cells (RBCs) should

be used.48,49 The introduction of erythro-

poietin has reduced the need for red cell

transfusions in patients awaiting a renal

transplant.

Liver Transplants

A liver transplant program presents one of

the greatest challenges to the donor cen-

ter and hospital transfusion service, de-

manding maximal support in terms of

preparedness, supply, and responsive-

ness. Massive blood loss and hypocoagu-

lability due to preexisting liver disease

and/or the anhepatic interval during the

procedure create complex problems for the

transfusion service. The liver is the major

site for synthesis of clotting factors and

other essential proteins and is a prime reg-

ulator of acid-base, electrolyte, and glucose

homeostasis. The three surgical phases of

the procedure—recipient hepatectomy,

anhepatic interval, and biliary reconstruc-

tion—seriously derange these functions.

Support Required

To achieve a successful liver transplant

program, a major commitment to this sup-

Chapter 26: Tissue and Organ Transplantation 627

port is required. A successful program re-

quires cooperation and communication

among the hospital administration; oper-

ating room and intensive care unit; respi-

ratory therapy, radiology, gastroentero-

logy, and anesthesiology services; the

coagulation and transfusion laboratories;

and the regional donor center. The insti-

tutional commitment must extend 24

hours a day, 365 days a year, because there

may be no more than a few hours’ ad-

vance notice of a liver transplant. Consis-

tent availability of blood bank staff on

short notice is essential to meet the trans-

fusion requirements. The surgical proce-

dure frequently takes place at night or on

weekends because of the availability of the

donor organ and in order to avoid dis-

rupting the operating room schedule. The

surgical procedure takes an average of 6

to 8 hours but may take up to 24 hours

and involve massive blood use and sev-

eral surgical teams.

The blood bank should be notified as

soon as the donor organ becomes available

and the decision for transplantation is

made. The blood bank obtains a generous

blood sample from the recipient for cross-

matching, but there may be more than one

patient waiting for a liver and the surgeons

may be undecided about the specific recip-

ient. Therefore, the blood bank may have to

perform numerous crossmatches for pa-

tients who may have different ABO groups

and/or Rh types. Liver transplant programs

initially used hundreds of units of blood

and components per patient. Although

blood use has steadily declined over the

years, liver transplant procedures fre-

quently use a volume of blood components

equal to one whole-body blood volume and

sometimes several blood volumes. Intra-

operative blood recovery frequently plays a

major role in the conservation of red cells

in such cases.

Considerations of ABO and Rh

Except in emergencies, donor livers

should be ABO-compatible with the re-

cipient. ABO-identical RBCs and Fresh

Frozen Plasma (FFP) are generally used

for transfusion support of group O and

group A recipients. Group B recipients

who need large quantities of red cells can

be switched to group O RBCs. Group AB

recipients needing massive transfusion are

often switched to group A RBCs to con-

serve group O RBCs for other patients. If

the supply of AB FFP is insufficient, early

use of group A RBCs followed by a switch

to group A FFP is appropriate. A general

rule for massive transfusions is to switch

red cells first, then switch plasma, and re-

verse the order when returning to the pa-

tient’s original blood group.46

Special considerations apply to the re-

cipient of an out-of-group but ABO-com-

patible liver transplant. In a group A patient

receiving a group O liver, lymphocytes of

donor origin may produce ABO antibodies

that cause hemolysis that begins several

days after the procedure and may continue

for 2 weeks or longer.50 Although passenger

lymphocytes may produce antibodies in

any out-of-group but compatible combina-

tion, significant hemolysis is seen most

often in the recipient of a group O liver.

Transfusion support of Rh-negative pa-

tients not immunized to the D antigen is

not standardized.46 Because successful

pregnancy has occurred after liver trans-

plantation, most programs consider it pref-

erable to provide D-negative units to

D-negative females with childbearing po-

tential, if needs are expected to be moder-

ate. Should massive blood loss occur, the

patient could be switched intraoperatively

to D-positive blood, if necessary. For pre-

menopausal females without anti-D, some

programs reserve 10 units of D-negative

RBCs; if more than 10 units are required,

628 AABB Technical Manual

they switch to D-positive blood.46 Produc-

tion of anti-D occurs less frequently in

D-negative liver transplant patients ex-

posed to the D antigen than in other

D-negative patients.51 In some programs,

patients without anti-D who are D-negative

postmenopausal females or D-negative

males are transfused exclusively with

D-positive blood.

Red Cell Alloantibodies

Liver transplant patients with clinically

significant red cell alloantibodies repre-

sent a special challenge to blood banks.

Sometimes, a sufficient quantity of anti-

gen-negative blood can be secured before

surgery. Some programs reserve a limited

number of antigen-negative units for use

at the beginning of surgery, when allo-

antibody is present, and at the end of

massive blood loss, when transfused cells

are expected to remain in circulation. An-

tibody screening during the interval of

massive blood loss can help guide use of

antigen-positive units during surgery.

Coagulation Considerations

During surgery, hemodilution, platelet

consumption, disordered thrombin regu-

lation, and fibrinolysis derange the hemo-

static process. The coagulopathy is espe-

cially severe during the anhepatic and

early reperfusion stage. The following tests

are useful: the hematocrit guides the use

of red cells, colloids, and crystalloids; the

platelet count guides transfusion of plate-

lets; the prothrombin time and activated

partial thromboplastin time guide FFP

use; and fibrinogen determinations guide

use of Cryoprecipitated AHF and anti-

fibrinolytic agents.46,52,53

Other Organ Transplants

Blood bank support for cardiac transplan-

tation is very similar to that routinely

used for other surgical procedures in

which cardiopulmonary bypass is em-

ployed. The blood bank may also provide

ABO testing and assist in the release

of ABO-compatible organs to prevent

ABO-mismatched organ transplantation.

Pancreatic transplants have compara-

tively low transfusion requirements, but a

specimen from the recipient should rou-

tinely be examined for clinically signifi-

cant unexpected red cell antibodies; in

some institutions, the protocol calls for

crossmatching several units.

Transfusion Service Support

for Organ Transplantation

The blood bank provides vital support for

a clinical transplantation program. Close

communication with the surgeons and

other professionals involved in the pro-

gram is essential. Transfusion practices in

the peritransplant period have a major ef-

fect on morbidity, mortality, and graft sur-

vival rates.

Potential recipients of solid organ trans-

plants are generally available well before

the procedure, so there is ample time to ob-

tain a history and perform laboratory tests.

It is important for the transfusion service to

know if there have been previous pregnan-

cies, transplants, or transfusions.

Laboratory tests routinely performed in-

clude: ABO group and Rh type, the direct

antiglobulin test (DAT), a screen for unex-

pected red cell antibodies, and determina-

tion of CMV serostatus. HLA typing and

HLA antibody studies are routine for organ

recipients.

Passenger lymphocyte hemolysis (typi-

cally “ABO”-incompatible hemolysis), as

Chapter 26: Tissue and Organ Transplantation 629

discussed previously in regard to liver

transplantation, can occur with other solid

organ transplants, such as lung, heart, and

kidney. In the case of a recipient receiving

an ABO-compatible but non-group-identi-

cal organ, prophylactic use of mutually

ABO-compatible erythrocytes (compatible

for the donor and the recipient) has been

suggested for intraoperative and postopera-

tive infusions, during the first postoperative

month, or at the appearance of an anti-

body. At present, there is no consensus on

this issue. It is important to remember that

if immediate-spin or computer cross-

matching is routinely performed after an

ABO-unmatched transplant, ABO incom-

patibility due to these IgG antibodies may

be missed. In such cases, the routine use of

a crossmatch with an antihuman globulin

phaseortheuseofaDAT(whichmayde-

tect such cases earlier than a crossmatch) is

recommended. If ABO hemolysis is present,

the patient should be transfused with group

ORBCs.

CMV infection, a serious and often fatal

complication in transplant recipients, is re-

lated to the presence of CMV in the donor

and recipient and the degree to which the

recipient is immunosuppressed. The pri-

mary test used to determine CMV status is

the demonstration of circulating antibody.

CMV-seronegative recipients of CMV-sero-

negative transplants characteristically re-

ceive transfusion components processed to

reduce risk of CMV transmission, either by

preparation from seronegative donors or by

leukocyte reduction to 5 × 106cells/compo-

nent or below.

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19. Human immunodeficiency virus transmitted

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20. Eastlund T. Hemodilution due to blood loss

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39. Alkhunaizi AM, de Mattos AM, Barry JM, et al.

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40. Eastlund T. The histo-blood group ABO sys-

tem and tissue transplantation. Transfusion

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41. Alexandre GPJ, Squifflet JP, DeBruyere M, et

al. ABO-incompatible related and unrelated

living donor renal allografts. Transplant Proc

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42. Breimer ME, Brynger H, Rydberg L, et al.

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43. Terasaki PI. Red-cell crossmatching for heart

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44. Opelz G, Sengar DPS, Mickey MR, Terasaki P.

Effect of blood transfusion on subsequent

kidney transplants. Transplant Proc 1973;5:

253-9.

45. Blumberg N, Heal JM. Transfusion immuno-

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46. Dzik WH. Solid organ transplantation. In:

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47. Lundgren G, Groth CG, Albrechtsen D, et al.

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fusions in cadaveric renal transplantation—a

changing picture with cyclosporin. Lancet

1986;ii:66-9.

48. Iwaki Y, Cecka JM, Terasaki PI. The transfu-

sion effect in cadaver kidney transplants, yes

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49. Opelz G, Vanrenterghem Y, Kirste G, et al.

Prospective evaluation of pretransplant

blood transfusions in cadaver kidney recipi-

ents. Transplantation 1997;63:964-7.

50. Triulzi DJ, Shirey RS, Ness PM, Klein AS. Im-

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unmatched liver transplants. Transfusion

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51. Casanueva M, Valdes MD, Ribera MC. Lack of

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immunosuppressed liver transplant recipi-

ents. Transfusion 1994;34:570-2.

52. Triulzi DJ, Bontempo FA, Kiss JE, Winkelstein

A. Transfusion support in liver transplanta-

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632 AABB Technical Manual

Chapter 27: Noninfectious Complications of Blood Transfusion

Chapter 27

Noninfectious

Complications of Blood

Transfusion

THIS CHAPTER ADDRESSES four

broad categories of transfusion re-

actions: 1) acute immunologic, 2)

acute nonimmunologic, 3) delayed immu-

nologic, and 4) delayed nonimmunologic

complications, as shown in Table 27-1.1,2

For each individual type of reaction, the

pathophysiology, treatment, and preven-

tion are discussed. More detailed cover-

age is available elsewhere.1Infectious

risks of transfusion are discussed in

Chapter 28.

Manifestations

All personnel involved in ordering and

administering transfusions must be able

to recognize a transfusion reaction so that

appropriate actions can be taken promptly.

Listed below are signs and symptoms that

are typically associated with acute trans-

fusion reactions and can aid in their rec-

ognition. In general, one should consider

any adverse manifestation occurring at

thetimeofthetransfusiontobeatransfu

sion reaction until proven otherwise.

■Fever with or without chills [gener-

ally defined for surveillance pur-

poses as a 1 C (2 F) increase in body

temperature] associated with trans-

fusion. Fever is the most common

symptom of a hemolytic transfusion

reaction (HTR),3but more frequently

it has other causes.

■Shaking chills (rigors) with or with-

out fever.

■Pain at the infusion site or in the

chest, abdomen, or flanks.

■Blood pressure changes, usually acute,

either hypertension or hypotension.

Circulatory shock in combination

with fever, severe chills, and high-

output cardiac failure suggests acute

633

634 AABB Technical Manual

Table 27-1. Categories and Management of Adverse Transfusion Reactions*

Type Incidence Etiology Presentation Diagnostic Testing

Therapeutic/Prophylactic

Approach

Acute (<24 hours) Transfusion Reactions–Immunologic

Hemolytic 1:38,000-

1:70,000

Red cell incompatibility Chills, fever,

hemoglobinuria,

hypotension, renal failure

with oliguria, DIC (oozing

from IV sites), back pain,

pain along infusion vein,

anxiety

■Clerical check

■DAT

■Visual inspection (free

Hb)

■Repeat patient ABO, pre-

and posttransfusion

sample

■Further tests as

indicated to define

possible incompatibility

■Further tests as

indicated to detect

hemolysis (LDH,

bilirubin, etc)

■Keep urine output >100

mL/hr with fluids and IV

diuretic (furosemide)

■Analgesics (may need

morphine)

■Pressors for

hypotension (low-dose

dopamine)

■Hemostatic components

(platelets, cryo, FFP) for

bleeding

Fever/chill,

nonhemoly-

tic

RBCs:

1:200-1:17

(0.5-6%)

Plts:

1:100-1:3

(1-38%)

■Antibody to donor WBCs

■Accumulated cytokines

in platelet unit

Fever, chills/rigors, head-

ache, vomiting

■Rule out hemolysis

(DAT, inspect for

hemoglobinemia, repeat

patient ABO)

■Rule out bacterial

contamination

■WBC antibody screen

■Antipyretic

premedication

(acetaminophen, no

aspirin)

■Leukocyte-reduced

blood

Urticarial 1:100-1:33

(1-3%)

Antibody to donor plasma

proteins

Urticaria, pruritis, flushing ■Rule out hemolysis

(DAT, inspect for

hemoglobinemia, repeat

patient ABO)

■Antihistamine, treatment

or premedication (PO or

IV)

■May restart unit slowly

after antihistamine if

symptoms resolve

Chapter 27: Noninfectious Complications of Blood Transfusion 635

Anaphylactic 1:20,000-

1:50,000

Antibody to donor plasma

proteins (includes IgA,

haptoglobin, C4)

Hypotension, urticaria,

bronchospasm (respira-

tory distress, wheezing),

local edema, anxiety

■Rule out hemolysis

(DAT, inspect for

hemoglobinemia, repeat

patient ABO)

■Anti-IgA

■IgA, quantitative

■Trendelenberg (feet up)

position

■Fluids

■Epinephrine (adult dose:

0.3-0.5 mL of 1:1000

solution SC or IM; in

severe cases, 1:10,000

IV)

■Antihistamines,

corticosteroids, beta-2

agonists

■IgA-deficient blood

components

Transfusion-

related acute

lung injury

1:5,000-

1:190,000

WBC antibodies in donor

(occasionally in recipient),

other WBC-activating

agents in components

Hypoxemia, respiratory

failure, hypotension, fever,

bilateral pulmonary edema

■Rule out hemolysis

(DAT, inspect for

hemoglobinemia, repeat

patient ABO)

■WBC antibody screen in

donor and recipient. If

positive, antigen typing

may be indicated

■WBC crossmatch

■Chest X-ray

■Supportive care until

recovery

■Defer implicated donors

Acute (<24 hours) Transfusion Reactions—Nonimmunologic

Transfusion-

associated

sepsis

Varies by

component

(see Chapter

28)

Bacterial contamination Fever, chills, hypotension ■Gram’s stain

■Culture of component

■Patient culture

■Rule out hemolysis

(DAT, inspect for

hemoglobinemia, repeat

patient ABO)

■Broad spectrum

antibiotics (until

sensitivities completed)

■Treat complications (eg,

shock)

(cont’d)

636 AABB Technical Manual

Table 27-1. Categories and Management of Adverse Transfusion Reactions* (cont’d)

Type Incidence Etiology Presentation Diagnostic Testing

Therapeutic/Prophylactic

Approach

Hypotension

associated with

ACE inhibition

Dependent

on clinical

setting

Inhibited metabolism of

bradykinin with infusion of

bradykinin (negatively

charged filters) or activa-

tors of prekallikrein

Flushing, hypotension ■Rule out hemolysis

(DAT, inspect for

hemoglobinemia, repeat

patient ABO)

■Withdraw ACE inhibition

■Avoid albumin volume

replacement for

plasmapheresis

■Avoid bedside leukocyte

filtration

Circulatory

overload

<1% Volume overload Dyspnea, orthopnea,

cough, tachycardia, hyper-

tension, headache

■Chest X-ray ■Upright posture

■Oxygen

■IV diuretic (furosemide)

■Phlebotomy (250-mL

increments)

Nonimmune

hemolysis

Rare Physical or chemical de-

struction of blood (heat-

ing, freezing, hemolytic

drug or solution added to

blood)

Hemoglobinuria,

hemoglobinemia

■Rule out patient

hemolysis (DAT, inspect

for hemoglobinemia,

repeat patient ABO)

■Test unit for hemolysis

■Identify and eliminate

cause

Air embolus Rare Air infusion via line Sudden shortness of

breath, acute cyanosis,

pain, cough, hypotension,

cardiac arrythmia

■X-ray for intravascular

air

■Place patient on left side

with legs elevated above

chest and head

Hypocalcemia

(ionized cal-

cium)

Dependent

on clinical

setting

Rapid citrate infusion

(massive transfusion of

citrated blood, delayed

metabolism of citrate,

apheresis procedures)

Paresthesia, tetany,

arrhythmia

■Ionized calcium

■Prolonged Q-T interval

on electrocardiogram

■Slow calcium infusion

while monitoring ionized

calcium levels in severe

cases

■PO calcium supplement

for mild symptoms

during apheresis

procedures

Chapter 27: Noninfectious Complications of Blood Transfusion 637

Hypothermia Dependent

on clinical

setting

Rapid infusion of cold

blood

Cardiac arrhythmia Central body temperature ■Employ blood warmer

Delayed (>24 hours) Transfusion Reactions–Immunologic

Alloimmuni-

zation, RBC

antigens

Alloimmuni-

zation, HLA

antigens

1:100 (1%)

1:10 (10%)

Immune response to

foreign antigens on

RBCs, or WBCs and

platelets (HLA)

Positive blood group

antibody screening test

Platelet refractoriness,

delayed hemolytic

reaction, hemolytic dis-

ease of the newborn

■Antibody screen

■DAT

■Platelet antibody screen

■Lymphocytotoxicity test

■Avoid unnecessary

transfusions

■Leukocyte-reduced

blood

■Avoid unnecessary

transfusions

■Leukocyte-reduced

blood

Hemolytic 1:11,000-

1:5000

Anamnestic immune

response to red cell

antigens

Fever, decreasing

hemoglobin, new

positive antibody screen-

ing test, mild jaundice

■Antibody screen

■DAT

■Tests for hemolysis

(visual inspection for

hemoglobinemia, LDH,

bilirubin, urinary

hemosiderin as clinically

indicated)

■Identify antibody

■Transfuse compatible

red cells as needed

Graft-vs-host

disease

Rare Donor lymphocytes en-

graft in recipient and

mount attack on host tis-

sues

Erythroderma,

maculopapular rash, an-

orexia, nausea, vomiting,

diarrhea, hepatitis,

pancytopenia, fever

■Skin biopsy

■HLA typing

■Corticosteroids,

cytotoxic agents

■Irradiation of blood

components for patients

at risk (including related

donors and HLA-

selected components)

(cont’d)

638 AABB Technical Manual

Table 27-1. Categories and Management of Adverse Transfusion Reactions* (cont’d)

Type Incidence Etiology Presentation Diagnostic Testing

Therapeutic/Prophylactic

Approach

Posttransfusion

purpura

Rare ■Recipient platelet

antibodies (apparent

alloantibody, usually

anti-HPA-1) destroy

autologous platelets

Thrombocytopenic

purpura, bleeding, 8-10

days after transfusion

■Platelet antibody screen

and identification

■IGIV

■HPA-1-negative platelets

■Plasmapheresis

Immunomo-

dulation

Unknown Incompletely understood

interaction of donor WBC

or plasma factors with re-

cipient immune system

Increased renal graft sur-

vival, infection rate,

postresection tumor recur-

rence rate (controversial)

■None specific ■Avoid unnecessary

transfusions

■Autologous transfusion

■Leukocyte-reduced red

cells and platelets

Delayed (>24 hours) Transfusion Reactions–Nonimmunologic

Iron overload Typically af-

ter >100 RBC

units

Multiple transfusions with

obligateironloadintrans-

fusion-dependent patient

Diabetes, cirrhosis,

cardiomyopathy

■Serum ferritin

■Liver enzymes

■Endocrine function tests

■Desferioxamine (iron

chelator)

*For platelet refractoriness, see Chapter 16; for septic transfusion reactions, see Table 28-1; for a recent summary of transfusion reactions, see Popovsky.1

ACE = angiotensin-converting enzyme; antibody screen = blood group antibody screening test; DAT = direct antiglobulin test; DIC = disseminated intravascular coagulation; FFP

= Fresh Frozen Plasma; Hb = hemoglobin; IV = intravenous; IGIV = intravenous immunoglobulin; IM = intramuscular; LDH = lactate dehydrogenase; PO = by mouth; RBC = Red

Blood Cell; SC = subcutaneous; WBC = White Blood Cell.

sepsis but may also accompany an

acute HTR. Circulatory collapse with-

out fever and chills may be the most

prominent finding in anaphylaxis.

■Respiratory distress, including dyspnea,

tachypnea, wheezing, or hypoxemia.

■Skin changes, including urticaria,

pruritis (itching), flushing, or local-

ized edema (angioedema).

■Nausea with or without vomiting.

■Darkened urine or jaundice. Dark urine

may be the earliest indication of an

acute hemolytic reaction in anesthe-

tized patients.

■Bleeding or other manifestations of

a consumptive coagulopathy.

Acute Transfusion Reactions

Immune-Mediated Hemolysis

Pathophysiology and Manifestations

The most severe hemolytic reactions oc-

cur when transfused red cells interact

with preformed antibodies in the recipi-

ent. In contrast, the interaction of trans-

fused antibodies with the recipient’s red

cells rarely causes symptoms. However,

there may be accelerated red cell destruc-

tion, and plasma-containing products with

high-titer ABO antibodies can cause acute

hemolysis. The interaction of antibody

with antigen on the red cell membrane

can initiate a sequence of complement

activation (see Chapter 11), cytokine and

coagulation effects, and other elements of

a systemic inflammatory response4that

result in the clinical manifestations of a

severe acute HTR. Severe symptoms can

occur after the infusion of as little as 10 to

15 mL of ABO-incompatible red cells. In

anesthetized patients who cannot report

symptoms, the initial manifestations of

an acute HTR may be hemoglobinuria,

hypotension, or diffuse bleeding at the

surgical site.

Such severe acute HTRs today are usu-

ally caused by ABO incompatibility5but oc-

casionally may be caused by antibodies

with other specificities.6In contrast,

hemolysis of an entire unit of blood can oc-

cur in the virtual absence of symptoms7

and may be a relatively slow process. In

such cases, hemolysis is typically extra-

vascular, without generation of significant

systemic levels of inflammatory mediators.

Complement Activation. The binding of

antibody to blood group antigens may acti-

vate complement, depending on the char-

acteristics of both the antibody and the an-

tigen, including antibody specificity, class,

subclass, titer, and antigen density (see

Chapter 11). C3 activation releases the ana-

phylatoxin C3a (see Chapter 11), and red

cells coated with C3b are removed by

phagocytes with complement receptors,

more rapidly than if antibody is present

alone. If the enzymatic cascade proceeds to

completion and a membrane attack com-

plex is assembled, intravascular hemolysis

results, with the production of C5a, which

is100timesaspotentananaphylatoxinas

C3a.3This sequence is characteristic of ABO

incompatibility and causes the cardinal

manifestations of hemoglobinemia and, if

the renal threshold for hemoglobin is ex-

ceeded, hemoglobinuria.8(p182) Anaphylatoxins

interact with a wide variety of cells, includ-

ing monocytes/macrophages, granulocytes,

platelets, vascular endothelial cells, and

smooth muscle cells, the latter leading to

hypotension and bronchospasm. Anaphy-

latoxins also cause the release or produc-

tion of multiple local and systemic media-

tors, including granule enzymes, histamine

and other vasoactive amines, kinins, oxy-

gen radicals, leukotrienes, nitric oxide, and

cytokines.3These mechanisms may cause

manifestations that mimic allergy, such as

flushing and rarely urticaria, wheezing and

Chapter 27: Noninfectious Complications of Blood Transfusion 639

chest pain or tightness, and abdominal

pain, nausea, and vomiting.

With most non-ABO blood group anti-

bodies, complement activation is usually

incomplete; hemoglobinemia is absent or

mild, but the consequences of complement

activation, most notably the release of ana-

phylatoxins and opsonization of red cells,

may still have adverse effects.

Cytokines. The role of cytokines in in-

flammatory responses (see Chapter 11), in-

cluding acute HTRs, is increasingly recog-

nized.9,10 The known activities of inflammatory

cytokines, such as tumor necrosis factor α

(TNFα), interleukin-1βand -6 (IL-1β,IL-6),

and chemokines such as IL-8 and mono-

cyte chemoattractant protein (MCP), sug-

gest that they mediate some of the effects of

alloimmune hemolysis. IL-1 and TNF cause

fever and hypotension (particularly in syn-

ergy), stimulation of endothelial cells to in-

crease expression of adhesion molecules

and procoagulant activity, and recruitment

and activation of neutrophils and platelets,

perhaps through the induction of IL-8 and

MCP. Incubation of whole blood with

washed, ABO-incompatible red cells in vi-

tro has been shown to cause dramatic in-

creases in TNF, IL-8, and MCP. This cyto-

kine response is complement dependent. A

similar model of hemolysis resulting from

anti-D (IgG) showed a different pattern of

cytokine production with “high-level” re-

sponses of IL-8 and MCP and “low-level”

responses of IL-1β,IL-6,andTNFα.9,10

The relevance of these in-vitro models to

HTRs in vivo is suggested by a case in

which TNFαand neutrophil elastase levels

werefoundtobeelevatedwhenagroupO

patient received 100 mL of group A red

cells; elevation of neutrophil elastase is

consistent with IL-8 activity.11 These find-

ings may lead to new therapeutic options

forpatients.However,thecompleteroleof

cytokines in the consequences of immune

hemolysis remains to be defined.

Coagulation Activation. Several mecha-

nisms, including those listed above, may be

responsible for abnormalities of coagula-

tion in HTRs.4The antigen-antibody inter-

action may activate the “intrinsic” clotting

cascade through Hageman factor. In addi-

tion, activated Hageman factor (Factor XIIa)

acts on the kinin system to generate brady-

kinin; bradykinin increases capillary per-

meability and dilates arterioles, causing a

decrease in systemic arterial pressure. Sev-

eral factors cited above may increase the

expression of tissue factor by leukocytes

and endothelial cells, including activated

complement components, TNFα, and

IL-1β. Tissue factor activates the “extrinsic”

coagulation pathway, and its release is as-

sociated with disseminated intravascular

coagulation (DIC), which may, in turn, cause:

1) formation of thrombi within the micro-

vasculature and ischemic damage to tissues

and organs; 2) consumption of fibrinogen,

platelets, and other coagulation factors; 3)

activation of the fibrinolytic system and

generation of fibrin degradation products.

The outcome can be a hemorrhagic diathesis

characterized by generalized oozing or un-

controlled bleeding.

Shock and Renal Failure. Considering

the absolute mass of antigen and anti-

body—and the list of mediators that may

be involved in HTRs, including anaphyla-

toxins, vasoactive amines, kinins, and cyto-

kines—it may not be surprising that shock

can occur. Hypotension provokes a com-

pensatory sympathetic nervous system re-

sponse that produces vasoconstriction in

organs and tissues with a vascular bed rich

in alpha-adrenergic receptors, notably, the

renal, splanchnic, pulmonary, and cutane-

ous capillaries, aggravating ischemia in

these sites.

Renal failure is another sequel of an

acute HTR. Although free hemoglobin, his-

torically considered the cause of renal fail-

ure, does impair renal function,12 current

640 AABB Technical Manual

thought attributes renal failure largely to

hypotension, renal vasoconstriction, anti-

gen-antibody complex deposition, and for-

mation of thrombi in the renal vasculature,

all of which compromise renal cortical

blood supply.

Frequency

Clerical and other human errors leading

to mistaken identity are the most common

causes of ABO-incompatible transfusion,

occurring either at pretransfusion sample

collection, within the transfusion service,

or at the time of blood administration. A

study of reported transfusion errors in

New York State over a 10-year period (the

1990s) estimated the incidence of ABO-

incompatible red cell transfusions at

1:38,000. Correction for the expected rate

of fortuitously compatible transfusions

led to an estimate of the rate of mis-

transfusion of 1:14,000.7A survey of 3601

institutions by the College of American

Pathologists found 843 acute HTRs re-

ported over a 5-year period, of which 50

(6%) were fatal.13 The Serious Hazards of

Transfusion (SHOT) initiative in the United

Kingdom and Republic of Ireland re-

ported 161 cases of ABO-incompatible

transfusion, with nine fatal cases (five def-

initely related deaths, one probably re-

lated death, and three possibly related

deaths) in 5 years.14 Although no precise

denominator is available for these confi-

dential reports, it is believed that over

90% of the total transfusions were re-

viewed and approximately 2.5 million

RBC units were issued each year, for a rate

of no less than 1 in 78,000. These values

probably underestimate the true fre-

quency, because even acute HTRs go un-

recognized or unreported. Estimates of

mortality rates from acute HTRs are gener-

ally in the range of 1 in 1,000,000 transfu-

sions.5,7,14

Treatment

The treatment of an acute HTR depends

on its severity.4Vigorous treatment of hypo-

tension and promotion of adequate renal

blood flow are the primary concerns. If

shock can be prevented or adequately

treated, progression to renal failure may

be avoided. Adequacy of renal perfusion

can be monitored by measurement of

urine output, with a goal of maintaining

urine flow rates above 100 mL/hour in

adults for at least 18 to 24 hours. The

usual first support is intravenous normal

saline, but underlying cardiac and/or re-

nal disease may complicate therapy, and

it is important to avoid overhydration. In-

vasive monitoring of pulmonary capillary

wedge pressure is recommended in guid-

ing fluid therapy in the face of hemo-

dynamic instability. Diuretics help to im-

prove blood flow to the kidneys and

increase urine output. Intravenous furo-

semide at a dose of 40 to 80 mg for an

adult or 1 to 2 mg/kg for a child not only

has a diuretic effect but also improves

blood flow to the renal cortex. This dose

may be repeated once, and the patient

should be adequately hydrated. Mannitol,

an osmotic diuretic, has been used in the

past, but furosemide is better for main-

taining renal cortical blood flow. If no di-

uretic response occurs within a few hours

of instituting fluid and diuretic therapy,

there is a strong likelihood that acute tu-

bular necrosis has occurred, and further

fluid administration may be harmful.

Treatment of hypotension with pressor

agents that decrease renal blood flow, such

as dopamine in higher doses, should be

avoided if possible. The use of low-dose do-

pamine (2-5 µg/kg/minute), as an agent to

protect renal function, has been recom-

mended in the management of acute HTRs.4

However, evidence suggests that it is not ef-

Chapter 27: Noninfectious Complications of Blood Transfusion 641

fective in this role, and it has many toxici-

ties.15

Consumptive coagulopathy, with resul-

tant bleeding or generalized oozing, may be

a prominent clinical finding in some HTRs

and may be the initial presentation in an

anesthetized patient. Heparin has been rec-

ommended by some, both to forestall DIC

when an ABO incompatibility is first dis-

covered and to treat the established coa-

gulopathy. Others believe the dangers of

heparin outweigh its potential benefits, es-

pecially because the immune event that

provoked the DIC is self-limited. Adminis-

tration of Platelets, Fresh Frozen Plasma

(FFP), and Cryoprecipitated AHF, a source

of fibrinogen and Factor VIII, may be nec-

essary. Red cell exchange may be consid-

ered in patients with a significant load of

circulating incompatible red cells.

Acute hemolytic reactions are rare and

few clinicians have first-hand experience

with their treatment. Because medical

management of an acute HTR is often com-

plicated and may require aggressive inter-

ventions such as hemodialysis, consultation

with physicians experienced in the organ

systems most damaged or specialists in

critical care medicine may be prudent when

treating a patient with a severe acute HTR.

Prevention

Because clerical errors cause the majority

of acute, immune-mediated HTRs, the

best hope for prevention lies in prevent-

ingordetectingerrorsineveryphaseof

the transfusion process. In each institu-

tion, there should be systems designed to

prevent and detect errors in patient and

unit identification at the time of phlebot-

omy (sample acquisition), at all steps in

laboratory testing, at the time of issue,

and when the transfusions are given. The

SHOT reports document multiple errors

in a majority of mistransfusion incidents

and particularly emphasize the impor-

tanceofthebedsidecheckatthetimeof

transfusion.14 Ensuring that all clinical staff

recognize the signs of acute reactions and

stop the transfusion before a critical vol-

ume of blood has been administered is es-

sential to preventing harm to the patient.

Crucial in the prevention of transfusion

mishaps are training and assessment of

personnel performing transfusions. Active

participation by physicians and manage-

ment, as well as by nursing, technical, and

clinical personnel, is essential.

Nonimmune-Mediated Hemolysis

Causes

Red cells may undergo in-vitro hemolysis

if the unit is exposed to improper temper-

atures during shipping or storage or is

mishandled at the time of administration.

Malfunctioning blood warmers, use of

microwave ovens or hot waterbaths, or in-

advertentfreezingmaycausetempera-

ture-related damage. Mechanical hemolysis

maybecausedbytheuseofrollerpumps

(such as those used in cardiac bypass sur-

gery), pressure infusion pumps, pressure

cuffs, or small-bore needles.16 Osmotic

hemolysis in the blood bag or infusion set

may result from the addition of drugs or

hypotonic solutions. Inadequate degly-

cerolization of frozen red cells may cause

the cells to hemolyze after infusion. Finally,

hemolysis may be a sign of bacterial

growth in blood units. In a patient with

transfusion-associated hemolysis for which

both immune and nonimmune causes

have been eliminated, the possibility might

be considered that the patient or donor has

an intrinsic red cell defect, such as glu-

cose-6-phosphatase dehydrogenase defi-

ciency, causing coincidental hemolysis.

642 AABB Technical Manual

Treatment

Treatment depends on the severity of the

reaction. If the patient develops a severe

reaction with hypotension, shock, and re-

nal dysfunction, intensive clinical man-

agement is required even before the cause

of the mishap is investigated. If the pa-

tient exhibits only hemoglobinemia and

hemoglobinuria, supportive therapy may

be sufficient.

Prevention

There should be written procedures for all

aspects of procuring, processing, and is-

suing blood, and administering transfusions.

All staff should be trained in the proper

use of equipment, intravenous solutions,

and drugs used during the administration

of blood and blood components. Equip-

ment must be properly maintained and

records kept of how and when items are

used. Intravenous medications shall not

be injected into blood bags, unless ap-

proved by the Food and Drug Administra-

tion (FDA) or documented to be safe for

that purpose,17(p48) and care must be exer-

cised in the selection and use of intrave-

nous access devices. Chapter 22 discusses

the details of administering transfusions.

Transfusion-Associated Sepsis

Bacterial contamination of transfused blood

should be considered if the patient expe-

riences severe rigors, especially if they are

accompanied by cardiovascular collapse

and/or fever over 40 C.18 For a more de-

tailed discussion of this potentially life-

threatening transfusion complication, see

Chapter 28.

Febrile Nonhemolytic Reactions

Pathophysiology and Manifestations

A febrile nonhemolytic transfusion reac-

tion (FNHTR) is often defined as a tem-

perature increase of >1 C associated with

transfusion and without any other expla-

nation. Such reactions are often associated

with chills or rigors. The 1 C definition is

arbitrary; the same events might cause

smaller temperature increments. Indeed,

some authors discuss reactions character-

ized by rigors or other symptoms, in the

absence of fever, as FNHTRs because of a

presumed common mechanism.2In one

study of 108 reactions characterized by

chills, cold, or rigors, only 18 involved a

rise in temperature.19 Febrile reactions

complicate 0.5% to 6% of nonleukocyte-

reduced red cell transfusions. Previous

opportunities for alloimmunization, es-

pecially pregnancies and multiple trans-

fusions, increase the frequency of FNHTRs

to red cells. The rate of such reactions is

much higher after platelet transfusion

(1-38%). Most FNHTRs are benign, al-

though some may cause significant dis-

comfort and hemodynamic or respiratory

changes. The temperature increase may

begin early in the transfusion or be de-

layed in onset for hours after completion

of the transfusion.

Many febrile reactions are thought to re-

sult from an interaction between antibod-

ies in the recipient’s plasma and antigens

present on transfused lymphocytes, granu-

locytes, or platelets, most frequently HLA

antigens. There is also evidence that febrile

reactions, particularly those due to plate-

lets, may be caused by the infusion of bio-

logic response modifiers, including cyto-

kines, that accumulate in the blood bag

during storage. Cytokine release in the re-

cipient undoubtedly contributes to those

reactions that begin with recipient antibody

against donor leukocytes.2,20-22 Because fever

maybeaninitialmanifestationofanacute

HTR or a reaction to transfusion of blood

contaminated with bacteria, any observa-

tion of an increase in temperature associ-

ated with transfusion warrants prompt at-

Chapter 27: Noninfectious Complications of Blood Transfusion 643

tention. The diagnosis of an FNHTR is

made after excluding other possible expla-

nations for the fever, particularly a

hemolytic or septic reaction. Guidelines for

evaluating a suspected acute transfusion

reaction are presented later in this chapter.

Treatment

Traditionally, transfusion was discontin-

ued when an FNHTR occurred.23 However,

some clinicians believe that fever should

not routinely cause discontinuation of a

transfusion,2,24 depending on whether the

patient has symptoms, signs, or labora-

tory data that suggest hemolysis, transfu-

sion-related acute lung injury (TRALI), or

bacterial contamination. The fever of an

FNHTR usually responds to antipyretics.

Acetaminophen is preferred to the use of

salicylates because the former drug does

not affect platelet function. Meperidine

injection may be useful in patients with

severe shaking chills. Antihistamines are

not indicated because most FNHTRs do

not involve histamine release.

Prevention

Febrile reactions in an alloimmunized

individual can often be prevented by

transfusion of leukocyte-reduced blood

components. Prevention of reactions

caused by cytokine accumulation during

storage requires that the leukocyte reduc-

tion be performed before storage,2,19 but

some patients will still react. With non-

leukocyte-reduced platelets, cytokine-

mediated reactions may be less frequent

when the component(s) are less than or

equal to 3 days old. Plasma removal may

also decrease febrile reactions. Aceta-

minophen is commonly given before

transfusion, but there is no evidence that

the premedication lessens the incidence

of FNHTR symptoms due to prestorage

leukocyte-reduced platelets.25

Allergy; Urticaria (Hives) to Anaphylaxis

Pathophysiology and Manifestations

Allergic reactions to transfusion form a

continuum, with the vast majority clus-

tered at the mild end, in the form of urti-

caria or “hives”—erythematous, sharply

circumscribed raised lesions, most often

present over the upper trunk and neck,

which may itch and which are not usually

accompanied by fever or other adverse

findings. At the other end of the spectrum

are anaphylactic reactions, in which there

are systemic symptoms including hypo-

tension, loss of consciousness, shock,

and, in rare cases, death. The latter may

begin after infusion of only a few millili-

ters, but less severe reactions tend to take

longer to develop. The term “anaphylac-

toid” is used in transfusion medicine to

denote reactions in between these ex-

tremes, but it is also used to denote reac-

tions that have clinical similarities to

anaphylaxis but different mechanisms.

Manifestations of these reactions may in-

volve one or several systems, notably, the

skin (urticaria, generalized flushing or

rash, localized swelling or “angioedema”),

respiratorytract(upperorlowerrespira

tory tract obstruction with cough, hoarse-

ness, stridor, wheezing, chest tightness or

pain, dyspnea), the gastrointestinal tract

(cramps, nausea, vomiting, diarrhea), or

the circulatory system (tachycardia and

other arrhythmias including cardiac ar-

rest).26 Fever is characteristically absent, a

feature that aids in differentiating these

reactions from hypotension due to a hemoly-

tic reaction or bacterial contamination,

and from respiratory compromise caused

by TRALI (see below). The severity of al-

lergic transfusion reactions may increase

with successive transfusions.

Allergic reactions are attributed to expo-

sure to a soluble substance in donor plasma

that binds to preformed IgE antibodies on

644 AABB Technical Manual

mast cells, resulting in the activation and

release of histamine. This presumption is

based on the facts that reactions tend to re-

cur in an affected recipient and that they

can be prevented by removal of the plasma

from cellular components or, in the case of

urticaria, by antihistamines. Anaphylactic

and anaphylactoid reactions are sometimes

associated with class, subclass, and allo-

type-specific antibodies against IgA, partic-

ularly in IgA-deficient patients.27 IgE anti-

IgA has been demonstrated in two patients

with common variable immunodeficiency

having reactions to immunoglobulin prep-

arations.28 However, most of the IgA anti-

bodies to which anaphylactic reactions are

attributed are of the IgG or IgM class,27 and

these antibodies are demonstrable in only a

minority of the anaphylaxis cases referred

for study (17.5% in the series of Sandler et

al27). Moreover, IgA antibodies are common

but anaphylactic reactions are not. There-

fore, demonstration of anti-IgA in an indi-

vidual who has not been transfused does

not predict anaphylaxis. Other allergens or

other mechanisms are likely.

Severe allergic reactions have been re-

ported in patients with antibodies directed

against C4 determinants,29,30 haptoglobin,31

and elements of nonbiologic origin such as

ethylene oxide used for sterilizing tubing

sets.32 However, the causative antigens have

not been identified in the vast majority of

cases. Reactions caused by passively trans-

ferred donor antibody have rarely been

documented.33,34

Hypotensive reactions mimicking ana-

phylaxis have been observed in patients

taking angiotensin-converting enzyme

(ACE) inhibitors who receive albumin dur-

ing plasma exchange.35 They were thought

to be due to inhibition of bradykinin catab-

olism by the ACE inhibitors combined with

bradykinin activation by low levels of pre-

kallikrein activator (a Hageman factor frag-

ment) in the albumin used for replacement.

Similarly, bradykinin activation by pre-

kallikrein activity in plasma protein fraction

has also been implicated in hypotensive re-

actions,36 and a similar mechanism is prob-

ably responsible for the many patients

taking ACE inhibitors reported to have

hypotensive reactions when receiving

blood components via bedside leukocyte

reduction filters.37-39 Similar reactions have

been observed in association with the con-

tact of plasma with charged dialysis mem-

branes, low-density lipoprotein adsorption

columns, and staphylococcal protein A

immunoadsorption columns. Other mech-

anisms that have been proposed include the

infusion of complement-derived anaphyla-

toxins and histamine.26 The differentiation

and appropriate classification of these dif-

ferent reactions will require additional re-

search and refined diagnostic tools.

Frequency

Urticaria may complicate as many as 1%

to 3% of transfusions, the observed fre-

quency depending on how vigorously it is

sought. The incidence of anaphylactic re-

actions fortunately is low, estimated to be

1 in 20,000 to 50,000 units. The SHOT data

suggest that anaphylaxis is much more

common as a complication of plasma and

platelet transfusions, than of red cells14;

although these reactions may have con-

tributed to the death of a few severely ill

patients, they were not a primary cause of

death. The mortality rate reported to the

FDA is about 1 per year.26

Treatment

If urticaria is the only adverse event noted,

the transfusion may be temporarily inter-

rupted while an antihistamine (eg, di-

phenhydramine, 25-50 mg) is adminis-

tered orally or parenterally. If symptoms

Chapter 27: Noninfectious Complications of Blood Transfusion 645

are mild and promptly relieved, the trans-

fusion may be resumed, provided the

interrupted infusion can be completed

within the acceptable time (see Chapter

22). If the patient develops severe urticaria,

a significant local swelling, respiratory or

gastrointestinal symptoms, or hypotension,

the transfusion should be discontinued.26

The immediate treatment of an anaphy-

lactic transfusion reaction should be to stop

the transfusion and treat hypotension by

placing the patient in the Trendelenberg

(feet up) position and administering a fluid

challenge. If the blood pressure does not

improve immediately, epinephrine should

be given. In mild to moderate cases, epi-

nephrine (1:1000) should be delivered sub-

cutaneously or intramuscularly in a starting

dose of 0.3 to 0.5 mL in adults, or 0.01

mL/kg in children. This dose may be re-

peated a second and third time at 5- to

15-minute intervals. In severe reactions (eg,

systolic blood pressure below 80 mm Hg,

laryngeal edema with upper airway com-

promise, or respiratory failure), the drug

should be given intravenously (1:10,000) for

the most rapid effect because drug absorp-

tion is unreliable in hypotensive patients.

Aerosolized or intravenous beta-2 agonists

and theophylline may be required in se-

lected patients in whom bronchospasm is

unresponsive to epinephrine treatment, or

in whom epinephrine is ineffective because

of pre-existing beta-blocker therapy. Oxy-

gen therapy should be administered as re-

quired, with endotracheal intubation if

there is significant upper airway obstruc-

tion. Continued hemodynamic instability

may require invasive hemodynamic moni-

toring. Under no circumstances should the

transfusion be restarted. Coincidental oc-

currence of myocardial infarction, pulmo-

nary embolism, or other medical catastro-

phes could present with hypotension and

respiratory compromise and should be

considered.26

Prevention

Recipients who have frequent transfusion-

associated urticarial reactions may re-

spond well to administration of antihista-

mine (eg, 25-50 mg of diphenhydramine)

one-half hour before transfusion. However,

diphenhydramine should not be given

routinely without a history of previous al-

lergic reactions, particularly to elderly pa-

tients.40 If antihistamine administration is

insufficient, 100 mg of hydrocortisone given

1 hour before transfusion may be useful.

If reactions are recurrent and severe or as-

sociated with other allergic manifestations

in spite of adequate premedication, trans-

fusion of washed red cell or platelet com-

ponents, or red cells that have been frozen,

thawed, and deglycerolized will usually be

tolerated.

Patients who have had a prior life-threat-

eninganaphylacticreactionandwhoare

IgA-deficient or have a demonstrated IgA

antibody should receive blood components

that lack IgA, either by washing or prepara-

tion of components from IgA-deficient

blood donors. Severe reactions that are not

caused by anti-IgA can be prevented only

by maximal antiallergy immunosuppres-

sion or washing. A need for red cells may be

met by the use of washed or frozen, thawed,

and deglycerolized units.26 Washed platelets

are generally not readily available and may

result in decreased platelet recovery, func-

tion, and survival41; therefore, after the first

reaction, if there is no evidence that the re-

action is mediated by IgA, some centers

elect to rechallenge the patient under

closely controlled circumstances. Preven-

tion of anaphylactoid reactions in patients

such as those with thrombotic thrombocy-

topenic purpura who absolutely require

plasma components may be a tremendous

challenge if IgA-deficient donors will not

suffice. Pretreatment with antihistamines,

corticosteroids (starting with 100 mg of hy-

646 AABB Technical Manual

drocortisone), and ephedrine may help. Fi-

nally, it may be possible to collect and store

autologous blood components from patients

known to have experienced anaphylactic

reactions.

Transfusion-Related Acute Lung Injury

Pathophysiology and Manifestations

TRALI should be considered whenever a

transfusion recipient experiences acute

respiratory insufficiency and/or X-ray

findings are consistent with bilateral pul-

monary edema but has no other evidence

of cardiac failure or a cause for respiratory

failure. The severity of the respiratory dis-

tress is usually disproportionate to the

volume of blood infused. The reaction

typically includes fever, chills, and hypo-

tension, usually occurring during or with-

in 1 to 2 after transfusion, often with an

immediate and dramatic onset. Implicated

components always have contained plasma,

but the volume may be as small as that of

a unit of cryoprecipitate or RBCs in an ad-

ditive solution.42,43

Because the manifestations of TRALI are

variable and may overlap with those of the

patient’s underlying medical problems, it is

useful to define the syndrome, particularly

for the purpose of conducting studies of its

epidemiology and pathogenesis. A consen-

sus conference of the blood services in

Canada developed such a definition.44 The

panel defined acute lung injury (ALI) as a

syndrome of: 1) acute onset; 2) hypoxemia

(PaO2/FIO2<300 mm Hg, or O2saturation

<90% on room air, or other clinical evi-

dence); 3) bilateral lung infiltrates on a

chest x-ray; and 4) no evidence of circula-

tory overload. TRALI is then defined as: 1)

new ALI occurring during transfusion or

within 6 hours of completion; and 2) no

other temporally associated ALI risk factors.

If the latter are present, the case is consid-

ered “Possible TRALI.” Risk factors for ALI

include aspiration, pneumonia, toxic inha-

lation, lung contusion, near drowning,

severe sepsis, shock, multiple trauma, burn

injury, acute pancreatitis, cardiopulmonary

bypass, and drug overdose. It was noted

that such a definition will not include cases

of mild respiratory embarrassment having

a similar pathogenesis, cases of ALI in pa-

tients with circulatory overload, and cases

in which a transfusion-related process causes

worsening of pre-existing ALI.44

TRALI may result from multiple mecha-

nisms. Donor antibodies directed against

recipient HLA class I or II antigens, or

neutrophil antigens of the recipient, have

been demonstrated45-47 and are thought to

cause a sequence of events that increase the

permeability of the pulmonary microcir-

culation so that high-protein fluid enters

the interstitium and alveolar air spaces. In-

frequently, antibodies in the recipient’s cir-

culation against HLA or granulocyte anti-

gens initiate the same events.45,46,48 Although

one would expect causative antibodies to

be far more common in recipients than do-

nors, the rarity of TRALI due to recipient

antibody might be due to the fact that the

pool of target leukocytes is much smaller in

a cellular blood component than in a recip-

ient’s circulation. Monocyte activation, with

expression of cytokines including IL-1β,

TNFα, and tissue factor, has been demon-

strated, and these reactions were highly

specific for cells bearing the target antigens.48

Perfusion of neutrophils, complement, and

neutrophil-specific antibody into an ex-vivo

rabbit lung preparation causes severe edema,49

and autopsy studies demonstrate neutro-

phil aggregation in the lungs of patients

who have died of TRALI.50 These and other

observations suggest that pulmonary edema

in TRALI is caused by neutrophil-mediated

endothelial damage, initiated by antibodies

activating neutrophils directly or via activa-

tion of monocytes, pulmonary macrophages,

and/or endothelial cells.

Chapter 27: Noninfectious Complications of Blood Transfusion 647

As the spectrum of antibodies implicated

in cases of TRALI broadens, more cases will

appear to be antibody-mediated. However,

other mechanisms have been proposed as

causes for transfusion-related respiratory

failure. Severe pulmonary reactions are re-

ported after granulocyte transfusions, par-

ticularly in patients with known or unap-

parent lung infections or with conditions

likely to promote prompt complement acti-

vation.51 Other factors may include anaphy-

latoxins C3a and C5a, aggregation of granu-

locytes into leukoemboli that lodge in the

pulmonary microvasculature, or transfusion

of cytokines that have accumulated in

stored blood components. Recently, reac-

tive lipid products from donor blood cell

membranes have been implicated as po-

tential granulocyte activators in the patho-

genesis of TRALI.52 These substances accu-

mulate during blood bank storage and

prime neutrophils to produce vasoactive

mediators in response to a second stimulus

such as infection. One nested case-control

study found that component age and levels

of bioactive lipids, but not leukocyte anti-

bodies, were associated with TRALI.53

The incidence rate of TRALI is not known,

but data from one institution in the 1980s

suggest that this complication may occur as

frequently as 1 in 5000 transfusions.45 The

passive surveillance data of 5 years of SHOT

reports14 include 70 TRALI cases (approxi-

mately 1 per 250,000 total components), of

which 18 cases were fatal (includes six defi-

nite,twoprobable,and10possibleTRALI-

related fatalities). In this series, TRALI was

the most common cause of morbidity and

mortality, ahead of transfusion-associated

graft-vs-host disease (TA-GVHD), ABO in-

compatibility, and bacterial contamination.

The SHOT data suggest that the rate of

TRALI is higher after plasma and platelet

transfusion. Conclusions regarding incidence

and fatality rates will, of course, depend on

the definition of TRALI used.

Treatment

If any kind of acute pulmonary reaction is

suspected, the transfusion should be

stopped immediately and the same unit

should not be restarted even if symptoms

abate. Clinical management focuses on

reversing progressive hypoxemia with ox-

ygen therapy and ventilatory assistance, if

necessary. The role of intravenous ste-

roids is unproved. Unlike other forms of

acute respiratory distress syndrome, most

patients recover adequate pulmonary

function within 2 to 4 days,42,45 and the ob-

served mortality is less than 6% to 23%

(Holness L, personal communication).

Prevention

If antibody in donor plasma can be shown

to have caused an acute pulmonary reac-

tion, blood from that donor should not be

used for plasma-containing components.

No special precautions are needed for the

patient if the problem was donor-specific

and components from other donors are

available. Current policy in the United

Kingdom is not to prepare plasma from

female donors.

Circulatory Overload

Pathophysiology and Manifestations

Transfusion therapy may cause acute pul-

monary edema due to volume overload,

and this can have severe consequences,

including death. Few data are available on

the incidence rate of transfusion-induced

circulatory overload in the general popu-

lation, but young children and the elderly

are considered most at risk, and incidence

rates of up to 1% have been observed in a

study of elderly orthopedic patients.54

Rapidincreasesinbloodvolumearees

pecially poorly tolerated by patients with

compromised cardiac or pulmonary sta-

tus and/or chronic anemia with expanded

648 AABB Technical Manual

plasma volume. The infusion of 25% albu-

min, which shifts large volumes of extra-

vascular fluid into the vascular space, may

also cause circulatory overload. Hyper-

volemia must be considered if dyspnea,

cyanosis, orthopnea, severe headache, hy-

pertension, or congestive heart failure oc-

curduringorsoonaftertransfusion.Ele

vated levels of brain natriuretic peptide

may be seen in cases of circulatory over-

load,55 and this test may be useful in sepa-

rating such cases from cases of TRALI.

Treatment

Symptoms usually improve when the in-

fusion is stopped, and it should not be re-

started until volume overload has been

addressed. Placing the patient in a sitting

position may help. Diuretics and oxygen

are often indicated and, if symptoms are

not relieved, multiple medical interven-

tions may be required, including phlebot-

omy.

Prevention

Except in conditions of ongoing, rapid

blood loss, anemic patients should re-

ceive blood transfusions slowly, with at-

tention to total fluid input and output.

The administration of diuretics before

and during the transfusion may be help-

ful.

Complications of Massive Transfusion

Among the numerous complications that

may accompany massive transfusion,

metabolic and hemostatic abnormalities

are matters of particular concern. Some

or all of the following metabolic derange-

ments can depress left ventricular func-

tion: hypothermia from refrigerated blood,

citrate toxicity, and lactic acidosis from

systemic underperfusion and tissue

ischemia, often complicated by hyper-

kalemia. Metabolic alkylosis due to me-

tabolism of citrate can occur after mas-

sive transfusion but is probably not clini-

cally significant. Patients who are losing

blood rapidly may have pre-existing or

coexisting hemostatic abnormalities or

develop them during resuscitation.

Hemostatic abnormalities may include

dilutional coagulopathy, DIC, and liver

and platelet dysfunction.

Citrate Toxicity

Pathophysiology and Manifestations.

When large volumes of FFP, Whole Blood,

or Platelets are transfused rapidly, partic-

ularlyinthepresenceofliverdisease,

plasma citrate levels may rise, binding

ionized calcium and causing symptoms.

Citrate is rapidly metabolized, however,

so these manifestations are transient.56

Hypocalcemia is more likely to cause clin-

ical manifestations in patients who are in

shock or are hypothermic. Prolonged

apheresis procedures put patients, and

occasionally blood donors, at some risk.

Exchange transfusion, especially in tiny

infants who are already ill, requires care-

ful attention to all electrolytes.

A decrease in ionized calcium increases

neuronal excitability, leading, in the awake

patient or apheresis donor, to symptoms of

perioral and peripheral tingling, shivering,

and lightheadedness, followed by a diffuse

sense of vibration, tetanic symptoms such

as muscle cramps, fasciculations and

spasm, and nausea. In the central nervous

system, hypocalcemia is thought to in-

crease the respiratory center’s sensitivity to

CO2, causing hyperventilation. Because

myocardial contraction is dependent on

the intracellular movement of ionized cal-

cium, hypocalcemia also depresses cardiac

function.57

Treatment and Prevention. Massively

transfused patients, particularly those with

severe liver disease or those undergoing

Chapter 27: Noninfectious Complications of Blood Transfusion 649

rapid apheresis procedures such as periph-

eral blood progenitor cell collections, may

benefit from calcium replacement. It should

be noted, however, that empiric replace-

ment therapy in the era before accurate

monitoring of ionized calcium was avail-

able was associated with iatrogenic mortal-

ity.58 Usually, however, unless a patient or

donor has a predisposing condition that

hinders citrate metabolism, hypocalcemia

due to citrate overload requires no treat-

ment other than slowing the infusion. Cal-

cium must never be added directly to the

blood container because the blood will clot.

Hypothermia

Pathophysiology and Manifestations.

Ventricular arrhythmias may occur in pa-

tients who receive rapid infusions of large

volumes of cold blood, and they can be

prevented by blood warming.59 The effect

of cold blood is presumed to be more

likely if the blood is administered via cen-

tral catheters positioned close to the car-

diac conduction system.60 Hypothermia

increases the cardiac toxicity of hypo-

calcemia or hyperkalemia and can result

in poor left ventricular performance.

Other complications of hypothermia in-

clude impaired hemostasis61 and in-

creased susceptibility to wound infec-

tions.62 Blood warming is a must during

massive transfusion of cold blood.

Treatment and Prevention. Hypother-

mia-induced arrhythmias are reduced by

avoiding rapid infusion of cold blood into

the cardiac atrium. Generalized effects of

hypothermia can be prevented by using

blood warmers. AABB Standards for Blood

Banks and Transfusion Services mandates

that warmers have a temperature monitor

and a warning system to detect malfunc-

tion and prevent hemolysis.17(p6) Attention to

proper protocol is critical during the use of

blood warming devices because overheat-

ing destroys red cells and has caused fatali-

ties.5

Hyperkalemia and Hypokalemia

Pathophysiology. When red cells are stored

at 1 to 6 C, the potassium level in the super-

natant plasma or additive solution in-

creases. Although the concentration in the

plasma/anticoagulant portion of an RBC

unit may be high (see Chapter 8), because

of the small volume, the total extracellular

potassium load is less than 0.5 mEq for

fresh units and only 5 to 7 mEq for units at

their outdate. This rarely causes hyperkale-

mic problems in the recipient because

rapid dilution, redistribution into cells, and

excretion blunt the effect. Hypokalemia is

probably more often observed63 because

potassium-depleted red cells reaccumulate

this intracellular ion, and citrate metabo-

lism causes movement of potassium into

the cells in response to the consumption

of protons. Hyperkalemia may be a prob-

lem in patients with renal failure and in

premature infants and newborns receiv-

ing relatively large transfusions, such as in

cardiac surgery or exchange transfusion;

otherwise, it can be demonstrated only as

a transient effect in very rapid transfusion.

Treatment and Prevention. No treat-

ment or preventive strategy is usually nec-

essary, provided the patient is adequately

resuscitated from whatever condition re-

quired the massive transfusion.63 For large-

volume transfusion to sick infants or adults

at risk, many professionals prefer red cells

that are no more than 5 to 14 days old or

washed units. However, for infants receiv-

ing small-volume transfusions infused

slowly, units may be used safely until their

expiration date.64 Thereisnoevidencethat

routine red cell transfusions require manip-

ulation to lower potassium levels, even in

patients with no renal function.

650 AABB Technical Manual

Coagulopathy in Massive Transfusion

Pathophysiology. Of greater concern is the

occurrence of coagulopathy during mas-

sive transfusion. Classically, this coagulo-

pathy is ascribed to dilution of platelets

and clotting factors, which occurs as pa-

tients lose hemostatically active blood. The

lost blood is initially replaced with red

cells and asanguinous fluids. Classic stud-

ies of military65 and civilian66 trauma

patients receiving stored Whole Blood

demonstrated a progressive increase in the

incidence of “microvascular bleeding” (MVB)

characteristic of a coagulopathy with in-

creasing transfusion, typically occurring af-

ter replacement of two to three blood vol-

umes (20 to 30 Whole Blood units). Although

platelet counts, coagulation times, and lev-

els of selected clotting factors all correlated

with volume transfused, contrary to ex-

pectations from a simple dilutional model,

the relationship was marked by tremen-

dous variability. Inspection of laboratory

parameters in the patients developing a

bleeding diathesis, as well as the response

to various hemostatic components, sug-

gested that platelet deficits were more im-

portant in causing the bleeding than were

coagulation factor deficiencies. MVB typi-

cally occurred when the platelet count fell

below 50,000 to 60,000/µL. On the other

hand, no simple relationship could be de-

termined between a patient’s coagulation

tests and the onset of bleeding.

Subsequent studies have refined these

observations. Significant platelet dysfunc-

tion has been demonstrated in massively

transfused trauma patients.67,68 In the stud-

iesofCountsandcoworkers,

66,69 low fibrino-

gen and platelet levels were better pre-

dictors of hemostatic failure than elevations

of prothrombin time (PT) and partial

thromboplastin time (PTT), suggesting that

consumption coagulopathy was an impor-

tant factor in addition to dilution. A similar

conclusion was reached by Harke and

Rahman,70 who showed that the degree of

platelet and clotting abnormalities corre-

lated with the length of time the patient

washypotensive,ingroupsofpatientsre

ceiving similar transfusion volumes, also

suggesting that the most important cause

wasDICduetoshock.Takingthesedatato

gether, Collins71 concluded that “...coagulo-

pathy in heavily transfused patients was due

to hypoperfusion, not transfusion.”

These data may not be generalizable to

patients undergoing massive transfusion in

the “clean” setting of the operating room,

where hypotension due to volume loss is

prevented. In this setting, coagulation fac-

tor levels may indeed have priority over

platelet problems.72

Treatment and Prevention. The dilu-

tional model of coagulopathy in massive

transfusion would suggest that prophylactic

replacement of hemostatic components

based on the volume of red cells or whole

blood transfused would prevent develop-

ment of a bleeding diathesis. However, pro-

spective studies have consistently shown

that such regimens do not work,73 perhaps

due to patient variability. Instead, replace-

ment of platelets and coagulation factors in

the massively transfused trauma or surgical

patient should be based on characteriza-

tion of the specific abnormality by use of

platelet counts, the PT (international nor-

malized rate), aPTT, and fibrinogen levels.

Thromboelastography may also be useful.

It is imperative that the laboratory rapidly

complete testing. Empiric therapy with

platelets and/or plasma may be initiated

immediately after specimens are obtained.

Air Embolism

Air embolism can occur if blood in an open

system is infused under pressure or if air

enters a central catheter while containers

or blood administration sets are being

Chapter 27: Noninfectious Complications of Blood Transfusion 651

changed. It has been reported in associa-

tion with intraoperative and perioperative

blood recovery systems that allow air into

the infusion bag.7The minimum volume

of air embolism that is potentially fatal for

an adult is approximately 100 mL.74 Symp-

toms include cough, dyspnea, chest pain,

and shock.

If air embolism is suspected, the patient

should be placed on the left side with the

head down, to displace the air bubble from

the pulmonic valve. Aspiration of the air is

sometimes attempted. However, proper use

of infusion pumps, equipment for blood re-

covery or apheresis, and tubing couplers is

still essential to prevent this complication.

Evaluation of a Suspected

Acute Transfusion Reaction

The Role of Clinical Personnel Attending

the Patient

Medical personnel attending the patient

are generally the first to suspect that a

transfusion reaction has occurred and the

firsttotakeaction.Theappropriateactions

should be specified in the institution’s pa-

tient care procedures manual, and trans-

fusion service personnel should be pre-

pared to act as consultants.

1. If a transfusion reaction is suspected,

the transfusion should be stopped to

limit the volume of blood infused.

2. All labels, forms, and patient identi-

fication should be checked to deter-

mine whether the transfused compo-

nent was intended for the recipient.

3. An intravenous line should be main-

tained with normal saline (0.9% so-

dium chloride), at least until a medi-

cal evaluation of the patient has been

completed.

4. Thetransfusionserviceandthepa

tient’s physician should be notified

immediately. A responsible physician

should evaluate the patient to deter-

mine whether a transfusion reaction

is a possibility, what kind it might be,

and what immediate actions should

be undertaken. The possibilities of acute

hemolytic reaction, anaphylaxis, trans-

fusion-induced sepsis, and TRALI

should be kept in mind because these

conditions require aggressive medical

management and must be reported

promptly to the laboratory.

5. If the observed events are limited to

urticaria or circulatory overload, the

transfusion service need not evalu-

ate postreaction blood samples. If there

are signs and symptoms other than

urticaria or circulatory overload,

particularly if there is any possibility

of acute HTR, anaphylaxis, TRALI,

transfusion-induced sepsis, or other

serious problem, a postreaction blood

sample(s) should be sent to the labo-

ratory for evaluation. The speci-

men(s) must be carefully drawn to

avoid mechanical hemolysis and

must be properly labeled. In addi-

tion, the transfusion container with

whatever contents remain, the ad-

ministration set (without the nee-

dle), and the attached intravenous

solutions should be sent to the labo-

ratory, following standard precau-

tions. In some cases, a postreaction

urine sample will be useful.

The Role of the Laboratory

Whenever hemolysis is a possibility, the

laboratory should perform three steps as

soon as possible after receiving notifica-

tion and the clinical material: check for

clerical errors; perform a visual check for

hemolysis; and check for evidence of blood

group incompatibility by performing a di-

rect antiglobulin test (DAT) and a recon-

652 AABB Technical Manual

firmation of the recipient’s ABO type. Some

laboratories do not follow this sequence

when the only manifestations are urtica-

rial or febrile reactions to ABO-compati-

ble platelets.

Check for Identification Errors

The identification of each patient’s sam-

ple and the blood component(s) must be

checked for errors. If an error is discov-

ered, the patient’s physician or other re-

sponsible health-care professional must

be notified immediately, and a search of

appropriate records should be initiated to

determine whether misidentification or

incorrect issue of other specimens or

components has put other patients at risk.

Once the acute crisis has passed, each step

of the transfusion process should be re-

viewed to find the source of error.

Visual Check for Hemolysis

The serum or plasma in a postreaction

blood specimen must be inspected for ev-

idence of hemolysis and compared with a

prereaction sample, if available. Pink or

red discoloration after, but not before, the

reaction suggests destruction of red cells

and release of free hemoglobin. Intravas-

cular hemolysis of as little as 2.5 mL of red

cells may produce visible hemoglobinemia.75

Hemolysis resulting from poor collection

technique or other medical interventions

can cause hemoglobinemia; if faulty sam-

pling is suspected, examination of a sec-

ond specimen should resolve the question.

Myoglobin, released from injured muscle,

may also cause pink or red plasma and

might be suspected if a patient has suf-

fered severe trauma or muscle injury.76(p369)

Ifthesampleisnotdrawnuntil5to7

hours after an episode of acute hemolysis,

hemoglobin degradation products, espe-

cially bilirubin, may be in the blood-

stream and cause yellow or brown discol-

oration. An increase in bilirubin may begin

as early as 1 hour after the reaction, peak

at 5 to 7 hours, and disappear within 24

hours if liver function is normal.

In examining a postreaction urine speci-

men, it is important to differentiate among

hematuria (intact red cells in the urine),

hemoglobinuria (free hemoglobin in the

urine), and myoglobinuria (free myoglobin

in the urine). In acute HTRs, free hemoglo-

bin released from damaged cells can cross

the renal glomeruli and enter the urine, but

hematuria and myoglobinuria would not

be expected. Urine examination should be

done on the supernatant fluid after centri-

fugation of a freshly collected specimen;

misinterpretation can occur if free hemo-

globin is released when previously intact

red cells in a specimen undergo in-vitro

hemolysis during transportation or storage.

Serologic Check for Incompatibility

A DAT must be performed on a postreac-

tion specimen, preferably one anticoagu-

lated with a chelating agent (such as EDTA)

to avoid in-vitro coating of red cells by

complement proteins. If the postreaction

DAT is positive, a DAT should be performed

on red cells from the pretransfusion spec-

imen (unless this was already done as part

of pretransfusion testing) and compared.

If transfused incompatible cells have been

coated with antibody but not immediately

destroyed, the postreaction specimen DAT

is likely to be positive, often with a mixed-

field agglutination pattern. If the trans-

fused cells have been rapidly destroyed,

the postreaction DAT may be negative,

particularly if the specimen is drawn sev-

eral hours later. If both the pre- and post-

reaction DATs are positive, further work-

up is required to rule out incompatibility.

Comparison of the graded strength of these

two tests is not a reliable method to rule

this out. Nonimmune hemolysis (eg, from

Chapter 27: Noninfectious Complications of Blood Transfusion 653

thermal damage or mechanical trauma)

causes hemoglobinemia but not a posi-

tive DAT. The recipient’s ABO type must

also be confirmed on the postreaction

specimen.

Additional Laboratory Evaluation

If any of the three initial checks and tests

(error check, visual inspection for hemo-

globinemia, DAT and ABO confirmation)

gives positive or suspicious results, the di-

agnosis of an acute HTR should be vigor-

ously pursued. Even if no error or apparent

incompatibility is found, the possibility of

an acute HTR should still be considered if

the patient’s clinical presentation is con-

sistent with such a reaction. The tests

listed below help characterize the cause

of the HTR, if one has occurred, or help

clarify the immunologic and serologic sta-

tusofpatientsinwhomthediagnosisis

unclear. Some or all may be performed

following a written institutional protocol

or at the discretion of the physician in

charge of the transfusion service.

1. If ABO and Rh typing on the prereac-

tion and postreaction samples do not

agree, there has been an error in pa-

tient or sample identification, or in

testing. If sample mix-up or misla-

beling has occurred, another pa-

tient’s specimen may also have been

incorrectly labeled; it is important to

check the records of all specimens

received at approximately the same

time.

2. Perform ABO and Rh testing on blood

from the unit or an attached seg-

ment. If blood in the bag is not of the

ABO type noted on the bag label,

there has been an error in unit label-

ing.

3. Perform antibody detection tests on

the prereaction and postreaction

samples and on the donor blood. If a

previously undetected antibody is

discovered, it should be identified.

Once the antibody has been identi-

fied, retained samples from transfused

donor units should be tested for the

corresponding antigen. If a previ-

ously undiscovered antibody is pres-

ent in a postreaction specimen but

not in a prereaction sample, the rea-

son may be 1) a sample identifica-

tion error, 2) anamnestic antibody

production after a recent transfusion,

or, less likely, 3) passive transfer of

antibody from a recently transfused

component. It may be desirable to use

enhancement techniques, such as an

increased serum-to-cell ratio, low-

ionic-strength saline, Polybrene, poly-

ethylene glycol, or enzyme techni-

ques, when retesting the prereaction

specimen.

4. Repeat crossmatch tests, with pre-

reaction and postreaction samples in

parallel using the antiglobulin tech-

nique. A positive crossmatch in the

face of a negative antibody screen-

ingtestmayindicatethepresenceof

an antibody directed against a low-

incidence blood group antigen.

5. Perform DAT and antibody detection

tests on additional specimens ob-

tained at intervals after the transfu-

sion reaction. A first postreaction

sample may have serologically un-

detectable levels of a significant allo-

antibody, especially if all the anti-

body molecules have attached to the

incompatible transfused cells. In this

event, antibody levels would rise

rapidly, and antibody detection and

identification would become possi-

ble within a few days.

6. Perform frequent checks of the pa-

tient’s hemoglobin values, to see

whether the transfused cells produce

the expected therapeutic rise, or

654 AABB Technical Manual

whether a decline occurs after an

initial increase. In patients with sickle

cellanemia,thesurvivaloftrans

fused red cells can be followed by

evaluation of the levels of hemoglo-

bin A. In complex cases, phenotypic

differences between autologous and

transfused cells quantitated by flow

cytometry have been used to follow

survival.77

7. In-vivo red cell survival studies have

been used to demonstrate the rare

occurrence of an acute HTR in the

absence of detectable alloantibody.78

When the patient is phenotyped in

preparation for such studies, it is im-

portant that the sample be one that

contains only the patient’s red cells.

This may be difficult if the patient

has received transfusions within the

previous several weeks. Method 2.15

gives a technique for obtaining auto-

logous red cells from a patient who

has been transfused. If an antigen is

present on the donor’s red cells and

absent from those of the patient, its

presence or absence in postreaction

samples indicates whether the trans-

fused cells have survived and re-

mained in the circulation.

8. Markers of hemolysis, including lac-

tate dehydrogenase, unconjugated

bilirubin, and haptoglobin levels,

may be useful, particularly if pre-

and multiple postreaction measure-

ments are available.

9. Examine the blood remaining in the

unit and the administration tubing

for evidence of hemolysis, especially

if no immune explanation for hemo-

lysis can be demonstrated. Depending

on how the blood was handled and

administered, hemolysis may be

present in the container and the ad-

ministration tubing, or only in the

administration tubing. For example,

if the unit had been inappropriately

heated in the container, both the

blood in the container and in the

administration tubing would be

hemolyzed. If a faulty infusion de-

vice had been used during blood

administration, hemolysis might be

present in the administration tubing,

but not in the container.

Additional testing may also be useful for

significant nonhemolytic reactions.

1. If the presentation suggests an ana-

phylactic reaction, test the patient’s

serum for the presence of anti-IgA.

Preliminary information can be ob-

tained by quantitation of IgA because

most patients with IgA-related ana-

phylaxis have been IgA deficient.27

Note, however, that subclass- or allo-

type-specific antibodies may de-

velop in patients with normal IgA

levels.26 If additional transfusions are

required, cellular components can

be washed; if plasma (or platelets;

see above) is required, IgA-deficient

plasma can be used.

2. Examine the returned unit for any

abnormal appearance, including clots

or any brownish, opaque, muddy, or

purple discoloration. If the clinical

presentation suggests bacterial sep-

sis, a Gram stain and bacterial cul-

tures of the contents should be per-

formed, even if the unit looks normal.

Blood cultures should also be per-

formed on the patient’s blood.79

Treatment for suspected bacterial

contamination should be based on

clinical considerations because a de-

lay in therapy may result in severe

morbidity or death. Treatment in-

cludes prompt intravenous adminis-

tration of broad-spectrum antibiot-

ics after blood and other appropriate

cultures have been obtained, combined

with therapy for shock, if present.

Chapter 27: Noninfectious Complications of Blood Transfusion 655

3. If the clinical presentation suggests

TRALI, test the patient’s pretransfu-

sion sample and a sample of the do-

nor’s plasma for antibodies to HLA and

neutrophil antigens. Crossmatching

recipient lymphocytes or granulocytes

with implicated donor sera can pro-

vide supportive evidence for TRALI.

Delayed Consequences of

Transfusion

Alloimmunization to Red Cell Antigens

Pathophysiology

Primary alloimmunization, evidenced by

the appearance of newly formed antibod-

ies to red cell antigens, becomes apparent

weeks or months after transfusion. It has

been estimated that alloimmunization

occurs in unselected immunocompetent

recipients with a risk of 1% to 1.6% per

RBC unit, provided that D-negative recip-

ients receive D-negative cellular compo-

nents.80 Hemolysis has been reported in

cases of primary immunization, but these

reports are controversial,81 and, even if it

occurs, the phenomenon must be very

rare and usually subclinical.

Serologic Observations. Once allo-

immunization has occurred, blood group

antibodies can become undetectable, espe-

cially those of the Kidd system. One investi-

gator reported that this occurred to 29% of

antibodies after a median of 10 months82

and to 41% of antibodies after 5 or more

years.83 If red cells that express the antigen

are subsequently transfused, however, an

anamnestic response may cause the ap-

pearance, within hours or days, of IgG anti-

bodies that react with the transfused red

cells. In a prospective study, previously un-

detected alloantibodies were found in 58 of

2082 (2.8%) recipients (37% known previ-

ously transfused, 36% previously pregnant)

within 7 days of transfusion.84 In two of the

58, only the DAT was positive, but, in all

others, repeat antibody screening would

have detected the new antibody. Regard-

less, characterization of an eluate is neces-

sary because alloantibodies may be present

on the red cells that are not in the serum. If

the clinical laboratory discovers an anam-

nestic response, both the transfusion ser-

vice director and the patient’s clinician

should be notified and the possibility of a

delayed HTR (DHTR) should be investigated.

Delayed Reactions. In most cases, ana-

mnestic antibody production does not cause

detectable hemolysis, leading to the desig-

nation “delayed serologic transfusion reac-

tion” (DSTR).85 However, in some patients,

clinically apparent hemolysis will result

from the combination of significant levels

of antibody with hemolytic potential and

large numbers of transfused red cells in the

circulation; in the study cited above, only

one of the 58 recipients with a new anti-

body within 7 days of transfusion had clini-

cally evident hemolysis.84 This translated

into a DHTR rate of one per 2082 recipients,

or one for every 11,328 units transfused. As

would be expected, retrospective studies,

which would be similar to the routine expe-

rience of a transfusion service, yield a lower

rate of DSTRs, but the rate of clinically de-

tectable hemolysis may be roughly equiva-

lent.85-87 Themostcommonpresentationof

a DHTR is a declining hemoglobin and a

newly positive antibody screen, but fever,

leukocytosis, and mild jaundice may be

present. Some DHTRs present as the ab-

sence of the anticipated increase in hemo-

globin after transfusion. Other clinical

problems are infrequent; hemoglobinuria is

occasionally noted, but acute renal failure

is uncommon. However, DHTRs may be

particularly problematic in patients with

sickle cell disease. In these patients,

hemolysis may include autologous red

cells, a phenomenon termed sickle cell

656 AABB Technical Manual

hemolytic transfusion reaction syndrome

(see Chapter 24).88

If a DHTR is suspected, a freshly ob-

tained blood sample may be tested for un-

expected alloantibodies, both in the serum

and, by DAT, on the red cells. Discovery of a

new red cell alloantibody in a recently

transfused patient with hemolysis strongly

suggests a DHTR, and the diagnosis is sup-

ported by demonstration of the corre-

sponding antigen on the red cells from a

retained segment from one or more trans-

fused units. Antigen typing of the red cells

circulating in the patient may also suggest

whether the newly incompatible cells have

been eliminated, or whether some are still

circulating. Repeat antibody screening on

the patient’s previous specimen will rule

out technical errors.

Treatment

Specific treatment is rarely necessary, al-

though it may be prudent to monitor the

patient’s urine output and renal function

and observe for changes in coagulation

function. If transfusion is still necessary,

donor red cells should lack the antigen

corresponding to the newly discovered

antibody. Passenger lymphocyte hemolysis,

seen after solid organ transplantation, is a

variant of DHTR and is covered in Chap-

ter 26.

Prevention

Future transfusions for the patient should

lack the antigen(s) responsible for the

anamnestic response, even if the antibody

again becomes undetectable. Some facili-

ties issue a medical alert card with this in-

formation for the patient to carry and

present at the time of hospitalization or

transfusion in a different facility. It is to

prevent these problems that Standards for

Blood Banks and Transfusion Services

mandates permanent preservation of re-

cords of clinically significant antibodies,

and review of previous records before red

cells are issued for transfusion.17(pp39,72) Pro-

spective antigen matching may prevent

DHTRs in selected patients, particularly

those with sickle cell disease (see Chap-

ters 21 and 24).89

Posttransfusion Autoantibody

Occasionally, transfusion of allogeneic red

cells and platelets stimulates production

of autoantibodies; in some of these pa-

tients, hemolytic anemia or thrombocyto-

penia may occur.90 See Chapter 20 for more

details.

Alloimmunization to Leukocyte Antigens

and Refractoriness to Platelet

Transfusions

See Chapter 16 and Chapter 17.

Transfusion-Associated Graft-vs-Host

Disease

Transfusion-associated graft-vs-host dis-

ease is a usually fatal immunologic trans-

fusion complication caused by engraft-

ment and proliferation of donor lympho-

cytes in a susceptible host.91 The engrafted

lymphocytes mount an immunologic at-

tack against the recipient tissues, includ-

ing hematopoietic cells, leading to refrac-

tory pancytopenia with bleeding and

infectious complications, which are pri-

marily responsible for the 90% to 100%

mortality rate in afflicted patients. TA-

GVHD is rare in US transfusion recipients

and has been observed almost exclusively

in immunocompromised patients. In con-

trast, over 200 cases of TA-GVHD have

been described in Japan,92 with incidence

rates reaching 1:660 in patients undergo-

ing cardiovascular surgery.93 Greater ge-

netic homogeneity of the Japanese popu-

lation and frequent use of fresh Whole

Chapter 27: Noninfectious Complications of Blood Transfusion 657

Blood from related donors are thought to

be the primary reasons for the surpris-

ingly frequent occurrence of TA-GVHD in

that country. The SHOT data14 demon-

strate a significant decline in the inci-

dence of TA-GVHD since the introduction

of universal leukocyte reduction, but two

cases occurred despite leukocyte reduction.

In the first 3 years of this study, the rate of

TA-GVHD was approximately 1 per 600,000

cellular components transfused and, in

the first 5 years, accounted for a greater

number of deaths than acute HTRs and

only slightly fewer than did TRALI.

Pathophysiology and Manifestations

The pathophysiology of TA-GVHD is com-

plex and incompletely understood. The

overall mechanism includes the escape of

donor T lymphocytes present in cellular

blood components from immune clearance

in the recipient and subsequent prolifera-

tion of these cells, which then mount an

immune attack on host tissues. Manifes-

tations include fever, enterocolitis, rash,

hepatitis, and pancytopenia. The rash

typically begins as a blanching, macu-

lopapular erythema of the upper trunk,

neck,palms,soles,andearlobes,which

becomes confluent with additional find-

ings ranging from edema to widespread

blistering. Skin biopsy reveals infiltration

of the upper dermis by mononuclear cells

and damage to the basal layer of epithe-

lial cells. Hepatitis manifests as elevations

in alanine and aspartate aminotrans-

ferases, alkaline phosphatase, and biliru-

bin. Enterocolitis causes anorexia, nausea,

and up to 3 to 4 liters per day of secretory

diarrhea. Pancytopenia is associated with

a hypocellular marrow. Symptoms typi-

cally appear within 8 to 10 days of the

transfusion but may occur as early as 3

days and as late as 30 days. The diagnosis

is proven by demonstration of donor-de-

rived lymphocytes in the recipient’s pe-

ripheral blood or tissues by HLA typing.91

Factors that determine an individual pa-

tient’s risk for TA-GVHD include whether and

to what degree the recipient is immunode-

ficient, the degree of HLA similarity be-

tween donor and recipient, and the num-

ber and type of T lymphocytes transfused

that are capable of multiplication.91 TA-GVHD

may occur in an immunologically normal

recipient if the donor is homozygous for an

HLA haplotype for which the recipient is

heterozygous, a so-called “one-way” HLA

match, and if the component contains via-

ble T cells (the fresher the unit, the higher

the risk). Cytokine dysfunction, recruitment

of host cells into the immune reaction, and

release of biologic mediators, in particular

nitric oxide, all play a role in the pathogene-

sis.94 Of interest is the fact that TA-GVHD

has not been reported in an AIDS patient.

Treatment and Prevention

Treatment of TA-GVHD with immunosup-

pressive agents has been attempted but

rarely succeeds, so prevention is neces-

sary. Irradiation of cellular blood compo-

nents is the accepted standard method to

prevent TA-GVHD. The dose mandated by

the FDA is a minimum of 25 Gy targeted

to the midline of the container and a min-

imum dose of 15 Gy delivered to all other

parts of the component.95 This renders T

lymphocytes incapable of replication

without substantially affecting the function

of red cells, platelets, and granulocytes.

AABB Standards for Blood Banks and

Transfusion Services requires routine irradi-

ation of cellular components from units

collected from the recipient’s blood rela-

tives, and donors selected for HLA compat-

ibility by typing or crossmatching.17(p43) Poli-

cies should be in place to define the other

groups of patients who should receive irra-

diated cellular components, and there must

658 AABB Technical Manual

be a process for ensuring that once a pa-

tient has been determined to be at risk for

TA-GVHD, all cellular components will be irra-

diated as long as clinically indicated.

Published guidelines96 additionally rec-

ommend component irradiation for: 1)

hematopoietic progenitor cell (HPC) trans-

plant recipients (this includes allogeneic

and autologous HPC transplants), 2) pa-

tients with hematologic disorders who will

be undergoing allogeneic HPC transplanta-

tion imminently, 3) intrauterine transfu-

sions, 4) neonates undergoing exchange

transfusion or use of extracorporeal mem-

brane oxygenation, 5) patients with Hodg-

kin’s disease, and 6) patients with congeni-

tal cellular immunodeficiencies. TA-GVHD

has also been reported in patients with

acute lymphoid and myeloid leukemias,

chronic lymphocytic leukemia particularly

in patients receiving fludarabine phos-

phate,91 patients with B-cell malignancies

including non-Hodgkin’s lymphoma,

myeloma, and Waldenstrom’s macroglobu-

linemia,14 premature or low-birthweight in-

fants without specific immunodeficiency

disorders, and children being treated for

neuroblastoma and rhabdomyosarcomas.91

Posttransfusion Purpura

Pathophysiology and Manifestations

Posttransfusion purpura (PTP) is an un-

common event, although over 200 cases

have been published. It is characterized by

the abrupt onset of severe thrombocyto-

penia (platelet count usually <10,000/µL)

an average of 9 days after transfusion

(range, 1-24 days).97 Components provok-

ing the reaction have usually been RBCs

or Whole Blood, but PTP has also been

reported after platelet and plasma trans-

fusion, and after transfusion of frozen

deglycerolized RBCs. Most patients have

previously been pregnant or transfused.

“Wet purpura” is common, and fatal intra-

cranial hemorrhage can occur. The ratio

of affected patients is five women to one

man, and the median age is 51 years

(range, 16-83). Most cases (68%) involve

patients whose platelets lack the HPA-1a

(PlA1) antigen (<2% of the population) and

who form the corresponding antibody.

However, immunization to HPA-1b is re-

ported in 10%, and other platelet antibod-

ies, including HLA antibodies, have been

associated with the syndrome as well. PTP

is usually self-limited, with full recovery

within 21 days. Historically, 10% to 15% of

patients have been reported to die from

PTP, typically from intracranial bleeding,

so treatment is desirable.

The reason for destruction of the pa-

tient’s own platelets by what appears to be

a platelet alloantibody is controversial.

Three mechanisms have been proposed,

including: 1) formation of immune com-

plexes of patient antibody and soluble do-

nor antigen that bind to Fc receptors on the

patient’s platelets and mediate their de-

struction, 2) conversion of antigen-negative

autologous platelets to antibody targets by

soluble antigen in the transfused compo-

nent, and 3) cross-reactivity of the patient’s

antibodies with autologous platelets (ie, the

presence of an autoantibody component).

The last of these theories has received the

most support.

Treatment

Because PTP remits spontaneously, treat-

ment may appear falsely efficacious. Ste-

roids are frequently given but their role is

controversial. Plasma exchange can achieve

platelet counts of 20,000/µLin1to2days,

but the use of high-dose Immune Globu-

lin Intravenous (IGIV) is now supplanting

this therapy.98,99 With the use of IGIV, re-

covery to platelet counts of 100,000/µLis

typically achieved within 3 to 5 days. As it

does in other disorders such as immune

Chapter 27: Noninfectious Complications of Blood Transfusion 659

thrombocytopenic purpura, IGIV appears

to block antibody-mediated clearance of

the target cells, although the mechanism

of action has not been established. If ran-

domly selected platelets are transfused,

patients may experience a febrile transfu-

sion reaction, and, in the vast majority of

cases, such transfusions have not been ef-

ficacious. Antigen-negative platelets can

be of benefit in PTP, and, in conjunction

with IGIV, reversal of this disorder in 1 day

is now possible.100,101 Unfortunately, the

time necessary to procure such platelets

often limits their usefulness. After recov-

ery, some authors have suggested that fu-

ture transfusions should be from donors

lacking the offending antigen, or, if they

are not available, washed platelet units.97

Immunomodulatory Effects of Transfusion

Transfusion has been known to modulate

immune responses since the 1973 obser-

vation by Opelz and coworkers102 of im-

provedrenalallograftsurvivalintrans-

fused patients. This beneficial tolerance-

inducing effect of transfusion raised con-

cerns that transfusion may have other ad-

verse effects in different clinical settings,

including increased rates of postoperative

solid tumor recurrence and bacterial in-

fection.103 Despite numerous retrospective

and several large prospective studies, the

clinical significance of transfusion-associ-

ated immunomodulation and the useful-

ness of preventive strategies, such as leu-

kocyte reduction of transfused components,

remain controversial.104,105

Iron Overload

Every RBC unit contains approximately

200 mg of iron. Chronically transfused

patients, especially those with hemoglo-

binopathies, have progressive and contin-

uous accumulation of iron and no phy-

siologic means of excreting it. Storage oc-

curs initially in reticuloendothelial sites,

but when they are saturated, there is de-

position in parenchymal cells. The

threshold for clinical damage is lifetime

exposure to greater than 50 to 100 RBC

units in a nonbleeding person.106 Iron de-

position interferes with function of the

heart, liver, and endocrine glands (eg,

pancreatic islets, pituitary); hepatic fail-

ure and cancer, diabetes mellitus, and car-

diac toxicity cause most of the morbidity

and mortality. Elevated ferritin levels

demonstrate increased iron stores, and tis-

sue damage can be shown with organ-spe-

cific assays such as liver enzyme levels or

endocrine function tests (eg, glucose, thy-

roid-stimulating hormone).

Treatment is directed at removing iron

without reducing the patient’s circulating

hemoglobin. Metered subcutaneous infu-

sion of desferoxamine, an iron-chelating

agent, can reduce body iron stores in such

patients, but the regimen of nightly subcu-

taneous infusion by pump is arduous and

expensive, and compliance is often poor. In

transfusion-dependent patients with hemo-

globinopathies, red cell exchange can mini-

mize additional iron loads and can reduce

the total iron burden.107 An oral iron che-

lator has been studied but is not yet avail-

able in the United States.

Records of Transfusion

Complications

Each transfusion service must maintain

indefinitely the records of patients who

have had transfusion complications or ev-

idence of alloimmunization. Possible cases

of contaminated blood must be reported

to the institution where the blood was drawn.

Records must be kept, and consulted, to

prevent patients who have had a transfu-

sion reaction from having a recurrence with

660 AABB Technical Manual

subsequent transfusions. For example, pa-

tients with a history of IgA-related anaphy-

lactic reactions should be transfused with

plasma products that lack IgA. A history of

repeated or severe FNHTRs might prompt

the use of leukocyte-reduced cellular blood

components. Red cell alloantibodies may

become undetectable over time as dis-

cussed above,82,83 so records should be

checked and compatible blood issued in

order to prevent a DHTR. Routine checking

of previous results of ABO and Rh testing

may disclose an error in testing or in the

identification of a current sample.

Records of Patients with Special Needs

In addition to records of transfusion reac-

tions, transfusion services should maintain

records of patients who need specially

prepared or manipulated components.

This is especially important in institu-

tions where physicians rotate frequently,

and the need for irradiated, leukocyte-re-

duced, or IgA-deficient components may

not be known to a particular physician

writing an individual order.

Reporting Transfusion Fatalities

When a complication of blood transfusion

has been confirmed to be fatal, it must be

reported to the Director, Office of Compli-

ance, Center for Biologics Evaluation and

Research, FDA, as soon as possible, with a

written report within 7 days (see Chapter

28 for reporting information). Patients

who are critically ill and near death often

receive transfusions in close temporal

proximity to death, and clinical suspicion

of cause and effect may occasionally be

raised. The overwhelming majority of

such deaths are unrelated to transfusion,

but if there is a suggestion that a transfu-

sion might have contributed to death, it

may be prudent to pursue an investiga-

tion.

In the absence of such errors as adminis-

tration of ABO-incompatible blood or of

physiologic events clearly attributable to

acutehemolysis,anaphylaxis,TRALI,or

sepsis, transfusion is highly unlikely to be

acutely responsible for death. The review

should include all available medical and

laboratory records and the results of an au-

topsy, if performed. On the other hand, if

an investigation does reveal evidence or the

possibility of hemolysis, anaphylactic or

pulmonary events, unexplained sepsis, or

ambiguous identification records, the case

may warrant more extensive inquiry.

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44. Kleinman S, Caulfield T, Chan P, et al. Toward

an understanding of transfusion-related acute

lung injury: Statement of a consensus panel.

Transfusion 2004;44:1774-89.

45. Popovsky MA, Moore SB. Diagnostic and patho-

genetic considerations in transfusion-re-

lated acute lung injury. Transfusion 1985;25:

573-7.

46. Popovsky MA, Haley NR. Further character-

ization of transfusion-related acute lung in-

jury: Demographics, clinical and laboratory

features, and morbidity. Immunohematology

2000;16:157-9.

47. Kao GS, Wood IG, Dorfman DM, et al. Inves-

tigations into the role of anti-HLA class II

antibodies in TRALI. Transfusion 2003;43:

185-91.

48. Kopko PM, Paglieroni TG, Popovsky MA, et

al. TRALI: Correlation of antigen-antibody and

monocytes activation in donor-recipient pairs.

Transfusion 2003;43:177-84.

49. Seeger W, Schneider U, Kreusler B, et al. Re-

production of transfusion-related acute lung

injury in an ex vivo lung model. Blood 1990;

76:1438-44.

50. Dry SM, Bechard KM, Milford EL, et al. The

pathology of transfusion-related acute lung

injury. Am J Clin Pathol 1999;112:216-21.

51. McCullough J. Granulocyte transfusion. In:

PetzLD,SwisherSN,KleinmanS,etal,eds.

Clinical practice of transfusion medicine. 3rd

ed. New York: Churchill Livingstone, 1996:413-

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52. Silliman CC, Paterson AJ, Dickey WO, et al.

The association of biologically active lipids

with the development of transfusion-related

acute lung injury: A retrospective study.

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53. Silliman CC, Boshkov LK, Mehdizadehkashi

Z, et al. Transfusion-related acute lung injury:

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54. Audet AM, Popovsky MA, Andrzejewski C.

Transfusion-associated circulatory overload

in orthopedic surgery patients: A multi-insti-

tutional study. Immunohematology 1996;12:

87-9.

55. Bowman RJ, Laudi N, Aslan D, Burgher A. Use

of BNP to evaluate TRALI (abstract). Transfu-

sion 2004;44(Suppl):24A.

56. Dzik WH, Kirkley SA. Citrate toxicity during

massive blood transfusion. Transfus Med Rev

1988;2:76-94.

57. Olinger GN, Hottenrott C, Mulder DG, et al.

Acute clinical hypocalcemic myocardial de-

pression during rapid blood transfusion and

postoperative hemodialysis: A preventable

complication. J Thorac Cardiovasc Surg 1976;

72:503-11.

58. Howland WS, Jacobs RG, Goulet AH. An eval-

uation of calcium administration during rapid

blood replacement. Anesth Analg 1960;39:

557-63.

59. Boyan CP, Howland WS. Cardiac arrest and

temperature of bank blood. JAMA 1963;183:

58-60.

60. Iserson KV, Huestis DW. Blood warming: Cur-

rent applications and techniques. Transfusion

1991;31:558-71.

61. ValeriCR,FeingoldH,CassidyG,etal.Hypo-

thermia-induced reversible platelet dysfunc-

tion. Ann Surg 1987;205:175-81.

62. Sessler DI. Current concepts: Mild periopera-

tive hypothermia. N Engl J Med 1997;336:

1730-7.

63. Collins JA. Problems associated with the

massive transfusion of stored blood. Surgery

1974;174:274-95.

64. Liu EA, Manino FL, Lane TA. Prospective,

randomized trial of the safety and efficacy of

a limited donor exposure transfusion pro-

gram for premature neonates. J Pediatr 1994;

125:92-6.

65. Miller RD, Robbins TO, Tong MJ, Barton SL.

Coagulation defects associated with massive

blood transfusion. Ann Surg 1971;174:794-801.

66. CountsRB,HaischC,SimonTL,etal.Hemo

stasis in massively transfused trauma patients.

Ann Surg 1979;190:91-9.

67. Lim RC, Olcott C, Robinson AJ, Blaisdell FW.

Platelet response and coagulation changes

following massive blood replacement. J

Trauma 1973;13:577-82.

68. Harrigan C, Lucas CE, Ledgerwood KAM, et

al. Serial changes in primary hemostasis af-

ter massive transfusion. Surgery 1985;98:

836-43.

69. Ciavarella D, Reed RL, Counts RB, et al. Clot-

ting factor levels and the risk of microvascu-

lar bleeding in the massively transfused pa-

tient. Br J Haematol 1987;67:365-8.

70. Harke H, Rahman S. Haemostatic disorders

in massive transfusion. Bibl Haematol 1980;

46:179-88.

Chapter 27: Noninfectious Complications of Blood Transfusion 663

71. Collins JA. Recent developments in the area

of massive transfusion. World J Surg 1987;11:

75-81.

72. MurrayDJ,PennellBJ,WeinsteinSL,Olson

JD. Packed red cells in acute blood loss: Dilu-

tional coagulopathy as a cause of surgical

bleeding. Anesth Analg 1995;80:336-42.

73. Reed RL, Ciavarella D, Heimbach DM, et al.

Prophylactic platelet administration during

massive transfusion: A prospective, random-

ized, double-blind clinical study. Ann Surg 1986;

203:40-8.

74. O’QuinRJ,LakshminarayanS.Venousair

embolism. Arch Intern Med 1982;142:2173-6.

75. Elliott K, Sanders J, Brecher, ME. Transfusion

medicine illustrated. Visualizing the hemoly-

tic transfusion reaction. Transfusion 2003;43:

297.

76. Henry JB. Clinical diagnosis and management

by laboratory methods. 20th ed. Philadel-

phia: WB Saunders, 2001.

77. Nance ST. Flow cytometry in transfusion

medicine. In: Anderson KC, Ness PM, eds.

Scientific basis of transfusion medicine. Phil-

adelphia: WB Saunders, 1994:707-25.

78. Baldwin ML, Barrasso C, Ness PM, Garratty

G. A clinically significant erythrocyte antibody

detectable only by 51Cr survival studies.

Transfusion 1983;23:40-4.

79. Sazama K. Bacteria in blood for transfusion:

A review. Arch Pathol Lab Med 1994;118:350-

65.

80. Lostumbo MM, Holland PV, Schmidt PJ. Iso-

immunization after multiple transfusions. N

Engl J Med 1966;275:141-4.

81. Issitt PD, Anstee DJ. Applied blood group se-

rology. 4th ed. Durham, NC: Montgomery

Scientific Publications, 1998.

82. Ramsey G, Larson P. Loss of red cell antibod-

ies over time. Transfusion 1988;28:162-5.

83. Ramsey G, Smietana SJ. Long-term follow-up

testing of red cell alloantibodies. Transfusion

1994;34:122-4.

84. Heddle NM, Soutar RL, O’Hoski PL, et al. A

prospective study to determine the frequency

and clinical significance of alloimmunization

post-transfusion. Br J Haematol 1995;91:

1000-5.

85. Ness PM, Shirey RS, Thoman SK, Buck SA.

The differentiation of delayed serologic and

delayed hemolytic transfusion reactions: In-

cidence, long-term serologic findings, and

clinical significance. Transfusion 1990;30:

688-93.

86. Pinkerton PH, Coovadia AS, Goldstein J. Fre-

quency of delayed haemolytic transfusion re-

actions following antibody screening and im-

mediate-spin crossmatching. Transfusion 1992;

32:814-7.

87. Vamvakas EC, Pineda AA, Reisner R, et al. The

differentiation of delayed hemolytic and se-

rologic transfusion reactions: Incidence and

predictors of hemolysis. Transfusion 1995;35:

26-32.

88. Garratty G. Severe reactions associated with

transfusions of patients with sickle cell dis-

ease. Transfusion 1997;37:357-61.

89. Vichinsky EP, Luban NL, Wright E, et al. Pro-

spective RBC phenotype matching in a

stroke-prevention trial in sickle cell anemia:

A multicenter transfusion trial. Transfusion 2001;

41:1086-92.

90. Petz LD, Garratty G. Immune hemolytic

anemias. 2nd ed. Philadelphia: Churchill

Livingstone, 2004:335-40.

91. Webb IJ, Anderson KC. Transfusion-associated

graft-vs-host disease. In: Popovsky MA, ed.

Transfusion reactions. 2nd ed. Bethesda, MD:

AABB Press, 2001:171-86.

92. Ohto H, Anderson KC. Survey of transfusion-

associated graft-versus-host disease in im-

munocompetent recipients. Transfus Med

Rev 1996;10:31-43.

93. Juji T, Takahashi K, Shibata Y. Post-transfu-

sion graft versus host disease as a result of di-

rected donations from relatives (letter). N

Engl J Med 1989;321:56.

94. Ferrara JL, Krenger W. Graft-vs-host disease:

The influence of type 1 and type 2 cell cyto-

kines. Transfus Med Rev 1998;12:1-17.

95. Food and Drug Administration. Memorandum.

Recommendations regarding license amend-

ments and procedures for gamma irradiation

of blood products. (July 22, 1993) Rockville,

MD: CBER Office of Communication, Train-

ing, and Manufacturers Assistance, 1993.

96. Przepiorka D, LeParc GF, Stovall MA, et al. Use

of irradiated blood components. Practice pa-

rameter. Am J Clin Pathol 1996;106:6-11.

97. McFarland JG. Postransfusion purpura. In:

Popovsky MA, ed. Transfusion reactions. 2nd

ed. Bethesda, MD: AABB Press, 2001:187-212.

98. McLeod BC, Strauss RG, Ciavarella D, et al.

Management of hematological disorders and

cancers. J Clin Apheresis 1996;11:211-30.

99. Mueller-Eckhardt C, Kiefel V. High-dose IgG

for post-transfusion purpura revisited. Blut 1988;

57:163-7.

100. Brecher ME, Moore SB, Letendre L. Post-

transfusion purpura: The therapeutic value of

PlA1-negative platelets. Transfusion 1990;30:

433-5.

101. Win N, Matthey F, Slater NGP. Blood compo-

nents—Transfusion support in post-transfu-

sion purpura due to HPA-1a immunization.

Vox Sang 1996;71:191-3.

102. Opelz G, Senger DP, Mickey MR, Terasaki PI.

Effect of blood transfusions on subsequent

664 AABB Technical Manual

kidney transplants. Transplant Proc 1973;5:

253-9.

103. Blumberg N, Heal JM. Effects of transfusion

on immune function: Cancer recurrence and

infection. Arch Pathol Lab Med 1994;118:

371-9.

104. Blajchman MA. Allogeneic blood transfusions,

immunomodulation, and postoperative bac-

terial infection: Do we have the answers yet?

Transfusion 1997;37:121-5.

105. Vamvakas EC, Blajchman MA, eds. Immuno-

modulatory effects of blood transfusion.

Bethesda, MD: AABB Press, 1999.

106. Sharon BI, Honig GR. Management of con-

genital hemolytic anemias. In: Simon TL,

DzikWH,SnyderEL,etal,eds.Rossi’sprinci

ples of transfusion medicine. 3rd ed. Balti-

more, MD: Lipincott Williams and Wilkins,

2002:463-82.

107. Adams DM, Schultz WH, Ware RE, Kinney

TR. Erythrocytapheresis can reduce iron

overload and prevent the need for chelation

therapy in chronically transfused pediatric

patients. J Pediatr Hematol Oncol 1996;18:

46-50.

Chapter 27: Noninfectious Complications of Blood Transfusion 665

Chapter 28: Transfusion-Transmitted Diseases

Chapter 28

Transfusion-Transmitted

Diseases

MANY ADVANCES HAVE been

made in the testing of blood do-

nations for infectious diseases.

However, the risk of transmitting viral,

bacterial, and parasitic diseases via trans-

fusion still exists, and new agents may

appear at any time. Thus, infectious com-

plications of transfusion remain an im-

portant area of concern in transfusion

medicine.

Hepatitis

Hepatitis is inflammation of the liver that

can be caused by many different toxins,

immunologic processes, or infectious agents.

Hepatitis linked to transfusion is almost

exclusively caused by viruses. These viruses

include hepatitis viruses A-E (HAV, HBV,

HCV, HDV, HEV), cytomegalovirus (CMV),

Epstein-Barr virus (EBV), and possibly

newly described putative hepatitis viruses

(such as TTV and SEN-V). Infectious agents

pose a serious threat to transfusion recipi-

ents if they persist in the circulation of

asymptomatic blood donors and can

cause clinically significant acute or chronic

disease manifestations in recipients.

The vast majority of posttransfusion

hepatitis in the past was attributable to

HBV and HCV, both of which can establish

prolonged carrier states in donors charac-

terized by high-titer viremia in the absence

of symptoms. HBV and HCV also cause sig-

nificant long-term liver-related morbidity

and mortality.1,2 These viruses are consid-

ered in detail below.

HAV and HEV, which are enterically trans-

mitted viruses, circulate only transiently

during the acute phase of infection. Be-

cause the viremic individual is usually clini-

cally ill and not a candidate for donation,

HAV and HEV are not serious threats to trans-

fusion recipients. However, HAV viremia

667

maybepresentforupto28daysbefore

symptoms develop, and isolated cases have

been reported associated with transfusion

of cellular components3and outbreaks with

Factor VIII concentrate.4Because HAV lacks

a lipid envelope, it is not inactivated by sol-

vent/detergent treatment; additional inac-

tivation methods are under development to

prevent recurrence of such outbreaks. HEV

is rare in the United States, and there have

been no documented cases of transfusion

transmission in this country.

HDV, formerly called the delta agent, can

cause infection and serious hepatitis after

transfusion or other parenteral exposure.

However, because HDV is a defective virus

found only in HBV carriers, screening do-

nors for HBV infection simultaneously

eliminates the risk of HDV.5HGV, also called

GBV-C, is distantly related to HCV and has

ahighprevalencerate(>1%)amongasymp-

tomatic donors. Although HGV is unequiv-

ocally transfusion-transmissible,6acausal

relationship has not been established be-

tween HGV infection and hepatitis or any

other disease manifestation, despite inten-

sive study.

TTV appears similar to HGV with respect

to prevalence, transmissibility, and the lack

of clinical disease significance. Thus,

screening blood donors for HGV or TTV is

not currently recommended. Hepatitis as-

sociated with CMV or EBV is generally mild

in the absence of severe immunosuppres-

sion. The frequency and severity of such

hepatitis cases do not justify routine

screening measures.7SEN-V has been asso-

ciated with transfusion-associated non-A

through non-E hepatitis in one study,8but a

causal association has not been estab-

lished, nor has SEN-V been significantly as-

sociated with chronic non-A through non-E

hepatitis. SEN-V appears to be distantly re-

latedtoTTVandtobeamemberofafam

ily of small, circular DNA viruses called

Circoviridae. Screening for these agents is

not currently recommended because disease

associations have not been established.

Clinical Manifestations of Hepatitis

Most individuals who acquire HBV or

HCV infection have a subclinical primary

infection without obvious symptoms or

physical evidence of disease. Some de-

velop overt hepatitis with jaundice, nau-

sea, vomiting, abdominal discomfort, fa-

tigue, dark urine, and elevation of liver

enzymes. Signs and symptoms usually re-

solve spontaneously. Acute hepatitis C

tends to be milder than hepatitis B. Un-

commonly, the clinical course of HBV and,

rarely, HCV infections may be compli-

cated by fulminant hepatitis. Of greater

concern is a propensity of hepatitis C to

evolve to chronic hepatitis (75% to 85% of

affected individuals), with a significant

number demonstrating long-term pro-

gression to cirrhosis, liver failure, or hepa-

tocellular carcinoma. Hepatitis A tends to

be clinically mild in otherwise healthy

hosts and is not known to progress to

chronic hepatitis or a chronic carrier

state.9HEV infection may lead to severe

disease in pregnant women.10 Vaccination

is available for hepatitis A and for hepati-

tis B, and hepatitis B immune globulin

(HBIG) has proven useful for post-expo-

sure prophylaxis for hepatitis B and im-

mune serum globulin (ISG) for hepatitis

Chronic Carriers of HBV

After initial HBV infection, a proportion of

patients fail to clear infectious virus from

the bloodstream and become chronic car-

riers for years or life. HBV carriers produce,

in addition to the infectious viral particle,

large amounts of noninfectious envelope

protein detected by the assay for hepatitis

B surface antigen (HBsAg). The risk of be-

coming an HBsAg carrier is strongly age-

668 AABB Technical Manual

dependent; ≤5% of those infected with

HBV as adults become chronic HBsAg

carriers, whereas ≥95% recover completely

and develop protective antibody against

HBsAg (anti-HBs). In contrast, before rou-

tine prophylactic immune globulin infu-

sion and immunization, 90% or more of

infants infected perinatally became carri-

ers and became at risk for progressing to

cirrhosis and hepatocellular carcinoma.

According to World Health Organization

estimates, the number of HBsAg carriers is

approximately 400 million worldwide,11

withaprevalenceofupto10%insome

Asian countries, 0.1% to 0.5% in the gen-

eral US population, and 0.02% to 0.04% in

US blood donors. Small proportions (<10%)

of HBsAg carriers develop clinical mani-

festations, such as hepatic insufficiency,

cirrhosis, or hepatocellular carcinoma.

Chronic Carriers of HCV

Most people who are initially infected with

HCV become chronic HCV carriers, with

75% to 85% having persistent HCV RNA in

the serum and liver for years to decades.

At least 50% of such HCV carriers have

biochemical and histologic evidence of

chronic liver inflammation.12 Despite this

chronic inflammatory process, most HCV-

infected individuals remain asymptom-

atic. During the first 20 years after infec-

tion, HCV is usually indolent and associ-

ated with low mortality and morbidity.

Those with clinical liver disease repre-

sented about 10% of the entire infected

cohort in one study.13 The risk that and the

rate at which chronic hepatitis by itself

will progress to cirrhosis is unknown. It is

thought that alcohol may play a synergis-

tic role in exacerbating chronic hepatitis

C. Posttransfusion HCV in children in-

fected after cardiac surgery appears to re-

solvemorefrequentlythanthatinadults

and to be a mild infection at almost 20

years of follow-up.14

Recommendations for clinical manage-

ment of persons with chronic HCV infec-

tion were developed by a National Insti-

tutes of Health consensus development

conference15 and have been updated and

expanded by others.16

Markers of Viral Infection

Laboratory tests can identify markers of

previous exposure and probable current

infectivity for HBV and HCV, which are

useful for screening and diagnostic appli-

cations. Table 28-1 lists the molecular and

serologic markers commonly used in the

diagnosis of hepatitis. Figure 28-1 illus-

trates the sequence of test results typical

of individuals with acute HBV infection

that completely resolves.

The period between exposure to HBV

and emergence of circulating markers of in-

fection (HBV DNA or HBsAg) is usually

about 6 weeks.3,9 HBV DNA, detectable by

pooled nucleic acid amplification testing

(NAT) techniques, is the first marker to ap-

pear, followed by detectable HBsAg. Indi-

vidual donor NAT (ID-NAT) is able to detect

HBV DNA approximately 19 days before

minipooled NAT (MP-NAT).17 Antibody to

the HBV core protein (anti-HBc) usually ap-

pears several weeks later, first as IgM and

then as IgG. The clearance of HBsAg and

appearance of anti-HBs signal resolution of

infection. However, there has been a report

of a fatality, after reactivation of HBV in a

patient treated with rituximab, who was

previously anti-HBs-reactive.18 The specific

virus showed multiple mutations in major

antigenic sites and was thought to escape

the patient’s endogenous immunity.

Two additional HBV markers, HBeAg or

its antibody (anti-HBe), are useful diagnos-

tic and prognostic markers but are not em-

ployed in donor screening. An asymptom-

Chapter 28: Transfusion-Transmitted Diseases 669

670 AABB Technical Manual

Table 28-1. Molecular and Serologic Tests in the Diagnosis of Viral Hepatitis

Virus Test Reactivity Interpretation

HBV DNA HBsAg Anti-HBc

Total

IgM Anti-HBs HBeAg Anti-HBe

+ – – – – – – Window period

+ + +/– +/– – +/– – Early acute HBV infection/chronic carrier

+ + + + – + – Acute infection

+/– – + + – +/– +/– Early convalescent infection/possible early

chronic carrier

+/– + + – – +/– +/– Chronic carrier*

– – + – + – +/– Recovered infection

– – – – + – – Vaccinated or recovered infection

– – + – – – – Recovered infection? False positive?

HDV RNA HBsAg Anti-HBc Anti-HBs Anti-Delta

+ + + – + Acute or chronic HDV infection

– – + + + Recovered infection

HCV RNA Anti-HCV

(Screening EIA)

Recombinant Antigens (RIBA)

5-1-1 c100-3 c33c c22-3

+/– + Not available Probable acute or chronic HCV infection (if RNA

is positive)

– + – – – – False positive

Chapter 28: Transfusion-Transmitted Diseases 671

+/– + + + – – Probable false positive (if RNA is negative); pos-

sible acute infection (if RNA is positive)†

+/– + – – + + Early acute or chronic infection (if RNA is posi-

tive); false positive or late recovery (if RNA is

negative)†

+ + + + + + Acute or chronic infection

– + +/– +/– + + Recovered HCV†

HAV RNA Anti-HAV

Total IgM

++ + AcuteHAV

– + – Recovered HAV/vaccinated

HEV RNA Anti-HEV

Total IgM

++ + AcuteHEV

– + – Recovered HEV

*Those with HBeAg are more infectious and likely to transmit vertically.

†Anti-5-1-1 and anti-c100-3 generally appear later than anti-c22-3 and anti-c33c during seroconversion and may disappear spontaneously, during immunosuppression or after

successful antiviral therapy.

HBsAg = hepatitis B surface antigen; anti-HBc = antibody to hepatitis B core antigen; anti-HBs = antibody to HBsAg; HBeAg = hepatitis B e antigen; anti-delta = antibody to delta

antigen; anti-HAV = antibody to hepatitis A virus; anti-HCV = antibody to hepatitis C virus; anti-HEV = antibody to hepatitis E virus.

atic HBsAg-positive individual may either

be in the early phase of acute HBV infection

(without anti-HBc or with IgM anti-HBc) or

a chronic HBV carrier (with IgG anti-HBc).

HBsAg particles are produced in excess

during acute and chronic infection; blood

from individuals with circulating HBsAg

can infect others. Current screening im-

munoassays detect approximately 0.2 to 0.7

ng/mL HBsAg or ≥3×107particles.3The

number of HBsAg particles in most acute

and chronic infections exceeds this level,

but transmission of HBV from HBsAg

seronegative donors has been described.

NAT testing allows the detection of as few

as 10 genomic copies of HBV DNA.3The

value of NAT for the detection of sero-

negative donors infected with HBV is under

study but may be reduced by high sensitiv-

ity of current assays for HBsAg and the rela-

tively slow rise in HBV DNA levels con-

trasted with HIV and HCV. It is possible that

effective detection of seronegative HBV in-

fected donors by NAT will require testing at

the single donation level.19 HBV vaccines

contain noninfectious HBsAg protein, which

may result in false-positive HBsAg screen-

ing test results for a few days after the inoc-

ulation. Resulting protective antibodies are

directed against HBsAg; vaccination does

not produce anti-HBc.

Tests for antibodies to HCV are enzyme

immunoassays (EIAs) using recombinant

antigens of HCV coated on a solid phase as

the capture reagent. Current assays detect

antibodies to c200 (including c33c and

c100-3), c22-3, and NS-5. Anti-HCV is de-

tectable by third-generation EIAs approxi-

mately 10 weeks after infection. HCV RNA

is present at high concentrations in plasma

during most of the period from exposure to

antibody seroconversion. Anti-HCV is de-

tected in 40% to 50% of samples from pa-

tients at initial diagnosis of acute hepatitis,

672 AABB Technical Manual

Figure 28-1. Serologic markers in hepatitis B virus infection that resolved without complications. In the

acute phase, markers often appear before onset of liver function test (LFT) abnormalities and symptoms

(SYMP). Anti-HBs and anti-HBc persist after recovery and indicate immunity. In chronic carriers (not

shown), HBsAg persists and anti-HBc is usually present, but anti-HBs is absent (HBeAg and anti-HBe

may be present, see Table 28-1). HBV DNA (not shown) may be detected approximately 1 to 2 weeks be-

fore HBsAg.

either transfusion-transmitted or community-

acquired.20

The clinical significance of a positive

screening test for anti-HCV in healthy blood

donors is unclear without supplemental

testing. Approximately 0.21% of US blood

donors have repeatedly reactive EIA re-

sults.21 Several generations of recombinant

immunoblot assays (RIBA) have been li-

censedbytheFoodandDrugAdministra

tion (FDA) for further elucidation of repeat-

edly reactive EIA results. An individual who

is positive by RIBA is considered to have true

HCV antibody; in 70% to 90% of these

cases, HCV nucleic acid is detectable by

NAT methods. The infectivity of units that

are positive for HCV RNA approaches

100%.22 In contrast, EIA repeatedly reactive

donors with negative or indeterminate

RIBA 3.0 results, representing 37% of EIA

repeatedly reactive donors, are rarely in-

fected or infectious. Regardless of RIBA re-

sults, a donation with a repeatedly reactive

EIA result cannot be used for transfusion.

Donors with negative RIBA results may be

considered for reentry (Table 28-2).

In 1999, NAT for HCV RNA was imple-

mented as a donor screening assay under

FDA-sanctioned investigational new drug

(IND) protocols. The testing was performed

in minipools of samples from 16 to 24 whole

blood donations. The rapid increase in

viremia and high viral load of seronegative,

acutely infected donors allows sensitive de-

tectionofHCVRNAeveninthesediluted

pools. The window period for HCV detec-

tion with pooled NAT is reduced to 10 to 30

days.17 After testing over 39 million donors,

approximately 1:270,000 donors had been

identified as being in the seronegative win-

dow with a positive NAT result.23

NAT results can be used in lieu of sup-

plemental testing in specific circumstances.

An FDA variance is required.24 It is antici-

patedthatNATHCVtestingwillbeusedin

the future for reentry.

Surrogate Markers

Before HCV was identified and anti-HCV

testing became feasible, two nonspecific

or “surrogate” tests on donor blood were

introduced to reduce the risk of non-A, non-B

(NANB) hepatitis after transfusion. In 1986

and 1987, the AABB called for testing of

whole blood donations for alanine amino-

transferase (ALT) and anti-HBc as surro-

gates for the direct detection of the NANB

agent. Current very sensitive tests for anti-

HCV have essentially eliminated the value

of surrogate tests in preventing hepatitis,25

but anti-HBc testing continues as recom-

mended by FDA to prevent HBV transmis-

sion. HBV from liver transplant donors with

reactive anti-HBc and negative HBsAg test

results has been transmitted to their re-

cipients, and reports in the literature

show that transfusions of blood reactive

for anti-HBc and negative for HBsAg have

been associated with development of

hepatitis B in some recipients. There may

also be a small number of potential do-

nors infected with HBsAg mutants of HBV

that may not be optimally detected by

currently licensed HBsAg tests.26 Donors

who test repeatedly reactive for anti-HBc

on two occasions or who test repeatedly

reactive on tests from two different manu-

facturers should be deferred.

Current Risk of Posttransfusion Hepatitis

The risk of posttransfusion HBV or HCV

infection decreased dramatically, to an es-

timated 1 in 60,000 to 1 in 100,000 risk be-

fore implementation of HCV NAT.27 Not a

single new case of transfusion-associated

HCV has been detected by the CDC Senti-

nel Counties Viral Hepatitis Surveillance

System since 1994 (M. Alter, personal com-

munication, 3/04).1The development of

progressively improved HCV antibody

tests and stringent selection measures for

donors have contributed to this remark-

Chapter 28: Transfusion-Transmitted Diseases 673

674 AABB Technical Manual

Table 28-2. Reentry of Donors with Repeatedly Reactive Screening Tests

Repeatedly Reactive for

Reentry

Status

Anti-HIV-1

or -1/2 Anti-HIV-2 HIV-1-Ag HBsAg Anti-HCV

Initial sample

Licensed West-

ern blot posi-

tive or inde-

terminate or

IFA reactive

Different HIV-2

EIA RR

Confirmed by

neutraliza-

tion

Confirmed by

neutraliza-

tion or

anti-HBc

RIBA indeter-

minate or

positive

Not eligible

for reentry

Licensed West-

ern blot or IFA

Different HIV-2

EIA NR and li-

censed West-

ern blot or IFA

Not confirmed

by neutral-

ization

HBsAg speci-

ficity not

confirmed

by neutral-

ization and

anti-HBc

RIBA negative Evaluate for

reentry

Follow-up sample

(Drawn 6

months

later)

(Drawn 6

months

later)

(Drawn 8

weeks

later)

(Drawn 8

weeks

later)

(Drawn 6

months

later)

EIA RR or West-

ern blot posi-

tive or inde-

terminate or

IFA reactive

RR HIV-1 or dif-

ferent HIV-2

EIA RR or a li-

censed West-

ern blot or IFA

reactive or in-

determinate

HIV-1-Ag RR,

neutraliza-

tion con-

firmed or

not con-

firmed

HBsAg RR or

anti-HBc

EIA RR or

RIBA inde-

terminate

or positive

Not eligible

for reentry

Original EIA

method NR

and whole vi-

rus lysate

anti-HIV-1 EIA

NR and li-

censed West-

ern blot or IFA

Screening test

and a differ-

ent HIV-2 EIA

NR and li-

censed West-

ern blot or

IFA NR

HIV-1-Ag and

anti-HIV-1

EIA NR or

HIV-1-Ag

RR, not

confirmed

(temporary

deferral for

additional

8 weeks)

HBsAg NR

and

anti-HBc

Licensed

multiantigen

EIA

method NR

and RIBA

negative

Eligible for

reentry

NR = nonreactive; RR = repeatedly reactive; RIBA = recombinant immunoblot assay; IFA = immunofluorescence assay;

EIA = enzyme immunoassay; anti-HIV-1 = antibody to human immunodeficiency virus, type 1; anti-HIV-2 = antibody to

human immunodeficiency virus, type 2; HIV-1-Ag = HIV-1 antigen; HBsAg = hepatitis B surface antigen; anti-HCV = anti-

body to hepatitis C virus; anti-HBc = antibody to hepatitis B core antigen.

able decline, and transfusion is no longer

considered a major risk factor for HCV

transmission.28 Nucleic acid screening as-

says for HCV were implemented by blood

centers in 1999. In 3 years of NAT testing,

170 HCV NAT-positive, seronegative do-

nations were identified in the United

States among 39.7 million screened dona-

tions.23 NAT has probably reduced the re-

sidual risk for HCV transmission to ≤1in

2,000,000 components transfused.29,30

Quarantine and Recipient Tracing

Donations with repeatedly reactive screen-

ing test results (HBsAg, anti-HBc, and/or

anti-HCV) cannot be used for transfusion.

In addition, in-date components from

collections preceding the current unsuit-

able donation may need to be quaran-

tined as follows, and consignee notifica-

tion for the purpose of recipient tracing

(ie, look-back) may be required31,32:

For HCV:

■Extending back indefinitely, to the

extent that computerized electronic

records exist for anti-HCV repeat-

edly reactive and confirmed dona-

tions or repeatedly reactive donations

for which supplemental tests were

not performed.

■Extending back to January 1, 1988 if

computerized electronic records are

not available.

For HBsAg and anti-HBc:

■In-date components, extending back

5years,or12monthsfromthemost

recent negative test result for units

that were repeatedly reactive or con-

firmed, or for which confirmatory

testing was not performed.

Depending on the results of licensed

supplemental tests and prior screening

tests, the quarantined units may be re-

leased for transfusion or further manufac-

ture, or may have to be destroyed. Recipi-

ents notified as a result of the HCV look-back

should be counseled regarding the nature

of the subsequent donor test results and of-

fered appropriate testing. If they test posi-

tive, life style changes (eg, abstinence from

alcohol consumption) and evaluation for

chronic liver disease justifying antiviral

therapy to reduce the likelihood of disease

progression may be warranted. Earlier ex-

periences from Canada and several Euro-

pean countries indicate that the number of

transfusion recipients who ultimately bene-

fit from look-back efforts is small.33 Asurvey

of US blood collection facilities and hospi-

tal transfusion services after implementa-

tion of targeted HCV look-back resulted in

an estimate that notification of the recipi-

ents of 98,484 components would result in

the identification of 1520 infected persons

who were previously unaware of their in-

fection.34 This would represent less than 1%

of the 300,000 still-living recipients who

may have acquired infection by blood

transfusion. More recently, 0.9% to 5.0% of

patients tested for hepatitis C were found to

be positive in a review of look-back studies

in Canada that notified recipients of any

previous transfusions of the risk of HCV

and then provided testing; 42% to 58% of

the cases were newly identified.35

Human Immunodeficiency

Viruses

The human immunodeficiency viruses type

1 (HIV-1) and type 2 (HIV-2) are the

etiologic agents of AIDS. The AIDS syn-

drome was recognized in 1981, well be-

fore the discovery of the causative virus in

1984. Wider implications of the immune

disorder were noted when, in 1982, AIDS

was reported in three patients with hemo-

philia,36 and in a 17-month-old infant

whosemultipletransfusionsatbirthin

cluded a unit of platelets from a donor

Chapter 28: Transfusion-Transmitted Diseases 675

who subsequently developed AIDS.37 With-

in a few years, studies established that well

over 50% of patients with hemophilia who

received clotting factor concentrates in

the early 1980s developed HIV-1 infec-

tion.38 In some regions of the United States,

up to 1% of single-donor unit transfusions

were infected with HIV in the early 1980s.39

Clinical Manifestations of HIV Infection

HIV is a cytopathic retrovirus that prefer-

entially infects CD4-positive T lympho-

cytes (helper T cells) in lymph nodes and

other lymphoid tissue.40 After primary in-

fection, HIV replicates and disseminates

initially as cell-free virions, and 10 days to

3 weeks after infection, viremia is first de-

tectable in the plasma. During this time,

about 60% of acutely infected persons de-

velop an acute retroviral syndrome, char-

acterized by a flu-like illness with fever,

enlarged lymph nodes, sore throat, rash,

joint and muscle pain—with or without

headache, diarrhea, and vomiting. As HIV-1

antibodies appear, the disease enters a

clinically latent stage; however, viral repli-

cation and dissemination continue. Dur-

ing this phase, the virus can be transmit-

ted by blood or genital secretions (Fig

28-2).

Persistent infection with an asymptom-

atic clinical status has been estimated to

lastamedianof10to12yearsintheab

sence of treatment.41 After years of asymp-

tomatic infection, both plasma viremia and

the percentage of infected T lymphocytes

676 AABB Technical Manual

Figure 28-2. Virologic events during primary HIV infection. After initial infection and propagation of

HIV in lymph nodes, a blood donor becomes infectious (defined as day 0), with HIV RNA being detect-

able in plasma on days 14-15, HIV DNA detectable in leukocytes at days 17-20, and HIV antibodies de-

tectable between days 20 and 25. Anti-HIV persists indefinitely but may be lost in the preterminal

stage of the disease, in parallel with a surge in viral burden, indicating collapse of the immune sys-

tem. HIV = human immunodeficiency virus; RT-PCR = reverse transcriptase polymerase chain reaction;

PBMC = peripheral blood mononuclear cells.

increase. Loss of the immune functions

served by helper T cells impairs immune

reactivity, and there may be inappropriate

immune activation and cytokine secretion.

Eventually, there is a sharp decline in the

number of CD4+ T lymphocytes, and the

vast majority of infected individuals suc-

cumb to opportunistic illnesses fostered by

profound immunosuppression.

Enumeration of viral load and CD4+ cells

is used to guide clinical and therapeutic

management of HIV-infected persons. The

AIDS classification system devised by the

Centers for Disease Control and Prevention

(CDC) is based on the number of CD4+ T

cells (<200/µL defines AIDS), the presence

orabsenceofsystemicsymptoms,andex

istence of any of the 26 clinical conditions

considered to be AIDS-defining illnesses.42

Among these conditions are otherwise un-

usual malignancies, such as Kaposi’s sar-

coma, central nervous system lymphoma,

and a wide array of devastating, potentially

lethal opportunistic infections with fungi

and parasites, the most common being

Pneumocystis carinii pneumonia.

Advances in treatment of HIV and op-

portunistic infections have dramatically en-

hanced the survival of infected persons.

Unfortunately, worldwide, the disease is

still spreading rapidly and, for the majority

of HIV-infected individuals in developing

countries, effective therapy is either not

available or not affordable.

Risk Factors for HIV Infection

HIV can be transmitted through sexual

contact, childbirth, breast-feeding, and

parenteral exposure to blood. Those iden-

tified early as being at highest risk were

men who had sex with other men; com-

mercial sex workers and their contacts;

needle-sharing drug users; patients with

hemophilia who received human-derived

clotting factor concentrates; and, to a les-

ser extent, recipients of blood transfusions.

By 1989, the rate of infection in each

group was no longer increasing exponen-

tially and appeared to have reached a pla-

teau in the populations most at risk.43 HIV

seroprevalence had stabilized in most US

cities. Heterosexual transmission of HIV

represented a progressively larger propor-

tion of US HIV infections and AIDS cases

reported in the 1990s.44 This is of impor-

tance in transfusion medicine because

screening for heterosexual high-risk be-

havior is more problematic than screen-

ing for male-to-male sex and parenteral

drug use.45

HIV-2 and HIV-1, Group O

First discovered in 1985, HIV-2 causes en-

demic infection in many countries in

West Africa. Although HIV-2 was initially

restricted to West Africa, recent studies in

European countries such as Great Britain

and France (which have significant immi-

gration from West Africa) have observed

increasing rates of HIV-2 and other HIV

subtypes.46,47

The first case of HIV-2 infection in the

United States was reported in March 1988

in a young West African who had recently

immigrated to the United States.48 The

spectrum of disease attributable to HIV-2 is

similar to that caused by HIV-1; however,

there appears to be a longer incubation pe-

riod and lower incidence of progression to

AIDS. HIV-2 is spread both sexually and

from mother to child, but transmission is

less efficient than for HIV-1.

Tests in the United States on parenteral

drug users, persons with sexually transmit-

ted diseases, newborn infants, and homo-

sexual men confirm the very limited preva-

lence and transmission of the agent. HIV-1/

HIV-2 combination tests were implemented

in the United States in 1992. Since then,

three HIV-2-infected donors have been

Chapter 28: Transfusion-Transmitted Diseases 677

identified; none appeared to have been in-

fected in the United States.49

To date, three groups of HIV-1 viruses

have been identified: group M (major

group); group O (outlier group); and, most

recently, group N. Further, there are 10 sub-

types (A-J) of group M. None of 97 donors

retrospectively identified as being HIV-1 in-

fected in 1985 and three (1%) of 383 donors

prospectively identified between 1993 and

1996 were found to have non-B subtypes

(two subtype As and one subtype C).50 Of

note, this study did find an increase in env

gene diversity among HIV-1 group B strains

overtimeandcalledforcontinuedsurveil

lance for emergence of non-B subtypes and

development of test systems for their de-

tection. A follow-up study from these same

investigators documented characterized

HIV subtypes in 291 infected US donors

identified from 1997 through 2000 and

identified that six (2%) were non-B sub-

types of HIV-1 and one was HIV-2.51 In

Cameroon and surrounding West African

countries, an estimated 1% to 2% of HIV in-

fections are caused by group O viral strains.52

As with HIV-2, group O isolates have rarely

been seen outside this geographic area.

Concern arose when studies demonstrated

that some group O viral isolates were not

reliably detected by several EIA tests used

for blood donor screening. Of the two

FDA-licensed tests for NAT, both were eval-

uated by the manufacturer for the detection

of non-B subtypes including group O and

N, using a limited number of specimens.

(although neither test detects HIV-2). How-

ever, until reliable detection of group O in-

fections is established, the FDA recom-

mends indefinite deferral of blood and

plasma donors who were born, resided, or

traveled in West Africa since 1977, or had

sexual contact with someone identified by

these criteria.53 The risk of group O infec-

tion in the United States is very low. In a

survey of HIV subtypes in US blood donors

over a period spanning two decades, only

three non-B subtypes were identified. Two

of these three donors were born in Africa.50

Transfusion Considerations

Transfusion-Transmitted HIV-1

All blood components can transmit HIV-1.

Although approximately 1% of all AIDS

exposures have resulted from transfusion

or organ or tissue transplantation, the in-

troduction of MP-NAT in 1999 virtually

eliminated the risk of transfusion-trans-

mitted HIV.54 The few cases of HIV infec-

tion that have been documented since

1999 were attributed to low-level viremic

units that likely would have been detected

by ID-NAT.55-57

Most but not all recipients of HIV-in-

fected blood transfusions become infected.

In one large study, HIV infection developed

in 89.5% of recipients who received blood

from anti-HIV-positive donors.58 Transmis-

sion rates correlated with component type

and viral load in the donation. With the ex-

ception of coagulation factor concentrates,

plasma derivatives (such as albumin and

immune globulins) have not been reported

to transmit HIV infection. No transmission

of HIV attributable to coagulation factors has

been documented in the United States

since implementation of full donor screening

and virus inactivation techniques in 1987.59

Transfusion-Transmitted HIV-2 and HIV-1,

Group O

There have been two reports of possible

HIV-2 transmission through blood com-

ponent use, both in Europe. Two women

were infected by Whole Blood obtained from

a donor who developed AIDS at least 16

years after becoming infected with HIV-2;

both women were asymptomatic 14 years

after transfusion.60 Two hemop hili a p a-

tients who received clotting factors were

also infected. Because of their extremely

678 AABB Technical Manual

low prevalence, no HIV-2 or HIV-1 group

O transmissions have been reported in

the United States by blood transfusion or

any other transmission route.

Current Risk of Posttransfusion HIV

With screening tests available before 1992,

the seronegative interval (“window pe-

riod”) averaged 45 days. More sensitive

screening tests for HIV antibody closed

the antibody-negative window to approxi-

mately 22 days.17 Introduced in 1996, p24

antigen screening further reduced the po-

tentially infectious window by an esti-

mated 6 days,61 although it appears that

fewer HIV-infected units were intercepted

by the introduction of this test than had

been expected based on the calculated re-

duction of the infectious window period.

Risk from seronegative donations will

vary in proportion to the incidence of HIV

infection in the donor community. Overall

estimates of posttransfusion HIV risk in the

United States since the implementation of

HIV NAT are approximately 1 in 2 million

screened donations.23,30

HIV Testing of Blood Donors

AABB Standards for Blood Banks and Trans-

fusion Services62(pp33,34) and FDA regulations63

require that all units of blood and compo-

nents be nonreactive for anti-HIV-1 and

anti-HIV-2 before they are issued for

transfusion. HIV-1-antigen (HIV-1-Ag)

testingisnomorerequiredbytheFDAor

AABBaslongaslicensedHIV-1NATisin

place.Figure28-3showsthesequenceof

screening and confirmatory testing for anti-

HIV-1/2.

Because the consequence of missing even

onetruepositiveisgreat,screeningtests

are designed to have high sensitivity both

to immunovariant viruses and to low-titer

antibody during seroconversion. EIA-de-

tectable antibody develops 2 to 4 weeks

after exposure,58 days to a week after the on-

set of symptoms in those who have any rec-

ognized acute illness,64 and about 12 days

after detectable viremia by ID-NAT or 9 to

10 days by MP-NAT.17,65

A few days later, HIV-1 antibodies become

detectable by the HIV-1 Western immuno-

blot. With very rare exceptions, all persons

infected with HIV develop anti-HIV reactiv-

ity detectable by EIA and Western blot that

persists for life.

More sensitive tests using NAT technol-

ogies have been shown to detect additional

potentially infectious donors. In February

2002, the FDA approved the first NAT sys-

tem for screening whole blood donors and

issued draft guidance for blood establish-

ments; the final guidance was issued in

October 2004.66,67 It has been estimated that

HIV NAT has reduced the window period

for HIV from 16 days to 10 days.17 The use

of NAT for donor testing has not only in-

creased sensitivity, but has also decreased

the number of false-positive tests, increas-

ing specificity. During the 3 years of inves-

tigational HIV NAT, 12 confirmed HIV-1

RNA-positive antibody-negative donors

were detected in 37 million donations

screened, or 1 in 3.1 million, of which only

two were detected by HIV-1 p24 antigen.23

Confirmatory Testing for Antibodies to

HIV-1/2

If a disease has low prevalence in the

tested population, the likelihood is high

that most positive screening test results will

be false positive. More specific supplemen-

tal tests are then required to confirm the

screening test results. The most commonly

used of these tests for antibodies to HIV-1/2

is the Western blot (see Chapter 7).

According to current FDA and CDC cri-

teria, a sample is defined as anti-HIV-posi-

tive if at least two of the following bands are

Chapter 28: Transfusion-Transmitted Diseases 679

present: p24, gp41, and/or gp120/160.68

Negative Western blot results have no

bands present. Western blot results classi-

fied as indeterminate have some bands

present but do not have the pattern defin-

ing HIV positivity. Individuals infected with

HIV may have indeterminate patterns when

initially tested but develop additional bands

within 6 weeks. Healthy individuals with

initial indeterminate patterns continue to

have negative or indeterminate results on

repeat samples and are negative on clinical

examination and additional tests, including

viral cultures and NAT. Healthy donors who

continue to show the same indeterminate

pattern for more than 3 months can be reas-

sured that they are unlikely to have HIV in-

fection, but they are not currently eligible

to donate blood. Several groups have iden-

tified Western blot patterns in blood donors

that were identified as false-positive results;

for these, testing (using NAT) is recom-

mended to resolve the infectious status of

the donor.69

Approximately 50% of all HIV screening

EIA repeatedly reactive donors test indeter-

minate by licensed HIV-1 Western blot as-

says. However, when these donations were

tested by ID-NAT, less than 0.1% were

shown to contain HIV-1 RNA (1:1450 RNA-

positive, indeterminate donors). When com-

bined with those donations that tested

Western blot negative, the frequency of a

donor testing HIV repeatedly reactive and

680 AABB Technical Manual

Figure 28-3. Decision tree for anti-HIV-1/HIV-2 testing of blood donors. IFA = immunofluorescence as-

say; WB = Western blot. If EIA testing is nonreactive, NAT testing must also be nonreactive before re-

lease of a donation.

then Western blot indeterminate or nega-

tive and demonstrating RNA was 1:4.27

million (S. Stramer, personal communica-

tion, 3/17/04).

The FDA has approved reentry protocols

to qualify donors with negative confirma-

tory test results as eligible for subsequent

donations (see Table 28-2).70 Reentry currently

requires retesting at least 6 months later, to

detect delayed seroconversion; the use of

EIA tests based on whole-virus lysate; and

use of either a licensed Western blot to en-

sure appropriate sensitivity of the methods

or an FDA-licensed immunofluorescence

assay.71 The later sample must also be

nonreactive in an EIA test for anti-HIV-2, if

standard testing does not include HIV-2.

The FDA recently published draft reentry

guidelines that will allow reinstatement of

donors with indeterminate Western blot re-

sults, eliminate the need for viral lysate EIA

testing, and include testing by HIV-1 NAT.

NAT results can be used in lieu of sup-

plemental testing in specific circumstances.

An FDA variance is required.24 It is antici-

pated that NAT HIV-1 testing will be used in

the future for reentry.

Positive Tests in Autologous Donors

Whether HIV EIA repeatedly reactive or

NAT-positive autologous donations should

be withheld from transfusion is contro-

versial.72 Theseunitsmaybesuppliedfor

autologous use if the following conditions

are met: 1) there is a written, signed, and

dated request from the patient’s physician

authorizing this shipment, 2) there is a

written statement from the transfusion

service indicating willingness to receive this

product, and 3) the transfusion service

takes responsibility for ensuring that there

is documented verification of the accurate

identity of the transfusion recipient. These

units must be labeled “BIOHAZARD” and

“FOR AUTOLOGOUS USE ONLY.”

Whether a facility elects to offer autolo-

gous services is an internal decision. Insti-

tutions should consider, however, that

where feasible for a patient, it is generally

accepted that the patient should have the

option to use his or her blood. In addition,

aUSSupremeCourtdecisionintheBrag-

don v Abbott case ruled that HIV-positive

individuals are protected under the Ameri-

cans with Disabilities Act.73 Whether this

would apply to HIV-positive donations that

could represent a risk to hospitalized trans-

fused patients remains controversial.

Recipient Tracing (Look-Back)

Identification of persons who have received

seronegative or untested blood from a do-

nor later found to be infected by HIV is

referred to as “look-back.” Because the in-

terval between receipt of an infected

transfusion and onset of AIDS can be very

long, recipients are usually unaware of their

infection and may be infectious to others.

To identify these individuals, blood cen-

ters must have procedures to notify recip-

ients of previous donations from any do-

nor later found to have a confirmed

positive test for anti-HIV or a confirmed

positive test for HIV using licensed NAT. If

a patient with AIDS is known to have do-

nated previously, recipients of blood or

blood components from these donations

should be traced and notified. Recipient

tracing and testing are usually accom-

plished through the patient’s physician, not

through direct contact with the patient. In

companion rules, the FDA and the Cen-

ters for Medicare and Medicaid Services

established timelines and standards de-

fining look-back.74-77 If recipients of units

that were donated at least 12 months be-

fore the last known negative test are tested

and found negative, earlier recipients are

probably not at risk because infectivity

Chapter 28: Transfusion-Transmitted Diseases 681

earlier than 12 months before a negative

screening test is extremely unlikely.

Human T-Cell Lymphotropic

Viruses

HTLV, Type I

Human T-cell lymphotropic virus, type I

(HTLV-I) was the first human retrovirus

isolated and the first to be causally associ-

ated with a malignant disease of humans,

adult T-cell lymphoma-leukemia (ATL).58

HTLV-I is also associated with the neuro-

logic condition HTLV-associated myelo-

pathy (HAM), often called tropical spastic

paraparesis (TSP). ATL was described be-

fore HAM. Both these conditions occur in

a small minority (no more than 2-4%) of

persons harboring the virus. Infection

during childhood is an important aspect

of, and possibly a requirement for, devel-

oping ATL many years later, whereas

childhood or adult infection can cause

HAM, with a variable latent period.

Prevalence of HTLV-I infection shows

striking geographic clustering, with pockets

of high endemicity in parts of southern

Japan and certain Pacific Islands; sub-Saha-

ran Africa; and the Caribbean basin, Cen-

tral America, and South America. Transmis-

sion is by mother to child through breast

milk, by sexual contact (predominantly

male-to-female), and by exposure to blood.

HTLV, Type II

Human T-cell lymphotropic virus, type II

(HTLV-II) was described several years af-

ter HTLV-I. There is at least 60% similarity

of genetic sequences to those of HTLV-I;

antibodies to either show strong cross-re-

activity in tests with viral lysates. HTLV-II

also shows clustering, but in different

populations. High prevalence has been

noted among some Native American pop-

ulations and in intravenous drug users in

the United States, in whom seropreva-

lence is 1% to 20%. Rare disease associa-

tions with HTLV-II include HAM; its

occurrence seems to be somewhat less

frequent than with HTLV-I.58

Epidemiologic data suggest that there is

an excess of infectious syndromes (eg,

bronchitis, urinary infections, and pneu-

monia) among blood donors infected with

HTLV-I or -II.78-81

Clinical Observations

For both HTLV-I and -II, infection persists

lifelong, as does the presence of antibody.

Studies of prevalence and transmission

use seroconversion as the endpoint for di-

agnosis. Infection does not cause any rec-

ognizable acute events, and with the ex-

ception of those developing ATL or HAM,

infected individuals experience few, if

any, health consequences. Most carriers

are asymptomatic and completely un-

aware of the infection.

Transmission

Both viruses are very strongly cell-associ-

ated. Contact with infected viable lym-

phocytes can cause infection, but plasma

appears to be not, or much less, infective.

Cellular components from infected donors

cause seroconversion in at least 50% of

recipients in Japan, but apparently in a

much smaller proportion of US recipi-

ents.58 After refrigerated storage for 10

days or more, red cells transfused from an

infected donor are far less likely to result

in seroconversion, presumably due to de-

gradation of lymphocytes that transmit

the virus.82,83 Transfusion-transmitted

HTLV-I infection has been associated with

HAM of rather rapid onset and at least

onecaseofATL.

682 AABB Technical Manual

Donor Tests

Donor screening for anti-HTLV-I began in

the United States in late 1988; at that time,

the rate of confirmed positive tests was

approximately 0.02%, or 1 in 5000 units

collected, a figure that has since declined

at least 10-fold as seropositive persons

have been removed from the donor pool.84

The combined risk of transfusion-trans-

mitted HTLV-I/II infection has been esti-

mated as about 1 in 641,000 units.84 Fur-

ther risk reduction can be expected from

the implementation of combination

HTLV-I/II EIA tests in blood donor screen-

ing. The first such test was licensed in

1998 in the United States. By using viral

lysatesfrombothHTLV-IandHTLV-IIvi-

ruses, the test offers sensitive detection of

both anti-HTLV-I and anti-HTLV-II. The

originally licensed anti-HTLV-I EIA

screening tests might have missed up to

50% of HTLV-II infections.85

A donation that is repeatedly reactive on

EIA may not be used for transfusion. If a

donor tests repeatedly reactive by the EIA

screening assay on two or more occasions,

he or she should be notified and indefi-

nitely deferred.85 Another recommended

approach is to test donor serum with a sec-

ond manufacturer’s EIA test kit.86 If that test

is also repeatedly reactive, then the donor is

indefinitely deferred on the basis of that

single donation. Further testing of serum

that is repeatedly reactive for anti-HTLV-I/II

against antigen preparations specific for

the two agents (HTLV-I or HTLV-II), or by

NAT on material from peripheral blood

mononuclear cells, can characterize the in-

fecting agent. Half or more of US blood do-

nors confirmed infected after EIA screening

provetohaveHTLV-IIinfections.Although

there is no FDA requirement to perform ad-

ditional testing (no confirmatory test for

HTLV is licensed), most centers do so using

an investigational supplemental test or se-

ries of tests in an algorithm if the donation

tests repeatedly reactive by both the test-

of-record and second HTLV-I/II EIAs; if

supplemental tests are positive, the donor

is indefinitely deferred (see Table 28-3). Re-

gardless of the results of these investiga-

tional supplemental tests, a donor meeting

the EIA criteria for indefinite deferral de-

scribed above must still be indefinitely de-

ferred.

Quarantine and Look-Back

In-date prior collections of blood or com-

ponents from donors who subsequently

are found repeatedly reactive for anti-

HTLV-I/II need to be quarantined. Be-

cause recipients of units from seroposi-

tive donors do not consistently sero-

convert and because many seropositive

donors have lifelong infection, the time

frame for look-back is not self-evident. No

requirement for recipient tracing and no-

tification has been established.85 Screen-

ing, in place since 1988, has probably re-

moved from the active donor pool most

donors with lifelong infection.

West Nile Virus

West Nile virus (WNV) is a flavivirus pri-

marily transmitted in birds through mos-

quito bites; humans are incidental hosts.

In humans, symptoms range from a mild

febrile illness to encephalitis, coma, and

death, although about 80% of infected in-

dividuals remain asymptomatic. WNV

outbreaks have been reported in Europe,

the Middle East, and Russia over the past

decade and have been associated with

human encephalitis and meningitis (in

<1%), although no transfusion-associated

cases were reported. WNV was observed

in the United States, in the metropolitan

Chapter 28: Transfusion-Transmitted Diseases 683

684 AABB Technical Manual

Table 28-3. Recommended Actions for HTLV-I/II Testing

First Donation to Be Tested for HTLV-I/II Antibodies Subsequent Donation(s)

EIA WB/RIPA Donation Donor EIA

Second

Manufacturer WB/RIPA Donation Donor

Repeatedly

reactive

Positive Destroy all

compo-

nents*

Defer and

counsel

Not applicable/Donor deferred

Repeatedly

reactive

Negative or

indeterm.

Destroy all

compo-

nents*

No action Repeatedly

reactive

Pos Any result Destroy all

compo-

nents*

Defer and

counsel

Nonreactive†Neg Not done All compo-

nents ac-

ceptable

No action

*Destroyed unless appropriately labeled as positive for HTLV-I/II antibodies, and labeled for laboratory research use or further manufacture into in-vitro diagnostic reagents.

†Assuming that separate prior donations have been repeatedly reactive for HTLV-I/II antibody no more than once. If separate prior donations had been repeatedly reactive for

HTLV-I/II antibodies on

two or more

occasions, the donor should have been indefinitely deferred.

HTLV-I = human T-cell lymphotropic virus, type I; HTLV-II = human T-cell lymphotropic virus, type II; EIA = enzyme immunoassay; WB = Western blot; RIPA = recombinant

immunoprecipitation assay.

New York City area, in 1999. Subsequently,

it dispersed rapidly westward throughout

the country, spread by infected birds. In

2001, 66 human cases occurred in 10

states. In 2002, 4161 cases of WNV illness

were reported in 39 states, including 284

deaths. In 2002, 23 cases were associated

with transfusion (with an infected donor

identified); Red Blood Cells (RBCs), Fresh

Frozen Plasma, and Platelets were impli-

cated.87 The implicated donations oc-

curred between July 22 and October 6,

2002. Statistical resampling of data avail-

able regarding case onset dates during the

2002 epidemic was used to generate esti-

mates of the mean risk of transfusion-as-

sociated WNV transmission (per 10,000

donations) for six states and six selected

metropolitan areas, with results ranging

from a mean of 2.12 to 4.76 and 1.46 to

12.33, respectively.88

An FDA guidance document in May

2003 recommended deferral of donors with

a diagnosis of WNV infection for 28 days af-

ter onset of symptoms or 14 days after reso-

lution, whichever is later, and recom-

mended inquiring whether donors had

experienced a fever with headache in the

week before donation.89 However, because

most infected persons are asymptomatic,

the yield of such measures would be ex-

pected to be modest, and testing was

sought as the best method to identify in-

fected donors. During the summer and fall

of 2003, almost 5 million donations consti-

tuting over 95% of collections in the United

States were tested for WNV by NAT in

minipools of six or 16 samples under one of

two IND protocols—one using a polymer-

ase chain reaction method and one using a

transcription-mediated amplification

method. During 2003, approximately 1000

donors were confirmed as viremic for WNV

and approximately 1500 likely infected

components were interdicted.90 However,

six probable or confirmed transfusion-as-

sociated WNV cases were reported in 2003;

four recipients had WNV encephalitis, one

had West Nile fever, and one critically ill

patient did not have discernible WNV-com-

patible illness despite confirmed WNV in-

fection.91 Some of the donors were identi-

fied through retrospective testing of

individual samples, and it appears that all

were related to specimens with very low vi-

ral titers. The number of transfusion-asso-

ciated cases undoubtedly would have been

much higher had widespread testing under

IND protocols not been initiated. There

were 9862 WNV cases and 264 deaths in the

United States overall during this time.

The persistent low-level transmission of

WNV by transfusion in 2003 led to the im-

plementation of targeted ID-NAT in high

epidemic regions in 2004. This effort suc-

cessfully interdicted low-level viremic units

that would have been missed by MP-

NAT.92,93

In 2004, the virus appeared predomi-

nantly in western states, with California ac-

counting for 31% of cases.94 Only three

states (Hawaii, Alaska, and Washington) re-

mained free of reports of infection in either

humans or animals (mammalian and

avian). A total of 2470 cases of human WNV

infection were reported in 40 states plus the

District of Columbia, including 88 fatal

cases—far fewer than the previous year. A

total of 192 WNV-positive donors were

identified, almost all from states west of the

Mississippi River.94 Of these donors, three

subsequently reported neuroinvasive WNV

illness and 55 subsequently developed

WNV fever.94 One probable transfusion-as-

sociated case was reported.95

Theexperiencetodateindicatesthat

blood screening for WNV has improved

blood safety. However, a small risk of WNV

transfusion-associated transmission re-

mains. If the number of WNV cases contin-

ues to dramatically decline, the need for

WNV testing will be reassessed. The FDA

Chapter 28: Transfusion-Transmitted Diseases 685

has agreed that asking the question con-

cerning fever with headache in the week

before donation may be discontinued (K

Gregory, personal communication,

3/30/05).

Herpesviruses and Parvovirus

Cytomegalovirus

CMV, a member of the human herpes-

virus family, is a ubiquitous DNA virus

that causes widespread infection; trans-

mission can occur through infectious

body secretions, including urine, oro-

pharyngeal secretions, breast milk, blood,

semen, and cervical secretions. About 1%

of newborns are infected, transplacentally

or through exposure to infected cervical

secretions at delivery or by breast milk. In

early childhood, CMV is often acquired

through close contact, especially in day-

care settings; in adulthood, through sex-

ual intercourse. The prevalence of CMV

antibodies ranges from 50% to 80% in the

general population.96 Therateincreases

with age and is generally higher in lower

socioeconomic groups, in urban areas,

and in developing countries.

Clinical Observations

Inpersonswithanintactimmunesystem,

CMV infection may be asymptomatic and

remain latent in tissues and leukocytes

for many years. Infection, either primary

or reactivation of latent infection, can be

associated with a mononucleosis-like

syndrome of sore throat, enlarged lymph

nodes, lymphocytosis, fever, viremia,

viruria, and hepatitis. Intrauterine infec-

tion may cause jaundice, thrombocyto-

penia, cerebral calcifications, and motor

disabilities; the syndrome of congenital

infection causes mental retardation and

deafness and may be fatal.

CMV infection can progress to CMV dis-

ease and cause serious morbidity and mor-

tality in premature infants, recipients of

organ, marrow, or peripheral blood progen-

itor cell transplants, and in AIDS patients.96

Pneumonitis, hepatitis, retinitis, and multi-

system organ failure are manifestations of

CMV disease. CMV infection can result from

blood transfusions. Other sources of infec-

tion, however, such as organ transplants

from CMV-positive donors or reactivation

of latent virus, may be as much or more of a

risk than transfusion.

Transfusion-Transmitted CMV

Infection with CMV varies greatly accord-

ing to socioeconomic status and geo-

graphic region. Although approximately

50% of blood donors can be expected to

be CMV seropositive, it has been esti-

mated that, currently, less than 1% of se-

ropositive cellular blood components are

able to transmit the virus.96,97 Rarely, post-

transfusion hepatitis may be due to CMV.

The postperfusion mononucleosis syn-

drome that first focused attention on

CMV in transfused components in the

early 1960s is now rarely seen. Posttrans-

fusion CMV infection is generally of no

clinical consequence in immunocompe-

tent recipients, and intentional selection

of CMV-reduced-risk blood (see below) is

not warranted.

In light of the potential for severe CMV

disease in immunocompromised patients,

several categories of recipients have been

identified who should be protected from

transfusion-transmitted CMV.7These in-

clude low-birthweight premature infants

born to seronegative mothers; seronegative

recipients of hematopoietic progenitor cells

from CMV-negative donors; seronegative

pregnant women, because the fetus is at

risk of transplacental infection; and recipi-

686 AABB Technical Manual

ents of intrauterine transfusions. In some

cases, seronegative recipients of organ

transplants from a seronegative donor;

seronegative individuals who are candi-

dates for autologous or allogeneic hemato-

poietic progenitor cell transplants; and

those few patients with AIDS who are free

of CMV infection are also included.

Preventive Measures

Blood from donors who test negative for

CMV antibody has very little risk of trans-

mitting CMV, but the supply of seronega-

tive blood is limited.96,97 Another approach

to reduce risk is to remove leukocytes

from donated blood (because leukocytes

are the principal reservoir for CMV).98-100

Although the precise leukocyte population

that harbors the virus has not been de-

fined, leukocyte removal with high-effi-

ciency filters, to 5 ×106leukocytes per

component or fewer, can significantly re-

duce, if not prevent, posttransfusion CMV

in high-risk neonates and transplant re-

cipients. Effectively leukocyte-reduced

cellular components are considered equiv-

alent to serologically screened compo-

nents by many experts, although this is

controversial.98-100 The incremental benefit

of serologic testing when added to leuko-

cyte reduction has not been established.

Prophylactic therapy with CMV immune

globulin and prophylactic use of antiviral

agents are being investigated as options

for high-risk immunosuppressed organ

transplant recipients.96,97

Epstein-Barr Virus

EBV causes most cases of infectious mono-

nucleosis and is closely associated with

the endemic form of Burkitt’s lymphoma

in Africa and with nasopharyngeal carci-

noma. Most persons have been infected

by the time they reach adulthood; al-

though usually asymptomatic, infection

persists. Infection is spread by contact

with infected saliva. Primary infection in

children is either asymptomatic or is

characterized by a sore throat and en-

larged lymph nodes. Primary infection in

older, immunologically mature persons

usually causes a systemic syndrome, infec-

tious mononucleosis, with fever; tonsillar

infection, sometimes with necrotic ulcers;

enlarged lymph nodes; hematologic and

immunologic abnormalities; and some-

times hepatitis or other organ involvement.

EBV infection targets B lymphocytes,

which undergo polyclonal proliferation and

then induce a T-lymphocyte response, ob-

served as “atypical lymphocytes.”

Transfusion-transmitted EBV infection is

usually asymptomatic, but it has been a

rare cause of the postperfusion syndrome

that follows massive transfusion of freshly

drawn blood during cardiac surgery and is

a rare cause of posttransfusion hepatitis.101

EBV plays a role in the development of na-

sopharyngeal carcinoma and at least one

form of Burkitt’s lymphoma and has the

in-vitro capacity to immortalize B lympho-

cytes. EBV contributes to the development

of lymphoproliferative disorders in im-

munosuppressed recipients of hemato-

poietic and organ transplants. Given a 90%

seropositivity rate for EBV among blood do-

nors and essentially no risk for clinical dis-

ease from transfusion-transmitted EBV in

immunocompetent recipients, serologic

screening for this virus has not been con-

sidered helpful. As is the case for CMV, leu-

kocyte reduction of cellular blood compo-

nents would be expected to reduce the risk

of EBV infection in severely immunosup-

pressed seronegative patients who may be

at risk for clinical disease. However, there

have been no studies to verify such a

reduced risk.

Chapter 28: Transfusion-Transmitted Diseases 687

Human Herpesviruses 6 and 8

As with CMV and EBV, human herpesvirus

6 (HHV-6) is a cell-associated virus that

integrates with the genome of lympho-

cytes.Theseroprevalenceinsomeadult

populations approaches 100%. Primary

infection in immunocompetent children

is recognized as exanthem subitum, a fe-

brile illness characterized by a rash; it is

rarely complicated by involvement of other

organ systems. Immunocompromised pa-

tients (eg, those with transplants or AIDS)

may experience manifestations of reacti-

vation of HHV-6 infection in multiple or-

gan systems. Studies have sought an asso-

ciation between multiple sclerosis and

HHV-6 infection, but this remains contro-

versial. With the ubiquity of antibodies to

HHV-6 and absence of disease associations

after transfusion transmission, no recom-

mendations have been made for protec-

tion of seronegative blood recipients from

transmission by blood components.102

HHV-8 (also known as Kaposi’s sarcoma-

associated herpesvirus or KSHV) is causally

associated with both Kaposi’s sarcoma and

body cavity-based lymphomas. It has been

found in apparently healthy blood donors,

but spread appears to be primarily by the

venereal route. Low titers of HHV-8 antibodies

were found in 11% of 91 healthy US blood

donors.103 Among HHV-8-seropositive women,

injection drug use and indices of sexual ac-

tivity were independent risk factors for

HHV-8 infection.104 In this study, the associ-

ation with injection drug use suggests that

transmission by infected blood is possible.

Transmission by organ donation has been

documented.105-107 Epidemiologic studies

suggest that blood transfusion is associated

with a small risk of HHV-8 transmission.108

Parvovirus

Parvovirus B19 is the cause of erythema

infectiosum or “fifth disease,” a contagious

febrile illness of early childhood. Infections

in adults may be associated with arthritis

but are generally benign. More ominou-

sly, parvovirus B19 can infect and lyse red

cell precursors in the marrow.109 This may

result in sudden and severe anemia in pa-

tients with underlying chronic hemolytic

disorders who depend on active erythro-

poiesis to compensate for shortened red

cell survival. Patients with cellular immu-

nodeficiency, including those infected with

HIV, are at risk for chronic viremia and as-

sociated hypoplastic anemia. Infection dur-

ing pregnancy predisposes to spontaneous

abortion, fetal malformation, and hydrops

from severe anemia and circulatory failure.110

The red cell P antigen is the cellular re-

ceptor for parvovirus B19, and people who

do not have the P antigen are naturally re-

sistant to infection.111 About 30% to 60% of

normal blood donors have antibodies to par-

vovirus B19, which indicates immunity

rather than chronic persistent infection.109

Viremia occurs only in the early phases of

infection and there is no evidence for a car-

rier state; the incidence of viremia in blood

donors has been estimated to range from 1

in 3300 to 1 in 40,000.110 Parvovirus B19 lacks

a lipid envelope and is therefore not inacti-

vated by solvent/detergent treatment or heat

inactivation using temperatures below 100

C.109 The virus has been found regularly in

clotting factor concentrates and has been

transmitted to persons with hemophilia. Rare

transmission through cellular blood compo-

nents and plasma, but not intravenous im-

munoglobulin and albumin, has been re-

ported.110

After the description of parvovirus B19

seroconversion (without clinical illness) in

volunteers during Phase IV clinical evalua-

tion of solvent/detergent-treated plasma,

derivative manufacturers (with the concur-

rence of FDA) began development and im-

plementation of NAT screening for high-ti-

ter parvovirus B19 viremic donations in

688 AABB Technical Manual

minipools. The rationale, supported by ob-

servations from the Phase IV evaluation, is

that high-titer donations overwhelm neu-

tralizing antibody in plasma pools, allowing

transmission of this highly resistant virus

by some derivatives. The FDA has classified

this MP-NAT as an in-process manufactur-

ing control rather than a donor screening

test. Screening of whole blood donations has

not been a high priority because of the be-

nign and/or transient nature of most par-

vovirus disease, the availability of effective

treatment (intravenous immunoglobulin)

for chronic hematologic sequelae, and the

extreme rarity of reports of parvovirus B19

transmission by individual components.112

Transmissible Spongiform

Encephalopathies

The transmissible spongiform encephalo-

pathies (TSEs) are degenerative brain dis-

orders caused by agents often called prions,

postulated to be infectious proteins. They

are characterized by long incubation peri-

ods, measured in years to decades, and by

the extreme resistance of the pathogens

to inactivation by physical and chemical

methods sufficient for classic pathogens.

Two TSEs, Creutzfeldt-Jakob disease (CJD)

and variant Creutzfeldt-Jakob disease

(vCJD), are of particular interest in trans-

fusion medicine.

Classic CJD

CJD is a degenerative brain disorder that

is rapidly fatal once symptoms of progres-

sive dementia and motor disturbances

develop. Approximately 85% of cases are

sporadic. Symptoms do not develop until

many years to several decades after the

initial infection. Ten to fifteen percent of

cases are familial, associated with inheri-

tance of one of at least 20 described mu-

tations in the prion gene that, in its non-

mutated form, encodes for a normal cel-

lular protein. Worldwide, there is about

one case of CJD per million people per

year, nearly all in older individuals. In

sporadic CJD, the vast majority of cases,

themodeofacquisitionisunknown.The

agent causing CJD is resistant to commonly

used disinfectants and sterilants. Iatro-

genic CJD has been transmitted by ad-

ministration of growth hormone and go-

nadotropic hormone derived from pooled

human pituitary tissue, through allografts

of dura mater, and through reuse of intra-

cerebral electroencephalographic elec-

trodes from infected patients.113

Early experimental studies in animals

raised the possibility that CJD could be

transmitted by blood transfusion. Addition-

ally, iatrogenic transmission from periph-

eral injection of human pituitary-derived

hormones has been observed. Neverthe-

less, several population-based, case-con-

trolled studies have shown no evidence that

blood transfusion is a risk factor for the de-

velopment of CJD.114-116

Individuals at increased risk for CJD are

excluded from donating blood; this group

includes persons who have received tissue

or tissue derivatives known to be a source

of the CJD agent (eg, dura mater allografts,

pituitary growth hormone of human origin)

and persons with a family history of

CJD.117-119 For the purposes of donor exclu-

sion, unit quarantine, and unit destruction,

family history has been defined as having

one blood relative who has had this diagno-

sis. However, a donor with CJD in a family

member may be accepted if gene se-

quences have been tested and found to be

normal.

Variant CJD

In 1996, the first cases of an unusual CJD

outbreak or cluster were described in the

Chapter 28: Transfusion-Transmitted Diseases 689

United Kingdom (UK). These cases were

later termed variant CJD (vCJD) and ap-

peared to be caused by the same prion

responsible for bovine spongiform en-

cephalopathy (BSE). This prion is distinct

from the prion found in classical CJD. A

donor infected by dietary exposure to BSE

during the incubation period of vCJD might

theoretically infect a transfusion recipi-

ent. Consequently, UK health authorities

prohibited the use of UK plasma for fur-

ther manufacture, restricted the use of UK

plasma for children (born on or after 1

January 1996), and implemented univer-

sal leukocyte reduction of cellular compo-

nents (to reduce the prions known to be

present in white cells).

Experimental transfusion transmission

of BSE to two sheep (and four cases of

transmission of natural scrapie—a sheep

prion illness) have been reported.120 Positive

transmissions occurred with blood taken at

preclinical and clinical stages of infection.

Two cases of transfusion-transmitted BSE

in humans have been observed121,122 as a re-

sult of surveillance in the UK of 48 individ-

uals identified as having received a labile

blood component from a total of 15 donors

who later developed vCJD. In the first possi-

ble case, the recipient developed symptoms

of vCJD 6.5 years after receiving a transfu-

sion of red cells donated by an individual

3.5 years before the donor developed

symptoms of vCJD. Although the source of

the infection could have been caused by

past dietary exposure to the BSE agent, the

age of the patient was well beyond that of

most vCJD cases, and the chance of observ-

ing a case of vCJD in a recipient in the ab-

sence of transfusion-transmitted infection

was estimated to be about 1 in 15,000 to 1

in 30,000, making dietary transmission un-

likely in this case.121 In the second possible

case, the person received a blood transfu-

sion in 1999 from a donor who later devel-

oped vCJD. This patient died of causes

unrelated to vCJD, but a postmortem exam-

ination revealed the presence of the

abnormal prion protein in the patient’s

spleenandinalymphnode.

122 Notably, un-

like previous cases of vCJD (by any method

of transmission), in which all involved peo-

ple were homozygous for methionine (MM)

at codon 129 of the prion protein gene

(PRNP), this individual was heterozygous

(methionine valine— MV). In the UK, the

population distribution of this gene is MM,

MV, or VV in 42%, 47%, and 11%, respec-

tively.123

In the United States, blood donors who

wereintheUKorEuropeduringtheyears

of potential exposure to the BSE agent are

deferred based on the duration of residence

there. Balancing the theoretical risk against

considerations of the adequacy of the blood

supply, the recommended deferral is for 3

months of cumulative residence in the UK

between 1980 and 1996 (and current and

former US military personnel, civilian mili-

tary employees, and their dependents who

were stationed at European bases for 6

months or more during this period) or 5

years of cumulative residence in Europe.

Potential donors who may have injected

bovine insulin from the UK or received

transfusions in the UK during the BSE epi-

demic are also excluded.119

Bacterial Contamination

Bacterial contamination remains an im-

portant cause of transfusion morbidity

and mortality. Bacterial contamination of

blood components accounted for 29 (16%)

of the transfusion fatalities reported to

the FDA between 1986 and 1991. How-

ever, in 2002 alone, there were 17 deaths

reported to the FDA from bacterial con-

tamination of blood components, most

commonly caused by contaminated

apheresis platelets and whole-blood-de-

690 AABB Technical Manual

rived platelets.124,125 Although the hepatitis

viruses, HIV, and WNV have been more

prominently featured in the media and

remain a primary concern of the public,

bacterial contamination is believed to be

the most common infectious source of

morbidity and mortality related to trans-

fusion. To place the risk of bacterial con-

tamination into some perspective, in 2002,

there were 23 transfusion-transmitted cases

of WNV identified in the United States.87

Of these 23 recipients, seven died, but

only five of these deaths were associated

with WNV meningoencephalitis. Thus, the

deaths from bacterial contamination were

more than three times more common than

those from WNV.

No matter how carefully blood is drawn,

processed, and stored, complete elimina-

tion of microbial agents is impossible. Bac-

teria are most often believed to originate

with the donor, either from the venipunc-

ture site or from unsuspected bacteremia.126

Bacterial multiplication is more likely in

blood components stored at room temper-

ature than in refrigerated components.126

Organisms that multiply in refrigerated

blood components are often psychrophilic

gram-negative organisms (such as Yersinia

enterocolitica, Serratia liquifaciens, and

Pseudomonas fluorescens). Gram-positive

organisms are more often seen in platelets

stored at 20 to 24 C.

For RBCs, the CDC estimates a sympto-

matic contamination rate of approximately

1 case per million units, primarily with Y.

enterocolitica, followed by S. liquifaciens.127

InNewZealand,theincidenceofsymp

tomatic Yersinia contamination of RBC

units has been reported to be as high as

one in 65,000 units, with a fatality rate of

one in 104,000.128 TransfusionofanRBC

unit heavily contaminated with a gram-

negative organism is often a rapid and cata-

strophic event, with a quick onset of sepsis

and a greater than 60% mortality rate.129,130

Because platelets are stored at 20 to 24 C

to retain their viability and function, they

serve as an excellent growth medium for

bacteria. Sepsis resulting from transfusion

of contaminated platelets is believed to be

both underrecognized and underreported.

Sepsis occurring after transfusion of con-

taminated platelets is usually not a cata-

strophic event, but it can occur several

hoursorlongeraftertransfusion,makingit

more difficult to connect the transfusion to

the sepsis. Because many of the patients in-

fected by bacteria from a platelet transfu-

sion are immunocompromised by their

underlying condition and treatment (eg,

chemotherapy), the event is frequently at-

tributed to other causes, such as an in-

fected catheter, which often involves the

same organisms.

In the United States, 4 million platelet

units are transfused annually (1 million

apheresis platelets and 3 million whole-

blood-derived platelet concentrates).130

Given that approximately 1:1000 to 1:2000

platelet units are contaminated with bacte-

ria (as measured by aerobic cultures done

in multiple studies before 2002), it would

be expected that 2000 to 4000 bacterially

contaminated units would be transfused.131

Estimates of the fraction of such units that

would result in signs or symptoms have

been as low as 1 in 10 cases. However, in

the only study that has prospectively cul-

tured platelets that were transfused, symp-

toms occurred in 3 of 8 (35.8%) patients

who received culture-positive but Gram’s-

stain-negative platelet pools.132 Notably, six

Gram’s-stain-positive pools were inter-

dicted and never transfused. Thus, of con-

taminated products, perhaps 1/10 to 2/5

would be expected to result in clinical sep-

sis (200 to 1600 cases) if transfused. Data

from national passive reporting studies in

the United States, Great Britain, and France

(Table 28-4) suggest that perhaps 1/5 to 1/3

would result in death (40 to 533 deaths per

Chapter 28: Transfusion-Transmitted Diseases 691

year).127,133,134 This translates to a risk of death

from a transfusion of a platelet unit con-

taminated with bacteria of between 1:7500

to 1:100,000. Clinical observations from

university hospitals with heightened aware-

ness of platelet-related sepsis confirm such

estimates. A fatality rate of 1:17,000 has

been reported by Ness et al, from Johns

Hopkins, with pooled whole-blood-derived

platelets and 1:61,000 with apheresis plate-

lets.135 University Hospitals of Cleveland

similarly observed a fatality rate of approxi-

mately 1:48,000 per whole-blood-derived

platelet concentrate.136 With the implemen-

tation of bacteria detection of platelets (see

below), it is anticipated that this rate will be

greatly reduced.

Clinical Considerations

Severe reactions are characterized by fe-

ver, shock, and disseminated intravascular

692 AABB Technical Manual

Table 28-4. Summary of Organisms Identified in the BaCon, SHOT, and BACTHEM

Studies*

Organism

United

States127

United

Kingdom133 France134 Total

Gram positive

Bacillus cereus

14(1)27(1)

Coagulase-negative

Staphylococci

9 6 (1) 5 20 (1)

Streptococcus

3(1) 2 5(1)

Staphylococcus aureus

4 2 (1) 6 (1)

Propionibacterium

acnes

Subtotal 17 (1 = 6%)†14 (3 = 21%) 10 (0 = 0%) 41 (4 = 10%)

Gram negative

Klebsiella

2(1) 2(1)

Serratia

2(2) 1(1) 3(3)

Escherichia coli

5(1) 2(1) 1 8(2)

Acinetobacter

Enterobacter

2(1) 1(1) 1 4(2)

Providencia rettgeri

1(1) 1(1)

Yersinia enterocolitica

Subtotal 11 (5 = 45%) 3 (2 = 67%) 6 (2 = 33%) 20 (9 = 45%)

Total 28 (6 = 21%) 17 (5 = 29%) 16 (2 = 13%) 58 (13 = 14%)

*Number of cases (fatalities) and the percent of the subtotal and total cases are listed. This table illustrates that although

gram-positive organisms are associated with the majority of reported cases (41/58 = 71/%), gram-negative organisms

account for the majority of deaths (9/11 = 82%). Modified with permission from Brecher and Hay.131

†There were 17 cases of gram-positive organisms identified in the US study; however, only one case (1/17 = 6%) re-

sulted in a fatality.

coagulation (DIC). If bacterial contamina-

tion is suspected, the transfusion should

be stopped immediately and a Gram’s stain

and blood culture should be obtained

from the unit (not an attached segment of

tubing because the bag may be contami-

nated but an isolated segment of tubing

may be sterile) and recipient as promptly

as possible after the reaction is observed.

Bacterial multiplication may cause the

oxygen in an RBC unit to be consumed,

resulting in hemoglobin desaturation and

erythrocyte lysis, both of which contrib-

ute to a darkening of the unit compared

to the color of the blood in the attached

sealed segments. Color change (to dark pur-

ple or black), clots in the bag, or hemoly-

sis suggest contamination, but the ap-

pearance of the blood in the bag is often

unremarkable. The presence of bacteria

on a Gram’s stain of the component is

confirmatory, but absence of visible or-

ganisms does not exclude the possibility.

Gram’s stain has a sensitivity of only 106to

107CFU/mL.Thepatient’sblood,thesus-

pect component, and intravenous solu-

tions in all the administration tubing used

should be cultured.

Treatment should not await the results of

these investigations and should include im-

mediate intravenous administration of an-

tibiotics combined with therapy for shock,

renal failure, and DIC, if present.

Preventive Measures

Prevention of septic reactions depends upon

reducing or preventing contamination of

components with bacteria. Careful selec-

tion of healthy blood donors is the first

and most important step.

The donor’s present appearance and re-

cent medical history should indicate good

health; additional questioning may be

needed if there is a present or recent his-

tory of antibiotic use, of medical or surgical

interventions, or of any constitutional

symptoms. Questions to elicit the possibil-

ity of bacteremia are especially important

for autologous donors, who may have un-

dergone recent hospitalization, antibiotic

therapy, or invasive diagnostic or therapeu-

tic procedures; there have been several re-

ports of Yersinia sepsis complications after

the infusion of stored autologous blood.

Scrupulous attention must be paid to se-

lecting and cleansing the donor’s phlebot-

omy site. Skin preparation reduces but does

not completely abrogate the contamination

of components by bacteria. Scarred or dim-

pled areas associated with previous derma-

titis or repeated phlebotomy can harbor

bacteria and should be avoided. Green

soap must not be used to prepare the

phlebotomy site.

Discarding the first aliquot of donor blood

removed (“diversion”) has been proposed

as a measure to reduce bacterial contami-

nation of blood components. This measure

would remove the skin core that may enter

the collection from the hollow bore needle

used in the phlebotomy. Systems have been

developed to facilitate the application of

this approach and would be expected to re-

duce skin contaminants (mostly gram-pos-

itive organisms).

Phagocytosis of contaminating bacteria

by donor white cells in blood components

may be important for the minimization of

clinical bacterial contamination. Leukocyte

removal, with coincident removal of adher-

ent or engulfed bacteria, has been advo-

cated as an approach to reducing Yersinia

contamination of RBCs.137,138

Care in the preparation of components

and handling of materials used in blood ad-

ministration is essential. If a waterbath is

used, components should be protected by

overwrapping, outlet ports should be in-

spected for absence of trapped fluid, and

the waterbath should be frequently emp-

tied and disinfected.

Chapter 28: Transfusion-Transmitted Diseases 693

Worldwide, screening of platelets for

bacteria is being implemented. Screening is

mandatory in several countries [eg, Bel-

gium (Flemish Red Cross), the Netherlands,

Hong Kong (Red Cross), and Wales].139

In the United States, the College of

American Pathologists (CAP) Commission

on Laboratory Accreditation has added a

question to the Transfusion Medicine

Checklist to assess the presence of a labora-

tory system to detect bacteria in platelet

components (TRM.44955).140 Similarly, the

AABB requires bacteria detection.62(p11) Cur-

rently, two culture techniques are approved

by the FDA for quality control of leuko-

cyte-reduced platelets and are available in

the United States. Because of expense and

logistics, whole-blood-derived platelets are

often tested in the United States with less

sensitive but more rapid detection strate-

gies, such as staining or the use of surrogate

markers of bacterial metabolism (eg, pH

and glucose). Several other more rapid and

sensitive detection strategies are under de-

velopment or are not readily available. One

possible investigative strategy after detect-

ing a confirmed culture-positive platelet

unit is outlined in Fig 28-4.

Prospect for Extended Storage

The extent of bacterial growth in platelet

components correlates with the duration

of storage. In 1983, in recognition of tech-

694 AABB Technical Manual

Figure 28-4. Possible investigative strategy for a positive culture in a platelet unit. Modified with per-

mission.141

nically improved storage conditions, the

FDA increased storage limits of platelets

at room temperature from 3 to 7 days.

However, it reduced the limits to a maxi-

mum of 5 days in 1986, responding to re-

ports of bacterial contamination after

more than 5 days of storage.142 The use of

bacteria detection systems has been given

as the rationale for an extension of plate-

let storage to 7 days in several European

countries and is being implemented in the

United States.139

Syphilis

Syphilis is caused by the spirochete Tre-

ponema pallidum and is characteristically

spread by sexual contact. The phase of

spirochetemia is brief and the organisms

survive only a few days at 4 C. Although

transmission by transfusion is possible,

its occurrence is exceedingly rare (the last

case reported in the United States oc-

curred in 1965). Syphilis transmission by

transfusion may not be effectively pre-

vented by subjecting the donor blood to

standard serologic tests for syphilis (STS)

because seroconversion often occurs after

the phase of spirochetemia. Most positive

STS results reflect immunologic abnor-

malities unrelated to syphilis (biologic

false-positives), inadequately treated non-

infectious syphilis that is more of a threat

to the individual being tested than to a

transfusion recipient, or the serologic re-

sidual of an effectively treated infection.

A recent series described T. pallidum

DNAandRNAtestingof169aliquotsfrom

platelet concentrates that were reactive by

STS and confirmed positive by fluorescent

treponemal antibody absorption.143 This se-

ries included 48 donors who were positive

by rapid plasma reagin tests (compatible

with recent or active disease). No sample

contained T. pallidum DNA or RNA, sug-

gesting a low probability that the blood of

donors who have a confirmed positive

syphilis test result is infectious for syphilis.

Nevertheless, a study from the CDC showed

that from 1995-2000, 22 primary, 81 sec-

ondary, and 413 early latent syphilis cases

were identified through blood or plasma

donor screening in the United States.144

Thus, screening of blood donors for syphilis

may have broader public health implica-

tions. Currently, performance of a STS is

still required.62(pp33,34)

Tick-Borne Infections

Because many tick-borne infectious agents

circulate in the blood, it is theoretically

possible that they will be transmitted by

transfusion of blood components.

Babesia

Clinical Events

In the United States, the most frequently

recognized transfusion-associated tick-borne

infection is babesiosis.145 Babesiosis is

usually transmitted by the bite of an in-

fected deer (black-legged) tick and is re-

ported most frequently in the coastal lands

and islands of northeastern United States,

including Martha’s Vineyard, Cape Cod, and

Long Island. Geographic areas of the hosts

and the vectors appear to be expanding.146

Transfusion-associated babesiosis has been

documented in more than 50 cases, caused

mostly by Babesia microti from the North-

east, but also by the recently recognized

WA1-type Babesia parasite, from asymp-

tomatic infected blood donors.145-147 As hu-

mans continue to encroach on the habitat

of vectors and natural reservoirs of infec-

tion [eg, deer (and other Cervidae)and

mice populations in the northeastern

United States], the incidence of transfu-

sion-transmitted babesiosis may increase.

Chapter 28: Transfusion-Transmitted Diseases 695

The vector and reservoir of the Babesia

more recently found in the northwestern

and western United States remain to be

defined. Babesia species survive blood

bank storage for up to at least 35 days and

canbetransmittedbybothRBCsand

platelet concentrates. Babesiosis classi-

cally causes a febrile illness with hemoly-

tic anemia, but infection can also cause

chronic asymptomatic or mildly symp-

tomatic parasitemia. Studies suggest that

untreated persons can harbor B. microti

DNA for long periods, despite mild or ab-

sent symptoms, and may transmit infec-

tion for months or possibly longer.148 Sym-

ptoms are often so mild that the infection

is not recognized, which likely explains

the low rate of reported transfusion-trans-

mitted babesiosis. Symptomatic patients

develop fever 2 to 8 weeks after transfu-

sion, sometimes associated with chills,

headache, hemolysis, or hemoglobinuria.

Rarely, life-threatening hemolytic anemia,

renal failure, and coagulopathy develop,

particularly in asplenic or severely im-

munocompromised patients.149

Preventive Measures

The Babesia carrier state may be asymp-

tomatic and may exceed a year in dura-

tion. Persons with a history of babesiosis

are indefinitely deferred, because lifelong

parasitemia can follow recovery from

symptomatic illness. Restrictive policies,

such as not collecting blood in areas

where the disease vectors are endemic

during spring and summer months when

tick bites are more common, are in prac-

tice in some locations but probably are of

limited value. No test is available for mass

screening to detect asymptomatic carriers

of Babesia species.

Other Agents

One case of transfusion transmission of

Rocky Mountain spotted fever (Rickettsia

rickettsii) and no cases of human mono-

cytic ehrlichiosis (caused by Erlichia chaf-

feensis) have been documented.150 Asingle

possible transfusion transmission of the

unnamed agent of human granulocytic

erlichiosis has been reported.151 In 1997,

Rocky Mountain spotted fever and/ or hu-

man monocytic erlichiosis developed in

National Guard trainees at Fort Chaffee,

AR. Ten components donated by infected

trainees had been transfused before a re-

call; however, none of the persons who re-

ceived blood from infected donors became

clinically ill.152

Lyme disease is the most common tick-

borne infection in the United States.

Borrelia burgdorferi, the causative spiro-

chete, is transmitted through bites of the

deer (black-legged) tick. No transfusion-

related cases have been reported, but

chronic subclinical infections do occur

and experimentally inoculated organisms

can survive conditions of frozen, refriger-

ated, and room temperature storage.153 On

the other hand, the phase of spirochete-

mia seems to be associated with symp-

toms that would render a potential donor

ineligible, and in two reported cases where

the donor became ill shortly after dona-

tion, the recipient did not develop infec-

tion.149 Potential donors who give a history

ofLymediseaseshouldbecompletely

asymptomatic and should have com-

pleted a full course of antibiotic therapy

before they are permitted to donate. Trans-

fusion transmission of tick-borne agents is

biologically plausible and, for some agents,

has been demonstrated. Nevertheless,

modifications to current donor screening

are not likely to be useful because of their

low predictive value and the potential

for nonspecific questions to defer large

696 AABB Technical Manual

numbers of donors for a small increment in

transfusion safety.153

Other Nonviral Infectious

Complications of Blood

Transfusion

Malaria

Malaria is caused by several species of

the intraerythrocytic protozoan genus

Plasmodium. Transmission usually results

fromthebiteofananophelesmosquito,but

infection can follow transfusion of para-

sitemicblood.Althoughveryrareinthe

United States, malaria is probably the most

commonly recognized parasitic complica-

tion of transfusion; the risk in the United

States is estimated to be <0.3 case per mil-

lion transfusions.154,155 From 1963 to 1999, 93

cases of transfusion-transmitted malaria (10

fatal) in the United States were reported to

CDC.155

The species involved in transfusion-

transmitted malaria in the United States are

P. f a l c i p a r u m (35%), P. m a l a r i a e (27%), P.

vivax (27%), and P. o v a l e (5%).155 Three per-

cent were mixed infections, and 2% were

caused by unidentified species. Fever,

chills, headache, and hemolysis occur a

week to several months after the infected

transfusion; morbidity varies but can be se-

vere, and deaths have occurred, especially

from P. f a l c i p a r u m . Adding to the risk of a

fatal outcome may be a lack of immunity in

the recipient, the patient’s underlying con-

dition(s), and delay in the diagnosis be-

cause of lack of suspicion and unfamiliarity

with the disease in areas where the parasite

is not endemic.

Malaria parasites survive for at least a

week in components stored at room tem-

perature or at 4 C. The parasites can also

survive cryopreservation with glycerol and

subsequent thawing. Any component that

contains red cells can transmit infection,

via the asexual form of the intraerythrocytic

parasite.

Asymptomatic carriers are generally the

source of transfusion-transmitted malaria,

although their parasite density is very low.

Asymptomatic infections rarely persist

more than 3 years, but asymptomatic P.

falciparum and P. v i v a x infections may per-

sist for 5 years, P. o v a l e for 7 years, and P.

malariae can remain transmissible for the

lifetime of the asymptomatic individual. In

extreme cases, transmission of P. v i v a x ,P.

ovale,P. f a l c i p a r u m , and P. m a l a r i a e have

been reported at 27, 7, 13, and 53 years, re-

spectively.156 There are no practical sero-

logic tests to detect transmissible malaria in

asymptomatic donors. Malaria transmis-

sion is prevented by deferral of prospective

donors with increased risk of infectivity,

based on their medical and travel history.

The AABB requires that prospective donors

who have had a diagnosis of malaria, or

who have traveled or lived in a malaria-en-

demic area and have had unexplained

symptoms suggestive of malaria, be de-

ferred for 3 years after becoming asymp-

tomatic.62(p65) Individuals who have lived for

at least 5 consecutive years in areas in

which malaria is considered endemic by

the CDC Malarial Branch shall be deferred

for 3 years after departure from that area.

Individuals who have traveled to an area

where malaria is endemic shall be deferred

for 12 months after departing that area.

These deferral periods apply irrespective of

the receipt of antimalarial prophylaxis. Up-

dated information on malaria risks world-

wide is available from the CDC, including

an on-line resource (http://www.cdc.gov/

travel/yb/outline.htm#2).

Chagas’ Disease

American trypanosomiasis, or Chagas’

disease, is endemic in South and Central

Chapter 28: Transfusion-Transmitted Diseases 697

America and is caused by the protozoan

parasite Trypanosoma cruzi. The human

host sustains infection after the bite of

reduviid bugs (called cone-nosed or “kiss-

ing” bugs), which usually exist in hollow

trees, palm trees, and in thatched-roofed

mud or wooden dwellings. Naturally ac-

quired Chagas’ disease in the United States

is exceedingly rare. Five such cases have

been recognized in the United States since

1955, the most recent in 1998 in Tennes-

see.157

Clinical Events

T. cruzi infectshumanswhoseskinormu

cosa comes in contact with feces of in-

fected reduviid bugs, usually as the result

of a bite. Recent infections are usually ei-

ther asymptomatic or the very mild signs,

and symptoms go undetected. Rarely, the

site of entry evolves into an erythematous

nodule called a chagoma, which may be

accompanied by lymphadenopathy. Fever

and enlargement of the spleen and liver

may follow. Recently infected young chil-

dren may experience acute myocarditis or

meningoencephalitis. Acute infection

usually resolves without treatment, but

persisting low-level parasitemia is usual

and up to one-third of infected individu-

als develop a chronic form associated with

cardiac or gastrointestinal symptoms

years or decades later.158

Transfusion Considerations

Blood transfusion has been a major source

of infection with T. cruzi in South Ameri-

can urban centers that receive large num-

bers of immigrants from rural areas where

the parasite is endemic. However, in many

countries, serologic screening has been

effective in reducing the risk of transfu-

sion-transmitted Chagas’ disease. Four

cases of transfusion-transmitted Chagas’

diseasehavebeenreportedintheUnited

States158:inNewYork,LosAngeles,Texas,

and Florida. All occurred in immuno-

compromised patients. Additionally, two

cases were reported in Manitoba, Canada.

In one interesting study, postoperative

blood specimens from 11,430 cardiac sur-

gery patients were tested by EIA and, if

repeatedly reactive, were confirmed by

radioimmunoprecipitation. Six postoper-

ative specimens (0.05%) were confirmed

positive. All six seropositive patients ap-

parently were infected with T. cruzi before

surgery; however, a diagnosis of Chagas’

disease was not known or even consid-

ered in any of these patients. No evidence

for transfusion-transmitted T. cruzi was

found.159

Reasonably sensitive and specific EIA

screening tests for antibodies to T. cruzi,as

well as confirmatory Western blot and

radioimmunoprecipitation assays, have

been developed.158 Testing in several US

blood centers located in geographic areas

with a large immigrant population from

Central or South America found a sero-

prevalence of 0.1% to 0.2% among at-risk

donors, who were identified by question-

naire.160,161 However, look-back studies iden-

tified no infected recipients; it is also likely

that not all at-risk donors can be identified

by questionnaire.160 As a consequence, if blood

donor screening were to be implemented,

testing of all donors might be necessary.

Other Parasites

Toxoplasmosis is caused by the ubiquitous

parasite Toxoplasma gondii, and infection

has been reported as an unusual transfu-

sion complication in immunocompromi-

sed patients.162 The disease has not been

considered a problem in routine transfu-

sion practice.

There have been occasional reports of

parasitic worm infections transmitted by

transfusion in countries other than the

698 AABB Technical Manual

United States.162 Microfilariasis is a potential

transfusion risk in tropical zones, acquired

by donors through bites by insects carrying

Wuchereria bancrofti.Transfusiontrans

mission of Leishmania species is a rare risk

in countries where such organisms are en-

demic. Currently, the AABB defers potential

donors who have been to Iraq in the previ-

ous12monthsasaresultofpossibleLeish-

mania exposure.163

Reducing the Risk of

Infectious Disease

Transmission

Overall, the risk per unit of transfusion-

transmitted disease is remarkably low

(Table 28-5). This low incidence is due to

both donor screening and specific disease

testing. Nevertheless, in pooled compo-

nents, which may contain elements from

thousands of donors, the risk of disease

transmission is increased. Therefore, sev-

eral strategies have been developed and

implemented to further reduce the risk of

disease transmission in pooled acellular

components and, in some cases, cellular

components.

Inactivation/Destruction of Agents in

Derivatives or Plasma Products

The first intervention specifically added

to reduce the risk of hepatitis transmis-

sion was heating (to 60 C for 10 hours),

which has been used for albumin prod-

ucts since at least 1948.167 In those rare in-

stances when infections occurred with

plasma protein fractions prepared with

this step, the processing had been com-

promised.

Immunoglobulins

The plasma fractionation process used for

most immunoglobulin products employs

cold ethanol precipitation after removal

of cryoprecipitate. Historically, when anti-

bodies to HCV were present in the plasma,

this process concentrated HCV in the Fac-

tor VIII-rich cryoprecipitate and other

fractions and left little in the immuno-

globulin fraction. The immunoglobulin

fraction also has a high concentration of

virus-neutralizing antibodies and the re-

sulting product for intramuscular appli-

cation has a remarkably low risk of virus

transmission.168

Preparations of immunoglobulin intended

for intravenous administration (IGIV) were

expected to be similarly free of disease

transmission. However, NANB hepatitis

transmission did occur in the 1980s during

early clinical trials of IGIV products in the

United States and with routinely manufac-

tured IGIV products in Europe.169 In late

1993 and early 1994, a worldwide outbreak

with more than 200 reported HCV infec-

tions was traced to a single IGIV prepara-

tion licensed in the United States.170,171 In

this case, transmission apparently occurred

because of lack of virus inactivation steps in

the specific manufacturing process for this

product172 and absence of complexing and

neutralizing anti-HCV subsequent to anti-

HCV screening of plasma donors, with re-

sultant accumulation of virus particles in

the immunoglobulin fraction.170 Anti-HCV-

positive source plasma has been excluded

from the manufacture of IGIV since 1992.

Theimportanceofthemanufacturing

method is underscored by outbreaks of HCV

infection from intravenous anti-D immu-

noglobulin in Germany in the late 1970s

and in Ireland from the late 1970s to the

early 1990s.173,174 Both products were pre-

pared by anion exchange chromatography

rather than cold-ethanol (Cohn) fraction-

ation.175 To prevent further HCV outbreaks,

the FDA has required, since 1994, virus

clearance steps in the manufacturing pro-

cess of immunoglobulin or proof of ab-

Chapter 28: Transfusion-Transmitted Diseases 699

700 AABB Technical Manual

Table 28-5. Infectious Risks of Blood Transfusion in the United States

Infectious Agent

or Outcome

Estimated Risk per

Unit Transfused

Estimated % of

Infected Units that

Transmit or Cause

Clinical Sequelae* Reference

Viruses

HIV-1 and -2 1:1,400,000-

1:2,400,000

90 23, 29, 30

HTLV-I and -II 1:256,000-

1:2,000,000

30 84

HAV 1:1,000,000 90 164

HBV 1:58,000-1:147,000 70 29

HCV 1:872,000-1:

1,700,000

90 23, 29, 30

B19 parvovirus 1:3,300-1:40,000 Low 110

Bacteria

RBCs 1:1000 1:10,000,000 fatal 165

Platelets

(screened with

Gram’s stain, pH,

or glucose con-

centration)

1:2000-1:4000 >40% result in clini-

cal sequelae

30, 132, 166

(screened with

early aerobic cul-

ture)

<1:10,000 Unknown

Parasites

Babesia and malaria <1:1,000,000†Unknown 145, 156, 164

Trypanosoma cruzi

Unknown <20 158

*Units that were confirmed test positive for the infectious agent.

Note: West Nile virus is not included in this table because of regional, temporal, and testing (eg, minipool vs individual

donation testing) variation; decreasing rates of infection; and the fact that all testing in the United States is being con-

ducted under an investigational new drug protocol.

†Risk is higher in areas where

Babesia

is endemic.

senceofHCVfromthefinalproductby

NAT. In addition, NAT technology is now

applied to screening of source plasma as an

additional layer of safety.

Coagulation Factors

Until the early 1980s, clotting factor con-

centrates frequently transmitted viral in-

fections. As the significance of HIV trans-

mission was recognized, virus inactivation

steps were applied more rigorously to

Factor VIII and other clotting factor con-

centrates, even though these steps were

initially introduced in the hope of reduc-

ing hepatitis transmission. Unfortunately,

a large proportion (over 50%) of the hemo-

philic population receiving concentrates

before processing was improved became

infected with HIV. Chronic hepatitis was

an additional complication in almost all

patients with hemophilia receiving older

clotting factor products.176

The thermal instability of Factor VIII

made it difficult to develop an effective heat

treatment, until a practical approach was

widely adopted in 1985. Since then, many

virus inactivation steps have been introduced,

and factor concentrates are now, in general,

very safe products. Each process has its

own set of advantages and disadvantages.

Application of organic solvents and deter-

gents inactivates viruses with a lipid-con-

taining envelope (eg, HIV, HBV, HCV, HTLV,

EBV, CMV, HHV-6, HHV-8) but is ineffective

against nonenveloped agents such as HAV

and parvovirus B19. Virus inactivation steps

have the potential drawback of reducing the

potency and biologic effectiveness of the

product. Another concern is whether virus

inactivation steps affect immunogenicity,

especially the induction of Factor VIII in-

hibitors in patients with hemophilia.

Current Risks of Human Plasma Deriva-

tives. Many methods are highly effective

against enveloped viruses, but sporadic re-

ports of viral transmission continue to

occur, possibly resulting from accident or

error during the manufacturing process.

The combination of heat treatment, sol-

vent/detergent treatment, and purification

steps with monoclonal antibodies provides

clotting factor concentrates with a risk of

transmitting hepatitis and HIV that is lower

than the risk associated with use of Cryo-

precipitated Antihemophilic Factor derived

from individual voluntary whole blood do-

nations. Documented transmission of HBV,

HCV, or HIV by US-licensed plasma deriva-

tivesisraresincetheintroductionofeffec

tive virus inactivation procedures and im-

proved viral screening. Although absolute

safety of products derived from human

plasma cannot be guaranteed, starting with

the safest possible donated plasma reduces

the viral load and has contributed to the ex-

cellent safety record of the products sub-

jected to virus inactivation/removal.177

Avoiding Human Plasma. Factor VIII

concentrates produced by recombinant

DNA technology are licensed for use and

have become the preparation of choice for

previously untreated patients with hemo-

philia.178 Batches are produced by culture of

mammalian cells engineered to secrete

Factor VIII into the supernatant medium,

which is purified by ion-exchange chroma-

tography and immunoaffinity chromatog-

raphy using a mouse monoclonal antibody.

Exceptforthefactthatexcipienthumanal

bumin is sometimes added to stabilize Fac-

tor VIII, the product is free of human pro-

teins, HIV, hepatitis viruses, and other

unwanted agents, thus avoiding many of the

risks associated with using human plasma.

On the other hand, recombinant products

have a relatively short history of use and

there is no guarantee that they are risk-free.

Plasma

Virus reduction steps, originally developed

for purified plasma protein fractions, have

Chapter 28: Transfusion-Transmitted Diseases 701

also been applied to plasma intended for

transfusion. Alternative approaches being

studied include organic solvents and de-

tergents and use of photochemicals. Sol-

vent/detergent treatment, which is effec-

tive against lipid-enveloped viruses, involves

addition of 1% Triton X-100 and 1% tri-n-

butyl phosphate (TNBP) to pooled plasma,

followed by oil extraction of the TNBP and

chromatographic adsorption of the Triton

X-100. After several years of experience

with this method in Europe, solvent/de-

tergent-treated plasma was transiently

available in the United States. However,

concern with the use of a pooled product,

expense, poor market penetration, and

possible thrombotic (or excessive bleed-

ing) events led to the discontinuation of

this product. A psoralen (S59) activated by

ultraviolet A light is undergoing US clini-

cal trials for pathogen inactivation in

platelets and plasma and is available in

some countries in Europe.

Processing Cellular Components

Photochemical and chemical pathogen

inactivation methods are theoretically ap-

plicable to cellular blood components;

another organic chemical (S-303) and a

nucleic acid targeting compound (PEN

110) have undergone initial clinical evalu-

ation for pathogen inactivation in red cells.

These methods have the potential for reli-

able inactivation of bacteria, viruses, and

parasites, including intracellular forms.179,180

However, both products have been shown

to result in the formation of antibodies in

recipients. Pathogen reduction trials in

platelets (eg, with S-59) have been associ-

ated with decreased cell recovery and sur-

vival and the need for increased platelet

transfusions. Such unintended consequences

of pathogen reduction have resulted in

some trials being halted and may ulti-

mately eliminate such molecules from

widespread clinical application.

Reporting Transfusion-Associated

Infections

Unexplained infectious disease reported

in a transfusion recipient must be investi-

gated for the possibility of transfusion-

transmitted illness.62(pp83,85) Hepatitis is ex-

pected to become apparent within 2 weeks

to 6 months if it resulted from transfu-

sion, but, even within this interval, the

cause need not necessarily have been

blood-borne infection. Blood centers and

transfusion services must have a mecha-

nism to encourage recognition and re-

porting of possible transfusion-associated

infections. HIV infection thought to be a

result of transfusion should also be re-

ported to the blood supplier, although the

interval between transfusion and the rec-

ognition of infection or symptoms may be

years.

Infectioninarecipientshouldbere-

ported to the collecting agency so that do-

nors shown or suspected of being infec-

tious can be evaluated and recipients of

other components from the implicated or

other donations can be contacted and, if

necessary, tested. A donor who proves to

have positive results on tests during the in-

vestigation must be placed on an appropri-

ate deferral list.

Reporting Fatalities

The Code of Federal Regulations [21 CFR

606.170(b)] requires that fatalities attrib-

uted to transfusion complications (eg,

hepatitis, AIDS, and hemolytic reactions)

be reported to the Director, Center for

Biologics Evaluation and Research

(CBER), Office of Compliance and

Biologics Quality, Attn: Fatality Program

Manager (HFM-650), 1401 Rockville Pike,

Suite 200N, HFM-650, Rockville, MD

702 AABB Technical Manual

20852-1448. A report should be made as

soon as possible by telephone

(301-827-6220), fax (301-827-6748), or

email to fatalities2@cber.fda.gov and a

written report should be submitted within

7 days. Current information can be found

on the Internet at http://www.fda.gov/

cber/gdlns/bldfatal.pdf.

Management of Posttransfusion Infections

Implicated Donors

If documented transfusion-associated

hepatitis, HIV, or HTLV-I/II occurs in a pa-

tient who received only a single unit, that

donor must be permanently excluded

from future donations, and the name

placed in a file of permanently deferred

individuals. If posttransfusion viral infec-

tion occurs after exposure to blood from

several donors, it is not necessary to ex-

clude all of the potentially implicated do-

nors.Ifonlyafewdonorsareinvolved,it

may be desirable to recall them to obtain

an interim medical history and to per-

form additional tests. Donors found to

have been implicated in more than one

case of transfusion-associated viral infec-

tion should be appropriately investigated

and possibly deferred permanently ac-

cording to procedures established by the

collecting agency.

Notification

A donor who will be permanently exclu-

ded as a future blood donor because of a

positive test implication in posttransfu-

sion viral infection must be notified of

this fact. Follow-up testing should, ideally,

be done by the donor’s own physician,

and the collecting agency should obtain

the donor’s consent to release available

information to a designated health-care

provider. If the donor does not have a

physician, a blood bank physician or

other trained staff member should pro-

vide initial counseling and appropriate

medical referral. The notification process

andcounselingmustbedonewithtact

and understanding, and the concerns of

the donor should be addressed. The do-

nor should be told clearly why he or she is

deferred and, when appropriate, about

the possibility of being infectious to oth-

ers. Notification should occur promptly

because a delay in notification can delay

initiation of treatment or institution of

measures to prevent the spread of infec-

tion to others.

Use of Immunoglobulins

It is not recommended practice to give in-

tramuscular or intravenous immune serum

globulin or HBIG prophylactically to pre-

vent posttransfusion hepatitis181;these

agents have not been shown to prevent

posttransfusion hepatitis B, and the avail-

able evidence is conflicting about their ef-

fect on posttransfusion hepatitis C.182,183 If

there has been inadvertent transfusion of

known marker-positive blood or needle-

stick exposure to infectious material, HBIG

may prevent or attenuate HBV infection.184

Prophylaxis with immunoglobulin is inef-

fective in preventing HCV transmission

following occupational exposures and is

not recommended for this indication.185

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117. Food and Drug Administration. Memoran-

dum: Revised precautionary measures to re-

duce the possible risk of transmission of

Creutzfeldt-Jakob disease (CJD) by blood and

blood products. (August 5, 1995) Rockville,

MD: CBER Office of Communication, Train-

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118. Food and Drug Administration. Memoran-

dum: Revised precautionary measures to re-

duce the possible risk of transmission of

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blood products. (December 11, 1996) Rock-

ville, MD: CBER Office of Communication,

Training, and Manufacturers Assistance, 1996.

119. Food and Drug Administration. Draft guid-

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duce the possible risk of transmission of

Creutzfeldt-Jakob disease (CJD) and variant

Creutzfeldt-Jakob disease (vCJD) by human

cells, tissues, and cellular and tissue-based

products (HCT/Ps). (June 25, 2002) Fed

Regist 2002;67:42789-90.

120. Hunter N, Foster J, Chong A, et al. Transmis-

sion of prion diseases by blood transfusion. J

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121. Llewelyn CA, Hewitt PE, Knight RS, et al. Pos-

sible transmission of variant Creutzfeldt-

Jakob disease by blood transfusion. Lancet

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122. Peden AH, Head MW, Ritchie DL, et al. Pre-

clinical vCJD after blood transfusion in a

PRNP codon 129 heterozygous patient. Lan-

cet 2004;364:521-9.

123. Nurmi MH, Bishop M, Strain L, et al. The nor-

mal population distribution of PRNP codon

129 polymorphism. Acta Neurol Scand 2003;

108:374-8.

124. Sazama K. Bacteria in blood for transfusion.

A review. Arch Pathol Lab Med 1994;118:350-65.

125. Food and Drug Administration. CBER annual

report FY 2002 (October 1, 2001 through Sep-

tember 30, 2002). Rockville, MD: CBER Office

of Communication, Training, and Manufac-

turers Assistance, 2002. [Available at http://

www. fda.gov/cber/inside/annrptpart3.htm.]

126. Morrow JF, Braine HG, Kickler TS, et al. Septic

reactions to platelet transfusions. A persistent

problem. JAMA 1991;266:555-8.

127. Kuehnert MJ, Roth VR, Haley NR, et al. Trans-

fusion-transmitted bacterial infection in the

United States, 1998 through 2000. Transfu-

sion 2001;41:1493-9.

128. Theakston EP, Morris AJ, Streat SJ, et al.

Transfusion transmitted Yersinia entero-

colitica infection in New Zealand. Aust N Z J

Med 1997;27:62-7.

129. Cookson ST, Arduino MJ, Aguero SM, Jarvis

WR, and the Yersinia Study Group. Yersinia

enterocolitica-contaminated Red Blood Cells

708 AABB Technical Manual

(RBCs)—an emerging threat to blood safety

(abstract). In: Program and abstracts of the

36th Interscience Conference on Antimicro-

bial Agents and Chemotherapy, New Or-

leans, September 15-18, 1996. Washington,

DC: American Society for Microbiology,

1996:237.

130. National Blood Data Resource Center. Com-

prehensive report on blood collection and

transfusion in the United States in 2001.

Bethesda, MD: AABB, 2003.

131. Brecher ME, Hay SN. Improving platelet

safety: Bacterial contamination of platelets.

Curr Hematol Rep 2004;3:121-7.

132. Dykstra A, Hoeltge G, Jacobs M, et al. Platelet

bacterial contamination (PBC) rate is sur-

veillance method (SM) dependent (abstract).

Transfusion 1998;38(Suppl):104S.

133. Serious Hazards of Transfusion (SHOT). An-

nual report 2000-2001. Manchester, UK:

SHOT Office, 2002. [Available at http://www.

shot.demon.co.uk/toc.htm.]

134. Perez P, Salmi LR, Follea G, et al. Determi-

nants of transfusion-associated bacterial

contamination: Results of the French

BACTHEM case-control study. Transfusion

2001;41:862-71.

135. Ness PM, Braine HG, King K, et al. Single do-

nor platelets reduce the risk of septic trans-

fusion reactions. Transfusion 2001;41:857-61.

136. Engelfriet CP, Reesink HW, Blajchman MA, et

al. Bacterial contamination of blood compo-

nents. Vox Sang 2000;78:59-67.

137. Kim DM, Brecher ME, Bland LA, et al. Pre-

storage removal of Yersinia enterocolitica

from red cells with white cell-reduction fil-

ters. Transfusion 1992;32:658-62.

138. Buchholz DH, AuBuchon JP, Snyder EL, et al.

Removal of Yersinia enterocolitica from AS-1

red cells. Transfusion 1992;32:667-72.

139. Pietersz RNI, Engelfriet CP, Reesink HW, et

al. Detection of bacterial contamination of

platelet concentrates. International Forum.

Vox Sang 2003;85:224-39.

140. Commission on Laboratory Accreditation.

Transfusion medicine checklist. TRM.44955

Phase I. Northfield, IL: College of American

Pathologists, January 2003. [Available at

http://www.cap.org/html/checklist_html/

transfusionmedicine_1202.html.]

141. Brumit MC, Hay SN, Brecher ME. Bacteria

detection. In: Brecher ME, ed. Bacterial and

parasitic contamination of blood compo-

nents. Bethesda, MD: AABB Press, 2003:57-

82.

142. Anderson KC, Lew MA, Gorgone BC, et al.

Transfusion-related sepsis after prolonged

platelet storage. Am J Med 1986;81:405-11.

143. Orton SL, Liu H, Dodd RY, Williams AE. Prev-

alence of circulating Treponema pallidum

DNA and RNA in blood donors with con-

firmed positive syphilis tests. Transfusion

2002;42:94-9.

144. Gardella C, Marfin AA, Kahn RH, et al. Per-

sons with early syphilis identified through

blood or plasma donation screening in the

United States. J Infect Dis 2000;185:545-9.

145. Leiby DA. Babesia and other parasites. In:

Brecher ME, ed. Bacterial and parasitic con-

tamination of blood components. Bethesda,

MD: AABB Press, 2003:179-200.

146. Herwaldt BL, Springs FE, Roberts PP, et al.

Babesiosis in Wisconsin: A potentially fatal

disease. Am J Trop Med Hyg 1995;53:146-51.

147. Herwaldt BL, Kjemtrup AM, Conrad PA, et al.

Transfusion-transmitted babesiosis in Wash-

ington State: First reported case caused by a

WA1-type parasite. J Infect Dis 1997;175:

1259-62.

148. Krause PJ, Spielman A, Telford SR 3rd, et al.

Persistent parasitemia after acute babesiosis.

N Engl J Med 1998;339:160-5.

149. Cable R, Trouern-Trend J. Tick-borne infec-

tions. In: Linden JV, Bianco C, eds. Blood

safety and surveillance. New York: Marcel

Dekker, 2001:399-422.

150. Wells GM, Woodward TE, Fiset P, Hornick RB.

Rocky Mountain spotted fever caused by

blood transfusion. JAMA 1978;239:2763-5.

151. Eastlund T, Persing D, Mathiesen D, et al.

Human granulocytic ehrlichiosis after red

cell transfusion (abstract). Transfusion 1999;

39(Suppl):117S.

152. Arguin PM, Singleton J, Rotz LD, et al. An in-

vestigation into the possibility of transmis-

sion of tick-borne pathogens via blood

transfusion. Transfusion-Associated Tick-

Borne Illness Task Force. Transfusion 1999;

39:828-33.

153. McQuiston JH, Childs JE, Chamberland ME,

TaborEfortheWorkingGrouponTransfu

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Transmission of tick-borne agents of disease

by blood transfusion: A review of known and

potential risks in the United States. Transfu-

sion 2000;40:274-84.

154. Mungai M, Tegtmeier G, Chamberland M,

Parise M. Transfusion-transmitted malaria

in the United States from 1963 through 1999.

N Engl J Med 2001;344:1973-8.

155. Centers for Disease Control and Prevention.

Probable transfusion-transmitted malaria—

Houston, Texas, 2003. MMWR Morb Mortal

Wkly Rep 2003;52:1075-6.

156. Katz LM. Transfusion-induced malaria. In:

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Chapter 28: Transfusion-Transmitted Diseases 709

tamination of blood components. Bethesda,

MD: AABB Press, 2003:127-55.

157. Herwaldt BL, Grijalva MJ, Newsome AL, et al.

Use of polymerase chain reaction to diagnose

the fifth reported US case of autochthonous

transmission of Trypanosoma cruzi,inTen

nessee, 1998. J Infect Dis 2000; 181:395-9.

158. Shulman IA. Transfusion of T. cruzi infection

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Bacterial and parasitic contamination of

blood components. Bethesda, MD: AABB

Press, 2003:157-78.

159. Leiby DA, Rentas FJ, Nelson KE, et al. Evi-

dence of Trypanosoma cruzi infection (Cha-

gas’ disease) among patients undergoing car-

diac surgery. Circulation 2000;102:2978-82.

160. Leiby DA, Read EJ, Lenes BA, et al. Seroepi-

demiology of Trypanosoma cruzi, etiologic

agent of Chagas’ disease, in US blood donors.

J Infect Dis 1997;176:1047-52.

161. Shulman IA, Appleman MD, Saxena S, et al.

Specific antibodies to Trypanosoma cruzi

among blood donors in Los Angeles, Califor-

nia. Transfusion 1997;37:727-31.

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3rd ed. Philadelphia: Lippincott Williams and

Wilkins, 2002:774-83.

163. Deferral for risk of Leishmaniasis exposure.

Association Bulletin #03-14. Bethesda, MD:

AABB, 2003.

164. US General Accounting Office. Blood supply:

Transfusion-associated risks. GAO/PEMD-97-1.

Washington, DC: US Government Printing

Office, 1997.

165. Brecher ME. Bacterial contamination of

blood products. In: Simon TL, Dzik WH,

Snyder EL, et al, eds. Rossi’s principles of

transfusion medicine. 3rd ed. Philadelphia:

Lippincott Williams and Wilkins, 2002:

789-801.

166. Rock G, Neurath D, Toye B, et al. The use of a

bacteria detection system to evaluate bacte-

rial contamination in PLT concentrates.

Transfusion 2004;44:337-42.

167. Suomela H. Inactivation of viruses in blood

and plasma products. Transfus Med Rev 1993;

7:42-57.

168. Centers for Disease Control. Safety of thera-

peutic immune globulin preparations with

respect to transmission of human T-lympho-

tropic virus type III/lymphadenopathy-asso-

ciated virus infection. MMWR Morb Mortal

Wkly Rep 1986;35:231-3. [Erratum in MMWR

Morb Mortal Wkly Rep 1986;35:607.]

169. Williams PE, Yap PL, Gillon J, et al. Non-A,

non-B hepatitis transmission by intravenous

immunoglobulin (letter). Lancet 1988;ii:501.

[Erratum in Lancet 1988;ii:584.]

170. Yu MW, Mason BL, Guo ZP, et al. Hepatitis C

transmission associated with intravenous

immunoglobulins (letter). Lancet 1995;345:

1173-4.

171. Centers for Disease Control. Outbreak of

hepatitis C associated with intravenous im-

munoglobulin administration: United States,

October 1993-June 1994. MMWR Morb Mor-

tal Wkly Rep 1994;43:505-9.

172. Farrugia A, Walker E. Hepatitis C virus trans-

mission by intravenous immunoglobulin

(letter). Lancet 1995;346:373-5.

173. Meisel H, Reip A, Faltus B, et al. Transmission

of hepatitis C virus to children and husbands

by women infected with contaminated anti-D

immunoglobulin. Lancet 1995;345:1209-11.

174. Power JP, Lawlor E, Davidson F, et al. Hepati-

tis C viraemia in recipients of Irish intrave-

nous anti-D immunoglobulin (letter). Lancet

1994;344:1166-7.

175. Foster PR, McIntosh RV, Welch AG. Hepatitis

C infection from anti-D immunoglobulin

(letter). Lancet 1995;346:372.

176. Makris M, Preston FE. Chronic hepatitis in

haemophilia. Blood Rev 1993;7:243-50.

177. Prowse C. Kill and cure. The hope and reality

of virus inactivation. Vox Sang 1994;67(Suppl

3):191-6.

178. Lusher JM, Arkin S, Abildgaard CF, Schwartz

RS. Recombinant factor VIII for the treatment

of previously untreated patients with hemo-

philia A. Safety, efficacy, and development of

inhibitors. Kogenate Previously Untreated

Patient Study Group. N Engl J Med 1993;328:

453-9.

179. Ben-Hur E, Moor AC, Margolis-Nunno H, et

al. The photodecontamination of cellular

blood components: Mechanisms and use of

photosensitization in transfusion medicine.

Transfus Med Rev 1996;10:15-22.

180. Zhang Q-X, Edson C, Budowsky E, Purmal A.

InactineTM—a method for viral inactivation

in red blood cell concentrates (abstract).

Transfusion 1998;38(Suppl):75S.

181. Seeff L. The efficacy of and place for HBIG in

the prevention of type B hepatitis. In:

Szmuness W, Alter H, Maynard J, eds. Viral

hepatitis: 1981 International Symposium.

Philadelphia: The Franklin Institute Press,

1982:585-95.

182. Sanchez-Quijano A, Pineda JA, Lissen E, et al.

Prevention of post-transfusion non-A, non-B

hepatitis by non-specific immunoglobulin in

heart surgery patients. Lancet 1988;i:1245-9.

183. Conrad ME. Prevention of post-transfusion

hepatitis (letter). Lancet 1988;ii:217.

710 AABB Technical Manual

184. Kobayashi R, Stiehm E. Immunoglobulin

therapy. In: Petz LD, Swisher SN, Kleinman S,

etal,eds.Clinicalpracticeoftransfusionmed

icine. 3rd ed. New York: Churchill Living-

stone, 1996:985-1010.

185. Centers for Disease Control. Recommenda-

tions for follow-up of health-care workers af-

ter occupational exposure to hepatitis C vi-

rus. MMWR Morb Mortal Wkly Rep 1997;46:

603-6.

Suggested Reading

Actions following an initial positive test for possi-

ble bacterial contamination of a platelet unit (As-

sociation Bulletin #04-07). Bethesda, MD: AABB,

2004.

Criteria for donor deferral in known or suspected

common source outbreaks of hepatitis A virus in-

fection (Association Bulletin #04-08). Bethesda,

MD: AABB, 2004.

Guidance on management of blood and platelet

donors with positive or abnormal results on bac-

terial contamination tests (Association Bulletin

#05-02). Bethesda, MD: AABB, 2005.

Chapter 28: Transfusion-Transmitted Diseases 711

Methods

The inclusion of methods in this edition

of the Technical Manual is a subjective

decision of the Technical Manual Pro-

gram Unit. Readers are encouraged to re-

fer to previous editions of the manual for

methods not appearing in this edition be-

cause exclusion from the current edition

does not necessarily indicate that their

use is prohibited. However, some proce-

dures, such as xylene and chloroform elu-

tion techniques, were removed because

the chemicals used in the procedures

could present a safety risk. Thus, readers

are cautioned when referring to proce-

duresinpreviouseditionsbecausethey

have not been reviewed for content and

safety.

There are often many different ways to

perform the same test procedure. Although

some workers may prefer other methods,

those given here are reliable, straightfor-

ward, and of proven value. Although the in-

vestigation of unusual serologic problems

often requires flexibility in thought and

methodology, the adoption of uniform

methods for routine procedures in the lab-

oratory is imperative. In order for labora-

tory personnel to have reproducible and

comparable results in a test procedure, it is

essential that everyone in the laboratory

perform the same procedure in the same

manner.

713

Methods

Methods Section 1: General Laboratory Methods

Methods Section 1

General Laboratory Methods

Introduction

The methods outlined in the following

sections are examples of acceptable pro-

cedures. Other acceptable procedures

may be used by facilities if desired. To the

greatest extent possible, the written pro-

cedures conform to the Guidelines for

Clinical Laboratory Technical Procedure

Manuals developed by the National Com-

mittee for Clinical Laboratory Standards.

As indicated in Title 21 of the Code of

Federal Regulations (CFR) Part 606.65(e),

the manufacturer’s instructions (eg, prod-

uct insert) for reagents and supplies li-

censed by the Food and Drug Administra-

tion (FDA) should be followed. Any deviation

should be validated using appropriate

controls and incorporated into a standard

operating procedure before approval by

the medical director. (Note: Deviations

may also require concurrence from the

FDA.) It is important to use Standard Pre-

cautions when appropriate (see Chapter

2).

Reagent Preparation

Many procedures include formulas for re-

agent preparation. Labels for reagents pre-

pared in-house must contain the follow-

ing:

■Name of solution.

■Date of preparation.

■Expiration date (if known).

■Storage temperature and/or conditions.

■Mechanism to identify the person pre-

paring the solution.

■Universal hazardous substance label.

Temperatures

Whenever specific incubation or storage

temperatures are given, the following ranges

are considered satisfactory:

Stated Temperature Acceptable Range

4C 2-8C

Room temperature 20-24 C

37 C 36-38 C

56 C 54-58 C

715

Section 1

Centrifugation Variables

Centrifugation speeds (relative centrifugal

force) and times should be standardized

for each piece of equipment. (See Meth-

ods Section 8.)

Reference

Guidelines for clinical laboratory technical proce-

dure manuals. 3rd ed. (NCCLS Document GP2-A3,

Vol. 12, No. 10.) Wayne, PA: National Committee

for Clinical Laboratory Standards, 1996.

Method 1.1. Transportation

and Shipment of Dangerous

Goods

Several agencies specify packaging and

shipping requirements for dangerous or

hazardous materials, depending on how

the material is shipped (mail, ground, or

air). For transport by mail of infectious

materials, clinical specimens, or biologic

products, the United States Postal Service

(USPS) Dangerous Goods Regulations

must be followed.1,2 For interstate trans-

port of infectious materials by ground or

air, the United States Department of

Transportation (DOT) regulations apply.3

Most air carriers apply the International

Air Transport Association (IATA)4regula-

tions and the technical instructions of the

International Civil Aviation Organization

(ICAO).5These agencies adopt the recom-

mendations of the United Nations (UN)

Committee of Experts on the Transport of

Dangerous Goods for the international

transport of infectious substances and

clinical specimens.

The Centers for Disease Control and Pre-

vention (CDC)6and the IATA7provide pack-

ing and labeling requirements for ship-

ments of infectious materials in order to

protect the public health by minimizing the

potential for direct contact with such mate-

rials, contamination of the environment,

and the spread of disease. The CDC also

serves as a Center for Applied Biosafety and

Training for the World Health Organization

(WHO)8andfortheUN.TheOccupational

Safety and Health Administration (OSHA)9

regulates worker safety related to handling,

packing, and transport. Both the DOT and

the IATA require active training for anyone

who packages infectious or toxic materials

for shipment.3,4 DOT and IATA documents

offer general shipping advice for all hazard-

ous materials because the regulations are

similar and most carriers follow the ship-

ping guidelines set forth in the IATA Dan-

gerous Goods Regulations4and the Infec-

tious Substances Shipping Guidelines.7

Facilities should also consult their local car-

riers for additional requirements. In gen-

eral, all dangerous goods regulations are

based on public risk from the materials and

therefore have specific packaging, labeling,

and documentation requirements. It is the

responsibility of the shipper of biologic or

infectious material to properly classify,

package, label, and document the sub-

stance being shipped.

Dangerous Goods Classifications

IATA classifies hazards into nine catego-

ries:

1. Explosives.

2. Compressed gases.

3. Flammable liquids.

4. Other flammable hazards.

5. Oxygen-rich material, oxidizers, and

organic peroxides.

6. Material affecting health, poisons,

and infectious substances.

7. Radioactive materials.

8. Corrosive material.

9. Miscellaneous hazards.4

The infectious category includes:

■Infectious substances: microbiologic

agents or their toxins that cause, or

716 AABB Technical Manual

may cause, disease; also called etio-

logic agents.

■Biologic products: products that are

prepared and shipped in compli-

ance with the provisions of 9 CFR

part 102 (Licensed Veterinary Bio-

logical Products), 9 CFR part 103 (Bi-

ological Products for Experimental

Treatment of Animals), 9 CFR part

104 (Imported Biological Products),

21 CFR part 312 (Investigational

New Drug Application), 21 CFR parts

600-680 (Biologics), 29 CFR Part

1910.1030 (Occupational Exposure

to Bloodborne Pathogens), 42 CFR

Part 72 (Interstate Shipment of Etio-

logical Agents), or 49 CFR Parts 171-

178 (Hazardous Materials Regula-

tions).

■Clinical or diagnostic specimens: hu-

man or animal material (including

excreta, secreta, blood and its com-

ponents, body fluids, tissue) being

shipped for the purpose of diagno-

sis.

■Genetically modified organisms.

■Clinical and medical waste.

Blood Bank Applications

Although the USPS regulates some bio-

logic products, such as live poliovirus vac-

cine, the DOT and IATA take the position

that biologic products and diagnostic

specimens may be shipped with less

stringent packaging and labeling require-

ments, unless they contain, or are reason-

ably believed to contain, an infectious

substance. The CDC has proposed regula-

tions to harmonize these requirements

with other federal and international re-

quirements.10 Units of blood that meet

disease testing requirements and un-

screened blood from healthy donors

meeting all FDA donor eligibility criteria

including specimens for screening may

be shipped without hazard restrictions.

Specimens being shipped for initial diag-

nosis purposes and with a low probability

that infectious agents are present are

packaged as diagnostic samples (IATA

packing instruction 650); specimens from

individuals known, or thought likely, to

have an infectious disease are shipped as

infectious substances (IATA packing in-

struction 602). The IATA Dangerous

Goods Regulations should be consulted

for the most recent amendments to ship

biologic specimens.

Method 1.1.1. Shipping Diagnostic

Specimens and Infectious Substances

Principle

The safe transport of hazardous materials

requires that they be packaged in a way to

protect the materials and those who han-

dle them. Prevention from leakage and

from being crushed in unexpected acci-

dents are required. Primary containers

should be leakproof and securely closed.

The primary container should be placed

in a watertight secondary container. Ab-

sorbent material capable of absorbing the

entire liquid contents in the package is

placed between the primary and second-

ary containers. The outer packaging should

have adequate strength for its intended

use and capacity and be labeled in accor-

dance with applicable regulations. Pack-

aging requirements for clinical specimens

are similar to those for infectious sub-

stances except that performance stan-

dards of the packaging materials are less

rigorous.

Materials

1. Leakproof, watertight primary con-

tainer (eg, test tube), heat sealed,

crimped, or otherwise reinforced to

prevent cap slippage.

Methods Section 1: General Laboratory Methods 717

2. Leakproof, watertight secondary pack-

age (eg, sealable plastic bag or con-

tainer with screw cap).

3. Nonparticulate absorbent material (eg,

paper towels, gauze, disposable dia-

per).

4. For infectious substances and for to-

tal shipment volumes greater than

50 mL (but less than 4000 mL or 4

kg), shock-absorbent material equal

in volume to the nonparticulate-ab-

sorbent material.

5. Outer packaging.

a. For infectious substances with a

shipping volume greater than 50

mL (but less than 4000 mL or 4

kg), an outer package of corru-

gated cardboard, fiberboard,

wood, metal, or rigid plastic meet-

ing UN strength requirements.

b. For biologic and diagnostic

specimens with a shipping vol-

ume less than 50 mL, a non-

certified outer container.

6. Coolant material (if necessary).

a. Wet ice, enclosed in sealed bags

to prevent leakage.

b. Dry ice, not to exceed 5 lb (for

air shipments).

7. Itemized listing of package contents.

8. Address label that includes the names,

addresses, and contact names and

telephone numbers of both the sender

and intended recipient.

9. Special labels, as applicable to cir-

cumstances (see Table 1.1.1-1).

a. Diagnostic specimens.

b. Infectious substances.

c. Dry ice.

d. Liquid nitrogen.

e. Cargo aircraft only.

10. Carrier-specific documents, such as

airbills or dangerous goods declara-

tions.

Procedure

1. Place the sealed primary container

into the secondary container.

2. Add sufficient absorbent material

around the primary container (be-

tween the primary and secondary

containers) to cover all sides of the

primary container and to absorb the

entire contents of the primary con-

tainer should breakage occur.

3. Seal the secondary container securely.

4. Place the secondary container into

the applicable outer package.

5. Forinfectioussubstanceswitha

shipping volume greater than 50 mL,

place shock-absorbent materials at

the top, bottom, and sides between

the secondary container and the

outer packaging. See Fig 1.1.1-1.

6. Place any necessary coolant material

(eg, wet ice or dry ice) between the

secondary container and the appli-

cable outer packaging. Provide inte-

rior support to secure the secondary

packaging in the original position

becausetheiceordryicemeltsor

dissipates.

718 AABB Technical Manual

Figure 1.1.1-1. Appropriate packaging of clinical

specimen material.

Methods Section 1: General Laboratory Methods 719

Table 1.1.1-1. Special Labels for Shipping

Clinical (Diagnostic) Specimens

Specimens collected for diagnosis, research, or other purposes must be labeled as biohazardous. Both the

primary container and the outer packaging must contain a biohazard label. The outer packaging label must

appear as follows:

1. The color of the material on which the label is printed must be

bright orange

, with the biohazard symbol

and printing in black.

2. The label must be a

rectangle

measuring 2 inches high by 4 inches long.

3. The biohazard symbol, measuring 1.56 inches in diameter, must be centered on a square measuring 2

inches on each side.

4. The size of the letters, printed in Helvetica, must be:

Biohazard 16 pt.

Clinical specimens 14 pt.

Packaged in compliance with 42 CFR part 72 6 pt.

In case of damage or leakage, notify 10 pt.

Shipper and receiver 10 pt.

Infectious Substances (Etiologic Agents)

For substances known to contain infectious agents or reasonably anticipated to contain such agents,

biohazardous labels must be placed on the primary container and outer packaging. The outer packaging

label must appear as follows:

1. The color of the material on which the label is printed must be

white

, with the biohazard symbol and

printing in black.

2. The label must be a

diamond-on-point

measuring, at a minimum, 4 inches on each side.

3. The biohazard symbol, measuring 0.81 inches in diameter, must be centered on a square measuring 2

inches on each side.

4. The Class 6 symbol for infectious substances must be centered at the bottom of the label.

5. The size of the letters, printed in Helvetica, must be:

Infectious substance 16 pt.

Packaged in compliance with 42 CFR part 72 5 pt.

In case of damage or leakage, notify 7 pt.

Immediately notify 7 pt.

Public Health Authority 7 pt.

In the United States 5 pt.

Centers for Disease Control and Prevention 5 pt.

Atlanta, GA 5 pt.

1-800-232-0124 5 pt.

624pt.

Dry Ice

Packages with dry ice must be labeled with the following information:

1. The diamond-shaped Class 9 symbol for miscellaneous hazardous materials.

2. The words “Dry Ice” or “Carbon Dioxide, Solid.”

3. “UN 1845,” the United Nations hazardous material category for dry ice (if shipped out of the country).

4. The name of the contents being cooled.

5. The weight of the dry ice in kg (not to exceed 5 lb if transported by air).

Liquid Nitrogen

Packages with liquid nitrogen must be labeled with a green IATA label stating “Contains Cryogenic Liquid.”

Cargo Aircraft Only

If the mode of transport is air and more than 50 mL of infectious substance is enclosed, the package must

have a warning label “Cargo Aircraft Only” to preclude the carrier from transporting the package on a

passenger plane.

7. Enclose the itemized listing of con-

tainer contents between the second-

ary and outer packaging.

8. Seal the outer packaging and label

with address label and applicable

special labels.

9. Complete applicable shipping forms

andsendthemwiththepackage.

Notes

1. Package specimens within a con-

tainer such that they will remain in

an upright position to help prevent

leakage.

2. Closures on primary containers can

be reinforced with adhesive tape or

paraffin. It is not necessary to rein-

force unopened evacuated specimen

collecting tubes.

3. For infectious substances, the name,

address, and telephone number of

the shipper must appear on both the

secondary and outer shipping con-

tainers.

4. Shipments of infectious substances

may contain multiple secondary con-

tainers, but the total volume of the

shipment may not exceed 4 L or 4

kg, excluding the packaging and

coolant weights.

5. Primary containers or secondary

packaging must be capable of with-

standing an internal pressure differ-

ential of 95 kPA (0.95 bar, 13.8 lb/in2)

between the temperatures of –40 C

and 55 C.

6. Styrofoam, plastic bags, and paper

envelopes are unacceptable for outer

packaging.

7. UN-certified containers must be able

to withstand a 30-foot “drop test,” as

specified in 49 CFR 178.609, without

breaking enclosed tubes. The UN

certification number must appear

on the container. Noncertified con-

tainersmustbeabletowithstandat

least a 1.2-meter drop on a hard un-

yielding surface without release of

the container’s contents.

8. Ensure that all state and local regu-

lations are followed.

9. Some exceptions may apply for

ground transportation.

10. If unsure about how to package and

ship materials, contact the CDC.

11. If breakage occurs during shipment,

the package should be handled with

extreme caution (wear personal pro-

tective equipment) and the entire

package (including containers, con-

tents, and packaging materials) should

be autoclaved before discard. Super-

visory personnel should be notified

if the contents are lost in transit or if

the damage appears related to inade-

quate packaging by the sender.

12. The carrier, the receiver, or anyone

handling damaged or leaking pack-

ages must isolate the package and

notify the shipper and intended re-

cipient immediately. In addition, for

infectious substances, notify the CDC

as soon as possible (1-800-232-0124).

When notifying the CDC, the caller

should provide a description of the

condition of the package, the name,

address, and telephone number of

the shipper, and any other pertinent

information, so that decontamina-

tion and disposal procedures can be

provided.

Additional Considerations with the Use of

Dry Ice

1. Solid carbon dioxide or “dry ice” is

classified as a hazardous material

because it can cause burns on con-

tact and gives off carbon dioxide gas

as it volatilizes.

720 AABB Technical Manual

2. Insulated gloves must be worn when

handling dry ice; eye protection must

be worn when breaking up chunks

of solid ice or when breaking apart

ice pellets.

3. Dry ice should be handled in a well-

ventilated area; its gas can cause light-

headedness and, in extreme cases, as-

phyxiation.

4. When kept in a tightly sealed ship-

ping container, dry ice could rupture

thepackaging.Ifdryiceisused,it

must be placed outside the second-

ary packaging or in an overpack with

one or more complete packages. The

outer packaging must be leakproof.

Procedures for packing with dry ice

must include instructions for sealing

the outer container in a manner that

allows the gas to escape and pre-

vents loosening of secondary con-

tainers as the dry ice dissipates.

Consult the Domestic Mail Manual

(section C023),249 CFR 173.217 and

175.10 (a)(13),3IATA Dangerous Goods

Regulations,4and IATA packing in-

struction 904.

5. HAZMAT training requirements for

dry ice are found in 49 CFR 172.704.3

Training for staff who come in con-

tact with dry ice should include in-

formation on:

■Potential hazards.

■Personal protective equipment

tousewhenhandlingorchip

ping.

■Procedures for packing, seal-

ing, labeling, and handling

boxes containing dry ice.

■Special considerations in oper-

atingavehicleusedtotrans

port boxes containing dry ice.

■Procedures for storing or dis-

posing of dry ice (eg, allowing

the gas to dissipate by natural

means in a well-ventilated

area) after a shipment is re-

ceived.

Additional Considerations with the Use of

Liquid Nitrogen

1. Liquid nitrogen is classified as a cryo-

genic liquid.

2. Insulated gloves, eye protection, and

a protective laboratory coat must be

worn when working with liquid ni-

trogen.

3. A watertight material capable of with-

standing cryogenic temperatures must

be used for the primary container.

This container must maintain its con-

tainment integrity at the tempera-

ture of the refrigerant as well as at

the temperature and pressure of air

transport if refrigeration is lost.

4. The secondary packaging must be

designed to withstand cryogenic tem-

peratures.

5. The design of the outer packaging

must allow for relief of pressure when

the liquid nitrogen evaporates. A

loose-fitting, but secured, container

cover or pressure relief valve may be

used.

References

1. Code of Federal Regulations. Title 39 CFR.

Washington, DC: US Government Printing

Office, 2004 (revised annually).

2. Etiologic agent preparations, clinical speci-

mens, and biological products. Domestic

Mail Manual (section C023), issue 57, July 10,

2003.

3. Code of Federal Regulations. Title 49 CFR Part

171-180. Washington, DC: US Government

Printing Office, 2004 (revised annually).

4. Dangerous goods regulations. 45th ed. Mon-

treal, Canada: International Air Transport As-

sociation, 2004 (revised annually).

5. Technical instructions for the safe transport

of dangerous goods by air. 2002-2004 ed.

Montreal, Canada: International Civil Avia-

tion Organization, Documents 9284 and

9284SU, 2002.

Methods Section 1: General Laboratory Methods 721

6. Code of Federal Regulations. Title 42 CFR Part

72. Washington, DC: US Government Printing

Office, 2004 (revised annually).

7. Infectious substances and diagnostic speci-

mens shipping guidelines. 4th ed. Montreal,

Canada: International Air Transport Associa-

tion, 2003.

8. World Health Organization. Guidelines for

thesafetransportofinfectioussubstances

and diagnostic specimens. Geneva, Switzer-

land: World Health Organization, 1997.

9. Code of Federal Regulations. Title 29 CFR

1910.1030. Washington, DC: US Government

Printing Office, 2004 (revised annually).

10. Centers for Disease Control and Prevention.

Packaging and handling of infectious sub-

stances and select agents, notice of proposed

rulemaking. Fed Regist 1999;64(208):58022-

31.

Method 1.1.2. Monitoring Temperature

During Shipment of Blood

Principle

Some form of temperature indication or

monitoring is desirable when shipping

blood. The temperature of the contents of

a shipping container used for whole blood

or liquid-stored red cell components can

be ascertained when the shipment is re-

ceived, as follows:

Procedure

1. Open the shipping container and

promptly place the sensing end of a

calibrated liquid-in-glass or elec-

tronic thermometer between two

bags of blood or components (labels

facing out) and secure the “sand-

wich” with two rubber bands.

2. Close the shipping container.

3. After approximately 3 to 5 minutes,

read the temperature.

4. If the temperature of red-cell-con-

taining components exceeds 10 C,

quarantine the units until their ap-

propriate disposition can be deter-

mined.

Notes

Other suitable methods for monitoring

shipments are:

1. Use time/temperature indicators,

one such indicator per shipping car-

ton. These indicators will change

colororshowanothervisibleindica

tion if the temperature has exceeded

10 C.

2. Place a “high-low” thermometer in

the shipping container. This simple,

reusable thermometer measures and

records the highest and lowest tem-

peratures during any period.

Method 1.2. Treatment of

Incompletely Clotted Specimens

Principle

Fibrin generation may continue in serum

separated from incompletely clotted blood,

especially during incubation at 37 C. This

produces strands of protein that entrap

red cells and make it difficult to evaluate

agglutination. Blood from patients who

have recently received heparin may not

clot at all, and blood from patients with

excessive fibrinolytic activity may relique-

fy or may contain protein fragments that

interfere with examination for agglutina-

tion.

Materials

1. Thrombin: dry human/bovine thrombin

or thrombin solution (50 units/mL

in saline).

2. Glass beads.

3. Protamine sulfate: 10 mg/mL in sa-

line.

4. Epsilon aminocaproic acid (EACA):

0.25 g/mL in saline.

722 AABB Technical Manual

Procedure

1. To accelerate clotting: Either of the

following techniques may be used:

a. Add to the specimen the amount

of dry thrombin that adheres to

the tip of an applicator stick or

1 drop of thrombin solution

permLofwholebloodorse

rum. Allow 10 to 15 minutes for

the clot to form. Use standard

centrifugation to separate the

clot and serum.

b. Gently agitate the separated se-

rum with small glass beads, at

37 C, for several minutes. Then,

use low speed centrifugation to

pellet the glass beads. Transfer

the serum to another tube.

2. To neutralize heparin: Protamine

sulfate can be added to the speci-

men to neutralize heparin; however,

excess protamine promotes rouleaux

formation and, in great excess, will

inhibit clotting. Add 1 drop of prota-

mine sulfate solution to 4 mL of

whole blood and wait 30 minutes to

evaluate the effect on clotting. If

clotting does not occur, add addi-

tional protamine sparingly. Note:

protamine sulfate may work more

rapidly when briefly incubated (5-10

minutes) at 37 C.

3. To inhibit fibrinolytic activity: Add 0.1

mL of EACA to 4 mL of whole blood.

Notes

1. The use of anticoagulated (eg, ACD

or EDTA) collection tubes may help

to avoid the problem of incom-

pletely clotted specimens. The use of

anticoagulated specimens must be

validated in accordance with each

standard operating procedure.

2. Because preparations of human

thrombin may contain red cell anti-

bodies, test results must be carefully

observed for false-positive reactions.

Quality control should be performed

on thrombin reagents before or con-

current with their use to identify

those with contaminating antibod-

ies.

Method 1.3. Solution

Preparation—Instructions

Principle

The basic definitions, calculations, and

instructions given below serve as a review

of simple principles necessary for solu-

tion preparation.

1. Mole, gram-molecular weight: Weight,

expressed in grams equal to the

atomic or molecular weight of the

substance.

2. Molar solution: A one molar (1 M)

solution contains one mole of solute

in a liter of solvent. The solvent is as-

sumedtobedistilledordeionized

water unless otherwise indicated.

3. Gram-equivalent weight: Weight, in

grams, of a substance that will pro-

duce or react with 1 mole of hydro-

gen ion.

4. Normal solution: A one normal (1 N)

solution contains one gram-equiva-

lent weight of solute in a liter of

solution.

5. Percentage solutions: The percent

designation of a solution gives the

weight or volume of solute present

in 100 units of total solution. Percent

can be expressed as:

a. Weight/weight (w/w), indicating

gramsofsolutein100gofsolu

tion.

b. Volume/volume (v/v), indicating

milliliters of solute present in

100 mL of solution.

Methods Section 1: General Laboratory Methods 723

c. Weight/volume (w/v), indicating

grams of solute in 100 mL of so-

lution. Unless otherwise speci-

fied, a solution expressed in

percentage can be assumed to

be w/v.

6. Water of crystallization, water of hy-

dration: Molecules of water that form

an integral part of the crystalline

structure of a substance. A given

substance may have several crystal-

line forms, with different numbers of

water molecules intrinsic to the en-

tire molecule. The weight of this wa-

ter must be included in calculating

molecular weight of the hydrated

substance.

7. Anhydrous: The salt form of a sub-

stance with no water of crystallization.

8. Atomic weights (rounded to whole

numbers): H, 1; O, 16; Na, 23; P, 31; S,

32; Cl, 35; K, 39.

9. Molecular weights:

HCl: 1 + 35 = 36; NaCl: 23 + 35 = 58

KCl: 39 + 35 = 74

H2O: (2 ×1) + 16 = 18

NaH2PO4:23+(2×1) + 31 + (4 ×16) =

120

NaH2PO4•H2O: 23 + (2 ×1) + 31 +

(4 ×16) + (2 ×1) + 16 = 138

KH2PO4:39+(2×1) + 31 + (4 ×16) =

136

H2SO4:(2×1) + 32 + (4 ×16) = 98

Examples

1. Molar solutions:

1MKH

2PO4=136gofsolutemadeup

to 1 L.

0.15 M KH2PO4= (136 ×0.15) = 20.4 g

of solute made up to 1 L.

0.5 M NaH2PO4= (120 ×0.5) = 60 g of

solute made up to 1 L.

2. Molar solution with hydrated salt:

0.5 M NaH2PO4•H2O = (138 ×0.5) =

69 g of the monohydrate crystals

made up to 1 L.

3. Normal solutions:

1NHCl=36gofsolutemadeupto1

L. One mole HCl dissociates into one

mole H+, so gram-equivalent weight

and gram-molecular weight are the

same.

12 N HCl = (36 ×12) = 432 g of solute

made up to 1 L.

1NH

2SO4=(98÷2)=49gofsolute

made up to 1 L. One mole H2SO4dis-

sociates to give two moles of H+,so

the gram-equivalent weight is half

the gram-molecular weight.

4. Percent solution:

0.9% NaCl (w/v) = 0.9 g of solute

made up to 100 mL of solution.

Notes

Accurate results require accurate prepara-

tion of reagents. It is important to care-

fully read and follow all instructions and

labels.

1. Weigh only quantities appropriate

for the accuracy of the equipment.

The operator’s manual should give

these specifications.

2. Prepare the largest volume that is

practical. There is greater accuracy

in measuring larger volumes than

smaller volumes. If a reagent bal-

ance is accurate to ±0.01 g, the po-

tential error in weighing 0.05 g (50

mg) will be 20%, whereas the poten-

tial error in weighing 0.25 g (250 mg)

will be only 4%. If the solution re-

tains its activity when stored appro-

priately, it is usually preferable to

preparealargevolume.Ifthesolu

tion deteriorates rapidly, smaller vol-

umes may be preferred to reduce

waste.

3. Note whether a substance is in the

hydrated or anhydrous form. If the

724 AABB Technical Manual

instructions give solute weight for

one form, and the available reagent

is in another form, be sure to adjust

the measurements appropriately.

For example, if instructions for 0.5 M

NaH2PO4call for 60 g, and the re-

agent is NaH2PO4H2O, find the ra-

tio between the weights of the

two forms. The molecular weight of

NaH2PO4H2O is 138 and the mo-

lecular weight of NaH2PO4is 120.

Therefore, the ratio is 138 ÷ 120 =

1.15. Multiply the designated weight

by the ratio (60 g ×1.15 = 69 g) to ob-

tain the final weight needed.

4. Dissolve the solute completely be-

fore making the solution to the final

volume. This is especially important

for substances, such as phosphates,

that dissolve slowly. For example, to

make 500 mL of 0.15 M KH2PO4:

a. Weigh 10.2 g of solute in a weigh-

ing boat or glass [(0.15 ×136) ÷

2] because only 500 mL will be

made.

b. Place 350 mL of water in a

500-mL volumetric flask on a

magnetic stirrer. Add the stir-

ring bar and adjust it to a slow,

steady stirring speed.

c. Add 10.2 g of salt, then rinse the

boat with several aliquots of

water until no salt remains. Nu-

merous small-volume rinses

remove adherent material more

effectively than a few larger

volumes. Add the rinse water to

the material in the flask and stir

until the salt has completely

dissolved.

d. If pH measurement is unneces-

sary, add water to the 500-mL

mark, adjusting the volume for

the stirring bar, and mix thor-

oughly. For solutions needing pH

adjustment, see the next step.

5. AdjustthepHofthesolutionbefore

bringing it to its final volume so that

the addition of water (or other sol-

vent) does not markedly change the

molarity. For example, to bring 500

mL of 0.1 M glycine to a pH of 3:

a. Add 3.75 g of glycine

(H2NCH2COOH: molecular

weight, 75) to 400-475 mL of

water in a beaker. Dissolve

completely, using a magnetic

stirrer.

b. Add a few drops of concentrated

(12 N) HCl and measure pH af-

ter acid is thoroughly mixed.

Continue adding HCl until pH

is 3.0.

c. Transfer the solution to a 500-mL

volumetric flask. Rinse beaker

and stirring bar with aliquots of

water, adding the rinse water to

the flask. Use the rinses to con-

tribute to the total 500-mL vol-

ume.

d. Measure the pH of the solution

at final volume.

References

1. Remson ST, Ackerman PG. Calculations for

the medical laboratory. Boston, MA: Little,

Brown & Co., 1977.

2. Henry JB, ed. Clinical diagnosis and manage-

ment by laboratory methods. 18th ed. Phila-

delphia: WB Saunders, 1991.

Method 1.4. Serum Dilution

Principle

Serum is sometimes diluted in saline or

other diluents to determine its relative

antibody concentration. It is customary to

express the dilution as 1 part of serum

contained in the total number of parts of

the dilution. For example, to test the se-

rum at one-tenth its original concentra-

tion, a dilution of 1 part in 10 may be

Methods Section 1: General Laboratory Methods 725

made by mixing 1 mL of serum with 9 mL

of saline. The final volume is 10,andthe

dilution is expressed as a 1 in 10 dilution.

The diluted material contains one-tenth

(1/10 or 0.1) of the unmodified serum. It

is often customary to report the titer of an

antibody as the reciprocal of the highest

dilution that retains a 1+ agglutination.

Therefore, serum that reacts at a dilution

of 1/32 is considered to have a titer of 32.

Note:A1in10dilutionis1partin9parts,

whereasa1to10or1:10is1partin10parts.

Procedure

1. Diluting an existing dilution:

a. A new higher dilution can be

prepared from diluted material

by adding more diluent. The

formula for calculating either

the new higher final dilution or

theamountofdiluenttoaddto

obtain a higher final dilution is:

reciprocal of present serum dilution

volume of serum dilution used

= reciprocal of new final dilution

total final volume

b. Example: Serum dilution is one

in two and volume of serum di-

lution is 1.0 mL. If 4.0 mL of sa-

line is added, what will be the

new final dilution?

2=X

X = 10 or 1 in 10 dilution

2. Diluting a dilution to a specified volume:

a. The formula for calculating the

volume of diluent to add to a

dilution to achieve a certain

quantity of a new higher final

dilution is:

reciprocal of present dilution

volume of present dilution needed

= reciprocal of final dilution

total final volume required

b. Example: Present serum dilu-

tion is one in two, total final

volume is 100 mL, and new fi-

nal serum dilution is 1 in 10.

How much serum (diluted one

in two) will have to be added to

make up a final volume of 100

mL of a 1 in 10 dilution?

2=10

X100

X = 20 or 20 mL of serum (dilu-

tion of one in two) must be added

to 80 mL of diluent to obtain

100 mL of a 1 in 10 dilution.

Method 1.5. Dilution of %

Solutions

Procedure

1. Dilutions can be prepared from more

concentrated solutions by use of the

following formula:

(Volume1×Concentration1)=

(Volume2×Concentration2)

V1×C1=V

2×C2

where V1and C1represent original

volume and concentration, and V2

and C2represent final desired vol-

ume and concentration.

726 AABB Technical Manual

2. Example: 30% albumin is available,

but 2 mL of 6% albumin is needed.

How should the albumin be diluted?

V1×30 = 2 ×6

30V1=12

V1=12÷30 = 0.4

Therefore, mix 0.4 mL of 30% albu-

minwith1.6mLsalinetoobtain2.0

mL of 6% albumin, or for small-vol-

ume use, mix 4 drops 30% albumin

with 16 drops saline to obtain 20

drops of 6% albumin.

Method 1.6. Preparation of a

3% Red Cell Suspension

Principle

A 3% red cell suspension is a common re-

agent in many serologic procedures. The

suspension need not be exactly 3%; an

approximation achieves the appropriate

serum-to-cell ratio for most test proce-

dures and an adequate number of red

cells to read and grade the reactions. The

following steps are intended to help an in-

dividual gain confidence in approximat-

ing a 3% red cell suspension visually, both

as a suspension of cells and in the appro-

priate size of the cell pellet achieved after

centrifugation.

Materials

1. Whole blood sample.

2. Test tubes.

3. Disposable pipettes (1 mL and 10 mL

serologic).

4. Saline.

5. Centrifuge (3000 rpm or equivalent).

6. Commercially prepared 3% reagent

red cell suspension.

Procedure

To prepare 10 mL of a 3% red cell suspen-

sion:

1. Transfer at least 1 mL of whole blood

to a 10-mL tube.

2. Wash the red cells in saline or phos-

phate-buffered saline (PBS), centri-

fuging for 5 minutes to pellet the

cells. Repeat two or three times. The

final supernate should be clear and

should be completely removed by

aspiration.

3. Transfer 0.3 mL of the washed red

cells to a tube with 9.7 mL of saline,

PBS, or Alsever’s solution.

4. Cap or cover the tube with parafilm.

Thoroughly mix the red cells and sa-

line by gently inverting the tube sev-

eral times.

5. To compare the color and density of

the suspension by eye, transfer a vol-

ume of the prepared suspension to a

10 ×75 mm tube. Also transfer a sim-

ilar volume of a known 3% red cell

suspension (eg, commercial reagent

red cell suspension) to another 10 ×

75 mm tube. Hold the two tubes in

front of a light source to compare them.

6. To compare the size of the cell pellet

expected from a 3% red cell suspen-

sion, transfer one drop of the pre-

pared suspension to a 10 ×75 mm

tube. Similarly, transfer one drop of

a known 3% commercial reagent red

cell suspension to another 10 ×75 mm

tube. Centrifuge the tubes in a sero-

logic centrifuge, using the spin time

designated for “saline.” The size of

the two cell pellets should be similar.

Note

For best results use red cell suspensions

on the day of preparation only, unless sta-

bility for a longer time has been validated.

Methods Section 1: General Laboratory Methods 727

Method 1.7. Preparation and

Use of Phosphate Buffer

Principle

Mixtures of acids and bases can be pre-

pared at specific pH values and used to

buffer (render) other solutions to that pH.

Thefollowingprocedureincludesamethod

for preparing phosphate-buffered saline

(PBS), which can be used as a diluent in

serologic tests.

Reagents

1. Prepare acidic stock solution (solu-

tion A) by dissolving 22.16 g of

NaH2PO4•H2O in 1 L of distilled wa-

ter. This 0.16 M solution of the mono-

basicphosphatesalt(monohydrate)

has a pH of 5.0.

2. Prepare alkaline stock solution (so-

lution B) by dissolving 22.7 g of

Na2HPO4in 1 L of distilled water.

This 0.16 M solution of the dibasic

phosphate salt (anhydrous) has a pH

of 9.0.

Procedure

1. Prepare working buffer solutions of

the desired pH by mixing appropri-

ate volumes of the two solutions. A

few examples are:

pH Solution A Solution B

5.5 94 mL 6 mL

7.3 16mL 84mL

7.7 7 mL 93 mL

2. CheckthepHoftheworkingsolu

tion before using it. If necessary, add

small volumes of acid solution A or

alkaline solution B to achieve the de-

sired pH.

3. To prepare PBS of a desired pH, add

one volume of phosphate buffer at

that pH to nine volumes of normal

saline.

References

1. Hendry EB. Osmolarity of human serum and

of chemical solutions of biologic importance.

Clin Chem 1961;7:156-64.

2. Dacie JV, Lewis SM. Practical haematology. 4th

ed. London, England: J and A Churchill, 1968:

540-1.

Method 1.8. Reading and

Grading Tube Agglutination

Principle

The purpose of grading reactions is to al-

low comparison of reaction strengths.

This is beneficial in detecting multiple

antibody specificities or antibodies exhib-

iting dosage. The grading of agglutination

reactions should be standardized among

all members of the laboratory staff, in the

interest of uniformity and reproducibility

of test results. Most laboratories define

their own version of a grading system,

which is described in a written procedure

available to all staff. Some systems use as-

signed numeric values (scores) for the

observed reactions.

Materials

1. Centrifuged serologic tests for agglu-

tination.

2. Agglutination viewer.

Procedure

1. Gentlyshakeortiltthetubeandre

suspend the red cell button in the

tube. The tilt technique uses the me-

niscus to gently dislodge the red cell

button from the wall of the tube.

2. Observe the way that cells are dis-

persed from the red cell button.

3. Record reactivity by comparing the

agglutinates to the descriptions in

Table 1.8-1. The reactivity should be

assessed when the red cells have been

728 AABB Technical Manual

completely resuspended from the

button.

Interpretation

Refer to Table 1.8-1.

Notes

1. An agglutination viewer may facilitate

the reading of tube tests. However,

the manufacturer’s test recommen-

dation must be followed for inter-

preting the test results.

2. Serum overlying the centrifuged cell

button must be inspected for

hemolysis, which is a positive sign of

an antigen-antibody reaction, pro-

vided the pretest serum was not

hemolyzed and no hemolytic agent

was added to the test.

3. The character of the agglutination

should be noted and recorded. This

information provides valuable clues

in the investigation such as the char-

acteristic refractile agglutination of

anti-Sda.

4. Mixed-field agglutination is ex-

pected when using pooled cells for

donor antibody detection and add-

ing check cells to negative antiglo-

bulin tests.

Reference

Race RR, Sanger R. Blood groups in man. 6th ed.

Oxford: Blackwell Scientific Publications, 1975.

Methods Section 1: General Laboratory Methods 729

Table 1.8-1. Interpretation of Agglutination Reactions

Macroscopically Observed Findings Designation Score

One solid agglutinate

Several large agglutinates

Medium-size agglutinates, clear background

Small agglutinates, turbid background

Very small agglutinates, turbid background

Barely visible agglutination, turbid background

No agglutination

Mixtures of agglutinated and unagglutinated red cells

(mixed field)

Complete hemolysis

Partial hemolysis, some red cells remain

w+ or +/–

Methods Section 2: Red Cell Typing

Methods Section 2

Red Cell Typing

Method 2.1. Slide Test for

Determination of ABO Type

of Red Cells

Principle

See Chapter 13 for a discussion of the

principles of testing for ABO groups.

Specimen

The reagent manufacturer’s instructions

must be consulted before slide tests are

performed; some manufacturers recom-

mend performing slide tests with whole

blood, whereas others specify the use of

red cell suspensions of lighter concentra-

tions prepared in saline, serum, or plasma.

Reagents

1. Anti-A.

2. Anti-B.

3. Anti-A,B (optional).

All reagents must be used in accordance

with the manufacturer’s instructions.

Procedure

1. Place 1 drop of anti-A on a clean, la-

beled glass slide.

2. Place 1 drop of anti-B on a separate

clean, labeled glass slide.

3. Place 1 drop of anti-A,B on a third

slide, if parallel tests are to be per-

formed with this reagent, or on a

single clean, labeled slide if this is

the only test performed.

4. Add to each drop of reagent on the

slides 1 drop of well-mixed suspen-

sion (in saline, serum, or plasma) of

the red cells to be tested. (Consult

the reagent manufacturer’s instruc-

tions to determine the correct cell

concentration to be used.)

5. Mix the reagents and red cells thor-

oughly, using a clean applicator stick

for each reagent. Spread the mixture

731

Section 2

over an area approximately 20 mm ×

40 mm.

6. Gently tilt the slide continuously for

up to 2 minutes. Do not place the slide

over a heated surface, such as an Rh

viewbox, during this period.

7. Read, interpret, and record the re-

sults of the reactions on all slides.

Interpretation

1. Strong agglutination of red cells in

thepresenceofanyABOtypingre

agent constitutes a positive result.

2. A smooth suspension of red cells at

the end of 2 minutes is a negative re-

sult.

3. Samples that give weak or doubtful

reactions should be retested using

Method 2.2.

Notes

1. Slide testing imposes a greater risk

of exposure to infectious samples.

Personnel should follow safety mea-

sures detailed in the facility’s proce-

dures manual.

2. Slide testing is not suitable for detec-

tion of ABO antibodies in serum/

plasma.

Method 2.2. Tube Tests for

Determination of ABO Group

of Red Cells and Serum

Principle

See Chapter 13 for a discussion of the

principles of testing for ABO groups. The

following procedure is an acceptable rep-

resentative method, but the manufacturer’s

instructions for the specific reagents must

be consulted.

Specimen

The reagent manufacturer’s package in-

sert must be consulted to determine spe-

cific specimen requirements. Generally,

clotted or anticoagulated blood samples

maybeusedforABOtesting.Theredcells

may be suspended in autologous serum,

plasma, or saline, or may be washed and

resuspended in saline.

Reagents

1. Anti-A.

2. Anti-B.

3. Anti-A,B. Note: Use of this reagent is

optional.

4. A1,A

2, and B red cells. They can be

obtained commercially or the testing

laboratory can prepare a 2% to 5%

suspension on each day of use. (Note:

The use of A2cells is optional.)

All reagents must be used in accordance

with the manufacturer’s instructions.

Procedures

Testing Red Cells

1. Place 1 drop of anti-A in a clean, la-

beled test tube.

2. Place 1 drop of anti-B in a clean, la-

beled tube.

3. Place 1 drop of anti-A,B in a clean,

labeled tube, if tests are to be per-

formed with this reagent.

4. Add to each tube 1 drop of a 2% to

5% suspension (in saline, serum, or

plasma) of the red cells to be tested.

Alternatively, the equivalent amount

of red cells can be transferred to each

tube with clean applicator sticks.

5. Mix the contents of the tubes gently

and centrifuge them for the cali-

brated spin time.

6. Gently resuspend the cell buttons

and examine them for agglutination.

732 AABB Technical Manual

7. Read, interpret, and record the test re-

sults. Compare the red cell test re-

sults with those obtained in the se-

rum/plasma tests (see below).

Testing Serum/Plasma

1. Label two clean test tubes as A1and

B. (Note: Label an additional tube if

an optional test with A2red cells is to

be performed.)

2. Add 2 or 3 drops of serum/plasma to

each tube.

3. Add 1 drop of A1reagent cells to the

tube labeled A1.

4. Add 1 drop of B reagent cells to the

tube labeled B.

5. Add A2cells to the appropriate tube,

if this optional test is being per-

formed.

6. Mix the contents of the tubes gently

and centrifuge them for the cali-

brated spin time.

7. Examinetheserumoverlyingthecell

buttons for evidence of hemolysis.

Gently resuspend the cell buttons

and examine them for agglutination.

8. Read, interpret, and record test results.

Compare serum test results with

those obtained in testing red cells

(see above).

Interpretation

1. Agglutination of tested red cells and

either hemolysis or agglutination in

tests with serum constitute positive

test results.

2. A smooth cell suspension after re-

suspension of the cell button is a

negative test result.

3. Interpretation of serum/plasma and

cell tests for ABO is given in Table

13-1.

4. Any discrepancy between the results

of the tests with serum/plasma and

cells should be resolved before an

interpretation is recorded for the

patient’s or donor’s ABO group (see

Chapter 13).

Note

Positive reactions characteristically show

3+ to 4+ agglutination by reagent ABO an-

tibodies; reactions between test serum

and reagent red cells are often weaker.

The serum tests may be incubated at

room temperature for 5 to 15 minutes to

enhance weak reactions. See Chapter 13

for a discussion of weakly reactive sam-

ples.

Method 2.3. Microplate Test

for Determination of ABO

Group of Red Cells and

Serum

Principle

See Chapter 13 for a discussion of the

principles of testing for ABO blood group.

Microplate techniques can be used to test

for antigens on red cells and for antibod-

ies in serum.

A microplate can be considered as a ma-

trix of 96 “short” test tubes; the principles

that apply to hemagglutination in tube tests

also apply to tests in microplates.

Microplates may be rigid or flexible, with

either U-shaped or V-shaped bottoms.

U-shaped bottom plates are more widely

used because results can be read either af-

ter centrifuging the plate and observing the

characteristics of resuspended red cells or

by observing the streaming pattern of the

cells when the plate is placed at an angle.

Either reading technique permits estima-

tion of the strength of agglutination.

Specimen

Refer to Method 2.2.

Methods Section 2: Red Cell Typing 733

Equipment

1. Dispensers (optional): Semiautoma-

ted devices are available for dispens-

ing equal volumes to a row of wells.

Special plate carriers can be pur-

chased to fit common table-top cen-

trifuges.

2. Microplate readers (optional): Auto-

mated photometric devices are avail-

able that read microplate results by

the light absorbance in U-shaped

bottom wells to differentiate be-

tween positive and negative tests.

The microprocessor component of

the reader interprets the reactions

and prints the blood testing results.

The manufacturer’s instructions for

the collection and preparation of se-

rum/plasma and cell specimens

must be followed.

3. Centrifuges: Appropriate conditions

must be established for each centri-

fuge. The following times and rela-

tive centrifugal forces, expressed as

g, are suggested. Consult the manu-

facturer’s directions for specific in-

formation.

For a flexible U-shaped bottom

microplate: 700 ×gfor 5 seconds for

red cell testing and serum/plasma

testing.

ForarigidU-shapedbottommicro

plate: 400 ×gfor 30 seconds for red

cell testing and serum/plasma testing.

Reagents

Many manufacturers supply ABO or Rh

typing reagents that are licensed by the

Food and Drug Administration (FDA) for

use as undiluted reagents in microplate

tests.

1. Anti-A.

2. Anti-B.

3. Anti-A,B. Note: Use of this reagent is

optional.

4. Group A1,A

2, and B red cells. They

can be obtained commercially or the

testing laboratory can prepare a 2%

to 5% suspension on each day of use.

(Note: The use of A2cells is optional.)

Procedure

Testing Red Cells

1. Place 1 drop of anti-A and anti-B in

separate clean wells of a U-bottom

microplate. If tests with anti-A,B are

to be performed, add this reagent to

a third well.

2. Add1dropofa2%to5%salinesus

pension of red cells to each well con-

taining blood typing reagent.

3. Mix the contents of the wells by

gently tapping the sides of the plate.

4. Centrifuge the plate at the appropri-

ate conditions established for the

centrifuge.

5. Resuspend the cell buttons by man-

ually tapping the plate or with the

aid of a mechanical shaker, or place

the plate at an angle for the tilt and

stream method.

6. Read, interpret, and record results.

Compare red cell test results with those

obtained in testing serum/plasma.

Testing Serum/Plasma

1. Add 1 drop of a 2% to 5% suspension

of reagent A1andBredcellstosepa

rate clean wells of a U-bottom micro-

plate. (Note: If an optional test on A2

cells will be performed, add A2cells

to a third well.)

2. Add 1 drop of serum or plasma un-

der test to each well.

3. Mix the contents of the wells by gently

tapping the sides of the plate.

4. Centrifuge the plate at the appropri-

ate conditions established for the

centrifuge.

734 AABB Technical Manual

5. Resuspend the cell buttons by man-

ually tapping the plate or with aid of

a mechanical shaker, or place the plate

at an angle for the tilt and stream

method.

6. Read, interpret, and record results.

Compare test results on serum/plasma

with those obtained in testing red

cells.

Note

To enhance weak serum/plasma reac-

tions, the plates may be incubated at

room temperature for 5 to 10 minutes,

then the centrifugation, reading, and re-

cording steps may be repeated.

Interpretation

1. Agglutination in any well of red cell

tests and hemolysis or agglutination

in any well of a serum test constitute

positive results.

2. A smooth suspension of red cells af-

ter resuspension of the cell button is

a negative test.

3. The interpretation of ABO tests is

given in Table 13-1.

4. Any discrepancy between results on

cell and serum/plasma tests should

be resolved before an interpretation

is recorded for the patient’s or donor’s

ABO group (see Chapter 13).

Method 2.4. Confirmation of

Weak A or B Subgroup by

Adsorption and Elution

Principle

See Chapter 13 for a discussion of the

principles of testing for ABO groups.

Specimen

Refer to Method 2.2.

Reagents

1. Human anti-A and/or anti-B. Be-

causesomemonoclonalABOtyping

reagents are sensitive to changes in

pH and osmolarity, they may not be

suitable for use in adsorption/elution

tests.

2. Eluting agent: See Methods Section

Procedure

1. Wash1mLoftheredcellstobe

tested at least three times with saline.

Remove and discard the supernatant

salineafterthelastwash.

2. Add 1 mL of reagent anti-A (if a weak

variant of A is suspected) or 1 mL of

anti-B (if a weak variant of B is sus-

pected) to the washed cells.

3. Mix the red cells with the reagent

antibody and incubate them at 4 C

for 1 hour, mixing occasionally.

4. Centrifuge the mixture to pack the

red cells. Remove all supernatant re-

agent.

5. Transfer the red cells to a clean test

tube.

6. Wash the cells at least eight times

with large volumes (10 mL or more)

of cold (4 C) saline. Save an aliquot

of the final wash supernatant fluid

and test it in parallel with the eluate.

7. Use an elution method suitable for

recovery of ABO antibodies, eg, heat

or Lui freeze-thaw elution techni-

ques can be used to remove anti-

body from the cells (see Methods

Section 4).

8. Centrifuge to pack the cells and trans-

fer the supernatant eluate to a clean

test tube.

Methods Section 2: Red Cell Typing 735

9. Testtheeluateandthefinalwash

solution (from step 6), in parallel,

against two examples of group O

cells and two examples of cells ex-

pressing the relevant antigen (A1

cells for suspected anti-A, B cells for

anti-B). Add 2 drops of eluate or

wash to 1 drop of cells and examine

them for agglutination after immedi-

ate centrifugation; if negative, incu-

bate 15 to 30 minutes at room tem-

perature. If these phases are both

negative, a 15-minute incubation at

37 C and the indirect antiglobulin

test may also be performed.

Interpretation

1. The presence of anti-A or anti-B in

the eluate, hence the presence of A

or B antigen on the test cells, is con-

firmed if: a) the eluate reacts with

both antigen-positive cells, at any

phase; b) the eluate is nonreactive at

all phases with all group O cells; and

c) the final wash solution is non-

reactive with all four cells.

Iftheeluatedoesnotreactwith

the A or B cells, it may indicate that

the test cells do not express the anti-

gen and cannot adsorb the relevant

antibody; alternatively, it could re-

flect failure to prepare the eluate

correctly.

Iftheeluatereactswithoneor

both of the A or B cells and also with

one or both or all of the O cells, it in-

dicates recovery of some other or

additional antibody in the adsorp-

tion/elution process.

2. If the final wash solution reacts with

the A or B cells, tests on the eluate

cannot be considered valid. This can

occur if unbound reagent antibody

was not adequately removed before

beginning the elution, if the cells

were not adequately washed, or if

there was dissociation of bound an-

tibody during the wash process.

3. A and B cells can be used in the ad-

sorption/elution procedure as posi-

tive/negative controls and tested in

parallel. Group O cells can also be

used as a negative control.

Note

The eluate may be stained by hemoglobin

and be difficult to read except at the indi-

rect antiglobulin phase.

Reference

Beattie KM. Identifying the causes of weak or

“missing” antigens in ABO grouping tests. In: The

investigation of typing and compatibility prob-

lems caused by red blood cells. Washington, DC:

AABB, 1975:15-37.

Method 2.5. Saliva Testing

for A, B, H, Lea, and Leb

Principle

Approximately 78% of all individuals pos-

sess the Se gene that governs the secretion

of water-soluble ABH antigens into all

body fluids with the exception of cere-

brospinal fluid. These secreted antigens

can be demonstrated in saliva by inhibi-

tion tests with ABH and Lewis antisera

(see Chapter 13).

Specimen

1. Collect 5 to 10 mL of saliva in a small

beaker or wide-mouthed test tube.

Most people can accumulate this

amount in several minutes. To en-

courage salivation, the subject may

be asked to chew wax, paraffin, or a

clean rubber band, but not gum or

736 AABB Technical Manual

anything else that contains sugar or

protein.

2. Centrifuge saliva at 900 to 1000 ×g

for 8 to 10 minutes.

3. Transfer supernatant to a clean test

tube and place it in a boiling water-

bath for 8 to 10 minutes to inactivate

salivary enzymes.

4. Recentrifuge at 900 to 1000 ×gfor 8

to 10 minutes, remove clear or slightly

opalescent supernatant fluid, and

discard the opaque or semisolid ma-

terial. Dilute the supernatant fluid

with an equal volume of saline.

5. Refrigerate, if testing is to be done

within several hours. If testing will

notbedoneonthedayofcollection,

freezethesampleandstoreitat–20

C. Frozen samples retain activity for

several years.

Reagents

1. Human (polyclonal) anti-A and anti-B.

Note: Some monoclonal reagents

may not be appropriate for use;

therefore, appropriate controls are

essential.

2. Anti-H lectin from Ulex europaeus

obtained commercially or prepared

by saline extraction of Ulex euro-

paeus seeds.

3. Polyclonal (rabbit/goat/human)

anti-Lea.Therearenopublisheddata

on the suitability of monoclonal

Lewis antibodies.

4. A1andBredcells,asusedinMethod

2.2.

5. Group O, Le(a+b–) red cells, as used

for antibody detection or identifica-

tion (see Chapter 19).

6. Specimens, frozen or fresh saliva,

from persons known to be secretors

or nonsecretors, to use as positive

and negative controls.

Procedures

Selection of Blood Grouping Reagent

Dilution

1. Prepare doubling dilutions of the ap-

propriate blood typing reagent.

2. To 1 drop of each reagent dilution,

add 1 drop of 2% to 5% saline sus-

pension of red cells. Use A, B, or O

cells to determine, respectively, A, B,

or H secretor status. Use Le(a+b–)

red cells to determine Lewis secretor

status.

3. Centrifuge each tube and examine

macroscopically for agglutination.

4. Select the highest reagent dilution

that gives 2+ agglutination.

Inhibition Test for Secretor Status

1. Add 1 drop of appropriately diluted

blood grouping reagent to each of

four tubes. For ABH studies, the

tubes should be labeled “Secretor,”

“Nonsecretor,” “Saline,” and “Un-

known.” For Lewis studies, they will

be “Lewis-positive,” “Lewis-nega-

tive,” “Saline,” and “Unknown.”

2. Add 1 drop of the appropriate saliva

to the “Secretor,” “Nonsecretor,” and

“Unknown” tubes, and 1 drop of sa-

line to the tube marked “Saline.”

3. Mix the contents of the tubes. Incu-

bate the tubes for 8 to 10 minutes at

room temperature.

4. Add 1 drop of 2% to 5% saline sus-

pension of washed indicator cells to

each tube, group A, B, or O for ABH

secretor status, as appropriate, or

Le(a+) for Lewis testing.

5. Mix the contents of the tubes. Incu-

bate the tubes for 30 to 60 minutes

at room temperature.

Methods Section 2: Red Cell Typing 737

6. Centrifuge each tube and inspect

each cell button macroscopically for

agglutination.

Interpretation

1. Agglutination of indicator cells by

antibody in tubes containing saliva

indicates that the saliva does not

contain the corresponding antigen.

2. The failure of known antibody to ag-

glutinate indicator cells after incu-

bation with saliva indicates that the

saliva contains the corresponding

antigen.

3. The failure of antibody in the saline

control tube to agglutinate indicator

cells invalidates the results of saliva

tests; this usually reflects use of re-

agents that are too dilute. Redeter-

mine the appropriate reagent dilu-

tion, as described above, and repeat

the testing.

4. For further interpretation, see Table

2.5-1.

Notes

1. Include, as controls, saliva from a

known secretor and nonsecretor. For

ABH status, use saliva from previ-

ously tested Se and sese persons. For

Lewis testing, use saliva from a per-

son whose red cells are Le(a+b–) or

Le(a–b+) as the positive control; use

saliva from a Le(a–b–) person as the

negative control. Aliquots of saliva

from persons of known secretor sta-

tus may be frozen for later use.

2. This screening procedure can be

adapted for the semiquantitation of

blood group activity by testing serial

saline dilutions of saliva. The higher

the dilution needed to remove inhib-

itory activity, the more blood group

substance is present in the saliva.

738 AABB Technical Manual

Table 2.5-1. Interpretation of Saliva Testing

Testing with Anti-H

Unknown

Saliva

(H Substance

Present)

Non-

Saliva

(H Substance Not

Present)

Saline

(Dilution Control) Interpretation

2+ 0 2+ 2+ Nonsecretor of H

0 0 2+ 2+ Secretor of H

Testing With Anti-Lea

Unknown

Saliva Le-positive Saliva

Le-negative

Saliva

Saline

(Dilution Control) Interpretation

2+ 0 2+ 2+ Lewis-negative

0 0 2+ 2+ Lewis-positive*

*A Lewis-positive person shown to be a secretor of ABH can be assumed to have Lebas well as Leain saliva. A Le(a+)

person who is

sese

and does not secrete ABH substance will have only Leain saliva.

Saliva should be diluted before it is

incubated with antibody. To detect

or to measure salivary A or B sub-

stance in addition to H substance,

thesameprocedurecanbeused

with diluted anti-A and anti-B re-

agents. The appropriate dilution of

anti-A or anti-B is obtained by titrat-

ing the reagent against A1or B red

cells, respectively.

3. A Lewis-positive person shown to be

a secretor of A, B, and H can be as-

sumed to have Lebas well as Leain

the saliva. A Le(a+) person who does

not secrete A, B, or H substances

lacks the Se gene and will have only

Leain the saliva.

4. Specimens with a high concentra-

tion of soluble antigen may give a

false-negative result and require di-

lution before testing.

Method 2.6. Slide Test for

Determination of Rh Type

Principle

See Chapter 14 for a discussion of the

principles of Rh typing.

Specimen

Refer to Method 2.2.

Equipment

View box.

Reagents

1. Reagent anti-D: Suitable reagents in-

clude polyclonal high-protein or low-

protein (eg, monoclonal) reagents.

Follow the instructions from the

manufacturer of the anti-D in use

before performing slide tests; the

method presented here is a repre-

sentative procedure.

2. Rh control reagent: The manufac-

turer’s instructions will indicate the

type of reagent to use, if needed.

Procedure

1. Place 1 drop of anti-D onto a clean,

labeled slide.

2. Place 1 drop of the appropriate con-

trol reagent, if needed, onto a sec-

ond labeled slide.

3. To each slide, add 2 drops of a well-

mixed 40% to 50% suspension (in auto-

logous or group-compatible serum

or plasma) of the red cells to be tested.

4. Thoroughly mix the cell suspension

and reagent. Using a clean applica-

tor stick for each test, spread (mix)

the reaction mixture over an area ap-

proximately20mm×40 mm.

5. Place both slides on the viewbox and

tilt the slides gently and continu-

ously to observe them for agglutina-

tion (see note 1). Most manufactur-

ers stipulate that the test must be

read within 2 minutes because dry-

ing of the reaction mixture may

cause the formation of rouleaux,

which may be mistaken for aggluti-

nation.

6. Interpret and record the results of

the reactions on both slides.

Interpretation

1. Agglutination with anti-D and a

smooth suspension on the control

slide constitute a positive test result

and indicate that the cells being

tested are D+.

2. No agglutination with either anti-D

or the Rh control suggests that the

cells are D–. Testing by the anti-

globulin procedure (see Method 2.9)

will show weak expression of D on

cells that are not agglutinated on

slide testing.

Methods Section 2: Red Cell Typing 739

3. If there is agglutination on the con-

trol slide, results of the anti-D test

must not be interpreted as positive

without further testing.

4. Drying around the edges of the reac-

tion mixture must not be confused

with agglutination.

Notes

1. Slide testing imposes a much greater

risk of biohazardous exposure. Per-

sonnel should follow safety mea-

sures detailed in the facility’s proce-

dures manual.

2. For slide tests using low-protein

anti-D, a negative result on slide

testing with either anti-A or anti-B

serves as the control reaction.

Method 2.7. Tube Test for

Determination of Rh Type

Principle

See Chapter 14 for a discussion of the

principles of Rh typing.

Specimen

Refer to Method 2.2.

Reagents

1. Reagent anti-D: Suitable reagents in-

clude polyclonal high-protein or low-

protein (eg, monoclonal) reagents.

Follow the instructions from the

manufacturer of the anti-D in use

before performing tube tests. The

method presented here is a repre-

sentative procedure.

2. Rh control reagent: The manufac-

turer’s instructions will indicate the

type of control to use, if needed.

Procedure

1. Place 1 drop of anti-D in a clean, la-

beled test tube.

2. Place 1 drop of the appropriate con-

trol reagent, if needed, in a second

labeled tube.

3. Add to each tube 1 drop of a 2% to

5% suspension (in saline, serum or

plasma) of the red cells to be tested;

alternatively, the equivalent amount

of red cells can be transferred to each

tube with clean applicator sticks.

4. Mix gently and centrifuge for the

time and at the speed specified by

the manufacturer.

5. Gently resuspend the cell button and

examine it for agglutination. If a

stick was used to transfer the red

cells, adding 1 drop of saline to each

tube will make it easier to resuspend

the cell button.

6. Grade reactions and record test and

control results.

Interpretation

1. Agglutination in the anti-D tube,

combined with a smooth suspension

in the control tube, indicates that

the red cells under investigation are

D+.

2. A smooth suspension of red cells in

both the anti-D and the control

tubes is a negative test result. Speci-

mens from patients may be desig-

nated as D– at this point. Donor

blood must be further tested for the

presence of weak D antigen. The se-

rum-and-cell mixture used in steps 1

through 5, above, may be used to test

for weak D, providing the manufac-

turer’s directions state that the re-

agent is suitable for the test for weak

740 AABB Technical Manual

Notes

1. Most commercially prepared antisera

provide a 2+ or greater agglutination

with D+ cells. A facility may choose

to do additional testing on results

with an agglutination of less than 2+.

Required testing must be defined in

the facility’s procedures manual.

2. A negative tube test with anti-A and/

or anti-B serves as a valid control

when a low-protein anti-D reagent

has been used.

Method 2.8. Microplate Test

for Determination of Rh Type

Principle

See Chapter 14 for a discussion of the

principles of Rh typing

Specimen

RefertoMethod2.2.Clottedoranticoagu-

latedsamplesmaybeusedforRhtesting.

Follow the manufacturer’s instructions for

specimen preparation when using semi-

automated microplate readers.

Reagents

Use only anti-D approved for use in micro-

plate tests (see the discussion in Method

2.3).

Procedure

The following is a representative method;

the manufacturer’s instructions should be

followed for specific reagents and equip-

ment.

1. Place 1 drop of the Rh reagent in a

clean well of the microplate. If the

reagent requires the use of an Rh

control, add 1 drop of the control to

a second well.

2. Add 1 drop of a 2% to 5% saline sus-

pension of red cells to each well.

3. Mix the contents of the wells by

gently tapping the sides of the plate.

4. Centrifuge the plate at the appropri-

ate conditions established for the

centrifuge.

5. Resuspend the cell buttons by man-

ually tapping the plate or with the

aid of a mechanical shaker, or place

the plate at an angle for the tilt and

stream method.

6. Read, interpret, and record the results.

7. Incubate negative tests at 37 C for 15

minutes.

8. Centrifuge the plate at the appropri-

ate conditions established for the

centrifuge.

9. Resuspend the cell buttons by man-

ually tapping the plate or with the

aid of a mechanical shaker, or place

the plate at an angle for the tilt and

stream method.

10. Read, interpret, and record the re-

sults.

Interpretation

Agglutination with anti-D reagent after

the immediate-spin or 37 C incubation

phase indicates a positive test provided

there is no agglutination with the control

reagent. See Table 14-3 for determining

Rh phenotypes from reactions obtained

with Rh blood typing reagents.

Note

Refer to the manufacturer’s instructions

for the necessity for weak D testing.

Method 2.9. Test for Weak D

Principle

Some red cells express the D antigen so

weakly that most anti-D reagents do not

Methods Section 2: Red Cell Typing 741

directly agglutinate the cells. Weak D ex-

pression can be recognized most reliably

by an indirect antiglobulin procedure af-

ter incubation of the test red cells with

anti-D.

Specimen

Refer to Method 2.2.

Reagents

1. Reagent anti-D: Suitable reagents in-

clude polyclonal high-protein or low-

protein (eg, monoclonal blend) re-

agents, but the manufacturer’s pack-

age insert should be consulted be-

fore any anti-D reagent is used for

this purpose.

2. Antihuman globulin reagent, either

anti-IgG or polyspecific.

3. IgG-coated red cells.

Procedure

If the original, direct test with anti-D was

performed by tube testing, the same tube

may be used for the weak D test, provid-

ing the manufacturer’s directions so state.

In this case, proceed directly to step 4, af-

ter recording the original anti-D tube test

as negative.

1. Place 1 drop of anti-D in a clean, la-

beled test tube.

2. Place 1 drop of the appropriate con-

trol reagent in a second labeled test

tube.

3. To each tube, add 1 drop of a 2% to

5% suspension in saline of the red

cells to be tested. It is permissible to

use a direct antiglobulin test (DAT)

on the test cells as a control, but an

indirect antiglobulin procedure with

an Rh control reagent is preferable

because this ensures that all reagent

components that might cause a

false-positive result are represented.

4. Mix and incubate both tubes accord-

ing to the reagent manufacturer’s di-

rections. Typically, this is 15 to 30

minutes at 37 C.

5. If a reading is desired after the 37 C

incubation phase, centrifuge the tubes

according to the reagent manufac-

turer’s directions.

6. Gently resuspend the cell buttons

and examine the tubes for agglutina-

tion. If the test red cells are strongly

agglutinated in the anti-D tube but

not in the control tube, record the test

sample as D+ and do not proceed with

the antiglobulin phase of the test.

7. If the test cells are not agglutinated

or the results are doubtful, wash the

cells three or four times with large

volumes of saline.

8. Add antiglobulin reagent, according

to the manufacturer’s directions.

9. Mix gently and centrifuge according

to the calibrated spin times.

10. Gently resuspend each cell button,

examine the tubes for agglutination,

and grade and record the test result.

11. If the test result is negative, add IgG-

coated red cells.

Interpretation

1. Either a diluent control or a direct

antiglobulin test (DAT) must accom-

pany the test for weak D. Agglutina-

tionintheanti-Dtubeandnonein

the control tube constitutes a posi-

tive test result. If the facility chooses

to perform the test for weak D, and

the result is clearly positive, the

blood should be classified as D+. It is

incorrect to report such red cells as

being “D–, weak D” or “D–, Du.”

2. Absence of agglutination in the tube

with anti-D is a negative result, indi-

cating that the cells do not express D

andshouldbeclassifiedasD–.

742 AABB Technical Manual

3. If there is agglutination at any phase

in the control tube, no valid inter-

pretation of the weak D test can be

made. If the specimen is from a po-

tential transfusion recipient, Rh-

negative blood should be given until

the D type can be resolved. If the

specimen is from a donor, the unit

should not be used for transfusion.

Note

Some facilities may elect to do an addi-

tional reading after the 37 C incubation

and before completing the antiglobulin

phase of testing. Refer to the manufac-

turer’s instructions. If this optional read-

ing is performed, the facility’s procedures

manual should indicate its policy on the

interpretation of this result and on the ad-

ditional testing requirements.

Method 2.10. Preparation

and Use of Lectins

Principle

Saline extracts of seeds react with specific

carbohydrates on red cell membranes and

make useful typing reagents that are

highly specific at appropriate dilutions.

Diluted extract of Dolichos biflorus agglu-

tinates A1red cells but not A2.Ulex

europaeus extract reacts with the H deter-

minant; it agglutinates in a manner pro-

portional to the amount of H present

(O>A2>B>A1>A1B red cells). Other lectins

useful for special purposes include

Arachis hypogaea (anti-T), Glycine max

(anti-T, -Tn), Vicia graminea (anti-N), and

the Salvia lectins (S. horminum,anti-

Tn/Cad; S. sclarea, anti-Tn). To investi-

gate red cell polyagglutination, prepare

and test the cells with Arachis,Glycine,

Salvia,andDolichos lectins. The antici-

pated reactions with various types of

polyagglutinable red cells are shown in

Table 2.10-1. If commercially made lectins

are used, follow the manufacturer’s in-

structions.

Reagents

Seeds may be obtained from health-food

stores, pharmacies, or commercial seed

companies. The seeds should be raw.

Procedure

1. Grind the seeds in a food processor

or blender until the particles look

like coarse sand. A mortar and pestle

may be used, or seeds can be used

whole.

2. In a large test tube or small beaker,

placegroundseedsandthreetofour

times their volume of saline. (Seeds

vary in the quantity of saline they

absorb.)

3. Incubate at room temperature for 4

to 12 hours, stirring or inverting oc-

casionally.

4. Transfer supernatant fluid to a cen-

trifuge tube and centrifuge it for 5

minutes, to obtain clear superna-

Methods Section 2: Red Cell Typing 743

Table 2.10-1. Reactions Between Lectins

and Polyagglutinable Red Cells

TThTkTnCad

Arachis hypogaea*

+++00

Dolichos biflorus

†000++

Glycine max (soja)

+00++

Salvia sclarea

000+0

Salvia horminum

000++

*T and Th cells give weaker reactions with

Arachis

after

protease treatment; Tk reactivity is enhanced after prote-

ase treatment.

†A and AB cells may react due to anti-A reactivity of

Doli-

chos

lectin.

tant. Collect and filter the super-

natant fluid and discard seed residue.

5. Test dilutions of the extract to find

dilution for the desired activity. De-

termine the activity of the extract

with the appropriate red cells, as be-

low.

For Dolichos biflorus:

a. Add 1 drop of 2% to 5% saline

suspension of known A1,A

A1B, A2B, B, and O red cells to

appropriately labeled tubes.

b. Add 1 drop of the extract to each

tube.

c. Centrifuge for calibrated time.

d. Inspect for agglutination and

record results.

e. The lectin should agglutinate

A1and A1B cells but not A2,A

2B,

B, or O cells. The native extract

often agglutinates all the cells

tested. To make the product

useful for reagent purposes, add

enough saline to the extract so

that there is 3+ or 4+ agglutina-

tion of A1and A1B cells, but not

of A2,A

2B, B, or O cells.

For Ulex europaeus:

a. Add 1 drop of 2% to 5% saline

suspension of known A1,A

A1B, B, and O cells to appropri-

ately labeled tubes.

b. Add 1 drop of extract to each tube.

c. Centrifuge for the calibrated

time.

d. Inspect for agglutination and

record results.

e. The strength of the agglutina-

tion should be in the order of

O>A2>B>A1>A1B.

f. Dilute extract with saline, if

necessary,toapointthatO

cells show 3+ or 4+ agglutina-

tion, A2and B cells show 1+ to

2+ agglutination, and A1or A1B

cells are not agglutinated.

Notes

1. To facilitate grinding hard seeds, the

seeds can be covered with saline and

soaked for several hours before

grinding. The container used for

soaking should not be tightly closed

becausesomebeansreleasegasdur

ing the soaking process, which could

cause the container to explode.

2. The saline extracts may be stored in

the refrigerator for several days; they

may be stored indefinitely if frozen.

3. Tests should include a positive and

negative control.

Method 2.11. Use of

Sulfhydryl Reagents to

Disperse Autoagglutination

Principle

See Chapter 20 for a discussion of auto-

agglutination dispersion.

Specimen

Immunoglobulin-coated red cells to be

evaluated.

Reagents

1. 0.01 M dithiothreitol (DTT): 0.154 g

of DTT dissolved in 100 mL of phos-

phate-buffered saline (PBS) at pH

7.3, or 0.1 M 2-mercaptoethanol

(2-ME), 0.7 mL of stock solution of

14 M 2-ME diluted in 100 mL of PBS

at pH 7.3.

2. PBS at pH 7.3.

Procedure

1. Dilute red cells to a 50% concentra-

tion in PBS.

2. Add an equal quantity of 0.01 M DTT

in PBS, or 0.1 M 2-ME in PBS, to the

cells.

744 AABB Technical Manual

3. Incubate at 37 C for 10 minutes (2-ME)

or 15 minutes (DTT).

4. Wash cells three times in saline and

resuspend them.

5. Dilute the treated red cells to a 2% to

5% concentration in saline for use in

blood grouping tests. Verify that red

cells do not spontaneously aggluti-

nate before typing or use.

Note

This procedure is normally used only for

ABO forward cell typing, Rh testing, and

the direct antiglobulin test. At this con-

centration of DTT, some antigens, in par-

ticular Jsaand Jsb, may be weakened or de-

stroyed by 0.01M DTT.

Reference

Reid ME. Autoagglutination dispersal utilizing

sulfhydryl compounds. Transfusion 1978;18:353-5.

Method 2.12. Gentle Heat

Elution for Testing Red Cells

with a Positive DAT

Principle

Red cells heavily coated with IgG may

spontaneously agglutinate in high-pro-

tein reagents and will cause false-positive

AHG test results. To perform red cell anti-

gen typing, it may be necessary to dissoci-

ate antibody from the cells by elution

without damaging membrane integrity or

altering antigen expression. The gentle

heat elution procedure employed to pre-

pare immunoglobulin-free red cells dif-

fers from procedures intended to recover

active antibody.

Reagent

Antihuman globulin.

Specimen

Test cells with a positive direct antiglo-

bulin test (DAT) result.

Procedure

1. Place one volume of washed anti-

body-coated red cells and three vol-

umes of normal saline in a test tube

of appropriate size. In another tube,

place the same volumes of saline

and washed red cells positive for the

antigen under test. This will provide

a check that the elution technique

does not destroy the antigen reactiv-

ity.

2. Incubate the contents of both tubes

atapproximately45Cfor10to15

minutes. The tubes should be agi-

tated frequently. The time of incuba-

tion should be roughly proportional

to the degree of antibody coating, as

indicated by strength of antiglobulin

reactivity.

3. Centrifuge the tubes and discard the

supernatant saline.

4. Test the person’s cells for degree of

antibody removal by comparing a

DAT on the treated cells with the

antiglobulin results on untreated red

cells. If the antibody coating is re-

duced but still present, steps 1

through 3 can be repeated; the con-

trol cells should be subjected to a

similar second treatment.

5. Test the treated cells for the desired

antigen.

Notes

1. This procedure may be unnecessary

if IgM monoclonal reagents are

available; these reagents cause di-

rect agglutination and are not usu-

ally affected by bound immunoglob-

ulin.

Methods Section 2: Red Cell Typing 745

2. As with untreated patient cells, re-

sults of antigen testing in recently

transfused patients should be inter-

preted with caution because of the

potential presence of donor cells.

Method 2.13. Dissociation of

IgG by Chloroquine for Red

Cell Antigen Testing of Red

Cells with a Positive DAT

Principle

Red cells with a positive direct antiglobulin

test (DAT) cannot be tested accurately

with blood typing reagents that require an

indirect antiglobulin technique. Under

controlled conditions, chloroquine dip-

hosphate dissociates IgG from the red cell

membrane with little or no damage to its

integrity. Use of this procedure permits

complete phenotyping of red cells coated

with warm-reactive autoantibody, includ-

ing tests with reagents solely reactive by

indirect antiglobulin techniques.

Specimen

Red cells with a positive DAT due to IgG

coating.

Reagents

1. Chloroquine diphosphate solution

prepared by dissolving 20 g of chloro-

quine diphosphate in 100 mL of sa-

line. Adjust to pH 5.1 with 1 N NaOH,

and store at 2 to 6 C.

2. Control red cells carrying a single-dose

expression of antigens for which the

test samples are to be phenotyped.

3. Anti-IgG antiglobulin reagent.

Procedure

1. To 0.2 mL of washed IgG-coated cells,

add 0.8 mL of chloroquine diphos-

phate solution. Similarly treat the

control sample.

2. Mix and incubate at room tempera-

ture for 30 minutes.

3. Remove a small aliquot (eg, 1 drop)

of the treated test cells and wash

them four times with saline.

4. Test the washed cells with anti-IgG.

5. If this treatment has rendered the

cells nonreactive with anti-IgG, wash

the total volumes of treated test cells

and control cells three times in sa-

lineandmakea2%to5%suspen

sion in saline to use in subsequent

blood typing tests.

6. If the treated red cells react with

anti-IgG after 30 minutes of incuba-

tion with chloroquine diphosphate,

steps 3 and 4 should be repeated at

30-minute intervals (for a maximum

incubation period of 2 hours), until

the sample tested is nonreactive

with anti-IgG. Then proceed as de-

scribed in step 5.

Notes

1. Chloroquine diphosphate does not

dissociate complement proteins

from the cell membrane. If red cells

are coated with both IgG and C3,

only anti-IgG should be used in tests

performed after chloroquine treat-

ment.

2. Incubation with chloroquine diphos-

phate should not be extended be-

yond 2 hours. Prolonged incubation

at room temperature or incubation

at 37 C may cause hemolysis and

loss of red cell antigens.

3. Some denaturation of Rh antigens

may occur.

4. Many serologists run chloroquine-

treated control cells for each antigen

tested. Select control cells that are

positive for the antigen correspond-

746 AABB Technical Manual

ing to the antisera that will be used

to type the patient’s cells.

5. Chloroquine diphosphate may not

completely remove antibody from

sensitized red cells. DAT results on

red cells from some persons, partic-

ularly those with a strongly positive

initial test, may only be diminished

in strength.

6. In addition to its use for removal of

autoantibodies, this method can be

used for removal of Bg (HLA)-related

antigens from red cells. Appropriate

Bg controls should be used.

7. If a commercial kit is used, manufac-

turer’s instructions should be followed

for testing and controls.

References

1. Edwards JM, Moulds JJ, Judd WJ. Chloro-

quine diphosphate dissociation of antigen-

antibody complexes: A new technique for

phenotyping rbcs with a positive direct

antiglobulin test. Transfusion 1982;22:59-61.

2. Swanson JL, Sastamoinen R. Chloroquine

stripping of the HLA-A,B antigens from red

cells (letter). Transfusion 1985;25:439-40.

Method 2.14. Acid Glycine/

EDTA Method to Remove

Antibodies from Red Cells

Principle

Acid glycine/EDTA can be used to dissoci-

ate antibody molecules from red cell

membranes. This procedure is routinely

used for blood typing tests or adsorption

procedures. All common red cell antigens

can be detected after treatment with acid

glycine/EDTA except antigens of the Kell

system, Bg antigens, and Er antigens.

Thus, cells treated in this manner cannot

be used to determine these phenotypes.

Specimen

Red cells with a positive direct antiglobulin

test (DAT).

Reagents

1. 10% EDTA prepared by dissolving 2 g

of disodium ethylenediamine tetra-

acetic acid (Na2EDTA) in 20 mL of

distilled or deionized water.

2. 0.1 M glycine-HCl buffer (pH 1.5)

prepared by diluting 0.75 g of glycine

to 100 mL with isotonic (unbuffered)

saline. Adjust the pH to 1.5 using

concentrated HCl.

3. 1.0 M TRIS-NaCl prepared by dis-

solving 12.1 g of tris (hydroxymethyl)

aminomethane (TRIS) and 5.25 g of

sodium chloride (NaCl) to 100 mL

with distilled or deionized water.

Procedure

1. Wash the red cells to be treated six

times with isotonic saline.

2. In a test tube, mix together 20 vol-

umes of 0.1 M acid glycine-HCl (pH

1.5) with five volumes of 10% EDTA.

This is the acid glycine/EDTA reagent.

3. Place 10 volumes of washed red cells

in a clean tube.

4. Add 20 volumes of acid glycine/EDTA.

5. Mix the contents of the tube thor-

oughly.

6. Incubate the mixture at room tem-

perature for no more than 2 to 3

minutes.

7. Add one volume of 1.0 M TRIS-NaCl

and mix the contents of the tube.

8. Centrifuge at 900 to 1000 ×gfor 1 to

2 minutes, then aspirate, and dis-

card the supernatant fluid.

9. Wash the red cells four times with

saline.

10. Test the washed cells with anti-IgG;

if nonreactive with anti-IgG, the cells

Methods Section 2: Red Cell Typing 747

are ready for use in blood typing or

adsorption procedures. If the DAT is

still positive, one additional treatment

can be performed.

Notes

1. Overincubation of red cells with acid

glycine/EDTA causes irreversible

damage to cell membranes.

2. Include a parallel control reagent,

such as 6% bovine albumin or inert

plasma, when typing treated red cells.

3. Use anti-IgG, not a polyspecific anti-

globulin reagent, in step 10.

4. Many serologists run acid glycine/

EDTA treated control cells for each

antigen tested. Select control cells that

arepositivefortheantigencorre-

sponding to the antisera that will be

used to type the patient’s cells.

5. If a commercial kit is used, manufac-

turer’s instructions should be fol-

lowed for testing and controls.

References

1. Louie JE, Jiang AF, Zaroulis CG. Preparation of

intact antibody-free red cells in autoimmune

hemolytic anemia (abstract). Transfusion

1986;26:550.

2. Champagne K, Spruell P, Chen J, et al. EDTA/

glycine-acid vs. chloroquine diphosphate

treatment for stripping Bg antigens from red

blood cells (abstract). Transfusion 1996;36

(Suppl):21S.

3. Reid ME, Lomas-Francis C. The blood group

antigen factsbook. New York: Academic Press,

2004.

Method 2.15. Separation of

Transfused from Autologous

Red Cells by Simple

Centrifugation

Principle

Newly formed autologous red cells gener-

ally have a lower specific gravity than

transfused red cells and may be separated

from the transfused population by simple

centrifugation. Newly formed autologous

cells concentrate at the top of the column

of red cells when blood is centrifuged in a

microhematocrit tube, providing a simple

method for recovering autologous cells in

a blood sample from recently transfused

patients. Note: Red cells from patients

with hemoglobin S or spherocytic disor-

ders are not effectively separated by this

method (see Method 2.16 for an alterna-

tive procedure).

Specimen

Red cells from whole blood collected into

EDTA.

Materials

1. Microhematocrit centrifuge.

2. Plain (not heparinized) glass or plas-

tic hematocrit tubes.

3. Sealant.

Procedure

1. Wash the red cells three times in sa-

line. For the last wash, centrifuge

them at 900 to 1000 gfor 5 to 15

minutes. Remove as much of the

supernatant fluid as possible with-

out disturbing the buffy coat. Mix

thoroughly.

2. Fill 10 microhematocrit tubes to the

60-mm mark with well-mixed washed

red cells.

3. Seal the ends of the tubes by heat or

with sealant.

4. Centrifuge all tubes in a microhema-

tocrit centrifuge for 15 minutes.

5. Cut the microhematocrit tubes 5

mm below the top of the column of

red cells. This 5-mm segment con-

748 AABB Technical Manual

tains the least dense, hence youn-

gest, circulating red cells.

6. Place the cut microhematocrit tubes

into larger test tubes (10 or 12 ×75

mm), add saline, and mix well to

flush the red cells from the micro-

hematocrit tubes. Then, either a)

centrifuge them at 1000 ×gfor 1

minute and remove the empty hema-

tocrit tubes or b) transfer the saline-

suspended red cells to a clean test

tube.

7. Wash the separated red cells three

times in saline before resuspending

them to 2% to 5% in saline for test-

ing.

Notes

1. Separation is better if 3 or more days

have elapsed since transfusion than

if the sample has been obtained

shortly after transfusion.

2. The red cells should be mixed con-

tinuously while the microhematocrit

tubes are being filled.

3. Separation techniques are only ef-

fective if the patient is producing

normal or above-normal numbers of

reticulocytes. This method will be

ineffective in patients with inade-

quate reticulocyte production.

4. Some red cell antigens may not be as

strongly expressed on reticulocytes

as on older cells. Particular attention

should be given to determinations of

theE,e,c,Fy

a,Jk

a, and Ge antigens.

References

1. Reid ME, Toy P. Simplified method for recov-

ery of autologous red blood cells from trans-

fused patients. Am J Clin Pathol 1983;79:364-6.

2. Vengelen-Tyler V, Gonzales B. Reticulocyte

rich RBCs will give weak reactions with many

blood typing antisera (abstract). Transfusion

1985;25:476.

Method 2.16. Separation of

Transfused Red Cells from

Autologous Red Cells in

Patients with Hemoglobin S

Disease

Principle

Red cells from patients with sickle cell

disease, either hemoglobin SS or SC, are

resistant to lysis by hypotonic saline, in

contrast to red cells from normal persons

and those with hemoglobin S trait. This

procedure permits isolation of autologous

red cells from patients with hemoglobin

SS or SC disease who have recently been

transfused.

Specimen

Red cells to be evaluated.

Reagents

1. Hypotonicsaline(0.3%w/vNaCl):

NaCl, 3 g; distilled water to 1 L.

2. Normal saline (0.9% w/v NaCl):

NaCl, 9 g; distilled water to 1 L.

Procedure

1. Place 4 or 5 drops of red cells into a

10 or 12 ×75-mm test tube.

2. Wash the cells six times with 0.3%

NaCl, or until the supernatant fluid

no longer contains grossly visible

hemoglobin. For each wash, centri-

fuge at 1000 ×gfor 1 minute.

3. Wash the cells twice with 0.9% NaCl

to restore tonicity. For each wash,

centrifuge at 200 ×gfor 2 minutes to

facilitate removal of residual stroma.

4. Resuspend the remaining intact red

cells to a 2% to 5% concentration for

phenotyping.

Methods Section 2: Red Cell Typing 749

Note

Larger volumes, for use in adsorption stud-

ies, can be processed in a 16 ×100-mm

test tube.

Reference

Brown D. A rapid method for harvesting autolo-

gous red cells from patients with hemoglobin S

disease. Transfusion 1988;28:21-3.

750 AABB Technical Manual

Methods Section 3: Antibody Detection/Identification and Compatibility Testing

Methods Section 3

Antibody Detection,

Antibody Identification, and

Serologic Compatibility

Testing

Method 3.1. Immediate-Spin

Compatibility Testing to

Demonstrate ABO

Incompatibility

Principle

See Chapter 18 for a discussion of the prin-

ciples of compatibility testing.

Specimen

Patient’s serum or plasma may be used.

The age of the specimen must comply with

the pretransfusion specimen requirements

in AABB Standards for Blood Banks and

Transfusion Services.1(p38)

Reagents

1. Normal saline.

2. Donor red cells, 2% to 5% suspen-

sion in normal saline or EDTA saline.

Some serologists prefer to suspend

the donor red cells in EDTA saline be-

cause high-titered anti-A or -B can

initiate complement coating, which can

cause steric hindrance of agglutina-

tion.2The use of a patient’s sample

collected in EDTA is an alternative

approach to prevent this phenome-

non.

Procedure

1. Label a tube for each donor red cell

suspension being tested with the pa-

tient’s serum.

2. Add2dropsofthepatient’sserumor

plasma to each tube.

3. Add 1 drop of the suspension of do-

nor red cells to the appropriate test

tube.

4. Mix the contents of the tube(s) and

centrifuge according to the calibra-

tion of the centrifuge.

751

Section 3

5. Examine the tube(s) for hemolysis,

gently resuspend the red cell button(s),

and examine for agglutination.

6. Read, interpret, and record test results.

Interpretation

1. Agglutination or hemolysis constitutes

a positive (incompatible) test result.

2. A smooth suspension of red cells af-

ter resuspension of the red cell but-

ton constitutes a negative result and

indicates a compatible immediate-

spin crossmatch.

References

1. Silva MA, ed. Standards for blood banks and

transfusion services. 23rd ed. Bethesda, MD:

AABB, 2005.

2. JuddWJ,SteinerEA,O’DonnellDB,Oberman

HA. Discrepancies in ABO typing due to

prozone; how safe is the immediate-spin

crossmatch? Transfusion 1988;28:334-8.

Method 3.2. Indirect

Antiglobulin Test (IAT) for

the Detection of Antibodies

to Red Cell Antigens

Principle

For a discussion of the principles of sa-

line, albumin, low-ionic-strength saline

(LISS), and polyethylene glycol (PEG) in-

direct antiglobulin testing, see Chapters 12,

18, and 19.

Specimen

Serum or plasma may be used. The age of

thespecimenmustcomplywithpretrans

fusion specimen requirements in AABB

Standards for Blood Banks and Transfusion

Services.

Reagents

1. Normal saline.

2. Bovine albumin (22% or 30%).

3. LISS made as follows:

a. Add 1.75 g of NaCl and 18 g of

glycine to a 1-liter volumetric flask.

b. Add 20 mL of phosphate buffer

prepared by combining 11.3 mL

of 0.15 M KH2PO4and 8.7 mL of

0.15 M Na2HPO4.

c. Add distilled water to the 1-liter

mark.

d. Adjust the pH to 6.7 ± 0.1 with

NaOH.

e. Add 0.5 g of sodium azide as a

preservative.

Note: LISS may be used as an addi-

tive (Method 3.2.2) or for the suspen-

sion of test red cells (Method 3.2.3).

LISS preparations are also available

commercially.

4. PEG, 20% w/v: To 20 g of 3350 MW

PEG, add phosphate-buffered saline

(PBS) pH 7.3 (see Method 1.7) to 100 mL.

PEG is also available commercially.

5. Antihuman globulin (AHG) reagent.

Polyspecific or anti-IgG may be used

unless otherwise indicated.

6. Commercially available group O an-

tibody detection cells. Pooled group

O antibody detection cells may be

used only for donor testing. Testing

of patients’ samples must be per-

formed with unpooled cells.

7. IgG-coated red cells.

Method 3.2.1. Saline Indirect Antiglobulin

Test

Procedure

1. Add 2 drops of serum or plasma to

properly labeled tubes.

2. Add 1 drop of 2% to 5% saline-sus-

pended reagent group O cells or do-

nor red cells to each tube and mix.

3. Centrifuge and observe for hemoly-

sis and agglutination. Grade and re-

cord the results.

752 AABB Technical Manual

4. Incubate at 37 C for 30 to 60 minutes.

5. Centrifuge and observe for hemoly-

sis and agglutination. Grade and re-

cord the results.

6. Wash the cells three or four times with

saline and completely decant the fi-

nal wash.

7. Add AHG to the dry cell button ac-

cording to the manufacturer’s direc-

tions. Mix well.

8. Centrifuge and observe for aggluti-

nation. Grade and record the results.

9. Confirm the validity of negative tests

by adding IgG-coated red cells.

Method 3.2.2. Albumin or LISS-Additive

Indirect Antiglobulin Test

Procedure

1. Add 2 drops of serum or plasma to

properly labeled tubes.

2. Add an equivalent volume of 22% or

30% bovine albumin or LISS additive

(unless the manufacturer’s directions

state otherwise.)

3. Add 1 drop of a 2% to 5% saline-sus-

pended reagent or donor red cells to

each tube and mix.

4. For albumin, incubate at 37 C for 15

to 30 minutes. For LISS, incubate for

10 to 15 minutes or follow the manu-

facturer’s directions.

5. Centrifuge and observe for hemoly-

sis and agglutination. Grade and re-

cord the results.

6. Perform the test described in Method

3.2.1, steps 6 through 9.

Method 3.2.3. LISS Indirect Antiglobulin Test

Procedure

1. Wash reagent or donor red cells three

times in normal saline and comple-

tely decant saline.

2. Resuspend the cells to a 2% to 3%

suspension in LISS.

3. Add 2 drops of serum to a properly

labeled tube.

4. Add 2 drops of LISS-suspended red

cells, mix, and incubate at 37 C 10 to

15 minutes or follow the manufac-

turer’s directions.

5. Centrifuge and observe for hemoly-

sis and agglutination by gently re-

suspending the cell button. Grade and

record results.

6. Perform the test described in Method

3.2.1, steps 6 through 9.

Method 3.2.4. PEG Indirect Antiglobulin

Test

Procedure

1. For each cell sample to be tested, mix

2 drops of test serum, 4 drops of 20%

PEG in PBS, and 1 drop of a 2% to

5% suspension of red cells.

2. Incubate at 37 C for 15 minutes.

3. DO NOT CENTRIFUGE.

4. Wash the cells four times with saline

and completely decant the final wash.

5. Perform the AHG test, using anti-IgG,

described in Method 3.2.1, steps 7

through 9.

Note: The manufacturer’s instructions

should be followed for the proper use

of commercial PEG solutions.

Interpretation (for Antiglobulin Tests,

Methods 3.2.1 through 3.2.4)

1. Thepresenceofagglutination/hem

olysis after incubation at 37 C con-

stitutes a positive test.

2. Thepresenceofagglutinationafter

addition of AHG constitutes a posi-

tive test.

3. Antiglobulin tests are negative when

no agglutination is observed after

initial centrifugation and the IgG-

coated red cells added afterward are

agglutinated. If the IgG-coated red

cells are not agglutinated, the nega-

Methods Section 3: Antibody Detection/Identification and Compatibility Testing 753

tive result is invalid and the test must

be repeated.

Controls

The procedure used for the detection of

unexpected antibodies in pretransfusion

testing should be checked daily with weak

examples of antibody. Control sera can be

prepared from reagent grade typing sera

diluted with 6% bovine albumin to give 2+

reactions by an IAT. Human sources of IgG

antibodies are also acceptable.

Notes

1. The incubation times and the volume

and concentration of red cells indi-

cated are those given in the literature.

Individual laboratories may choose to

standardize techniques with somewhat

different values. See Chapter 12 for

other limitations when modifying

procedures. In all cases, the manu-

facturer’s package insert should be

consulted before modifying a proce-

dure.

2. For the saline procedure, step 3 may

be omitted to avoid the detection of

antibodies reactive at room temper-

ature.

3. For the PEG procedure:

a. Omit centrifugation after 37 C

incubation because red cells will

not resuspend readily.

b. Use anti-IgG rather than poly-

specific AHG to avoid unwanted

positive reactions due to C3-

binding autoantibodies.

4. Steps 6 through 9 of the IAT (Method

3.2.1) must be performed without in-

terruption.

Reference

Silva MA, ed. Standards for blood banks and trans-

fusion services. 23rd ed. Bethesda, MD: AABB, 2005:

38.

Method 3.3. Prewarming

Technique

Principle

Prewarming may be useful in the detec-

tion and identification of red cell antibod-

ies that bind to antigen only at 37 C. This

test is particularly useful for testing sera

of patients with cold-reactive autoantibody

activity that may mask the presence of

clinically significant antibodies. However,

use of the prewarming technique for this

application has become controversial.1-2 It

has been shown to result in decreased

reactivity of some potentially significant

antibodies and weak antibodies can be

missed.3The technique should be used

with caution and not used to eliminate

unidentified reactivity.

Strong cold-reactive autoantibodies may

react in prewarmed tests; other techniques

such as cold allo- or autoadsorption or

dithiothreitol treatment of plasma may be

required to detect underlying clinically sig-

nificant antibodies.

Specimen

Serum or plasma may be used. The age of

thespecimenmustcomplywithpretrans

fusion specimen requirements in AABB

Standards for Blood Banks and Transfusion

Services.4(p38)

Reagents

1. Normal saline.

2. Anti-IgG.

3. Commercially available group O an-

tibody detection cells. Pooled group

O antibody detection cells may be used

only for donor testing. Testing of pa-

tients’ samples must be done with

unpooled cells.

4. IgG-coated red cells.

754 AABB Technical Manual

Procedure

1. Prewarmabottleofsalineto37C.

2. Label one tube for each reagent or

donorsampletobetested.

3. Add 1 drop of 2% to 5% saline-sus-

pended red cells to each tube.

4. Place the tubes containing red cells

and a tube containing a small volume

of the patient’s serum and a pipette

at 37 C; incubate for 5 to 10 minutes.

5. Using the prewarmed pipette, trans-

fer 2 drops of prewarmed serum to

each tube containing prewarmed red

cells. Mix without removing tubes

from the incubator.

6. Incubate at 37 C for 30 to 60 min-

utes.

7. Without removing the tubes from the

incubator, fill each tube with pre-

warmed (37 C) saline. Centrifuge

and wash three or four times with 37

C saline.

8. Add anti-IgG, according to the man-

ufacturer’s directions.

9. Centrifuge and observe for reaction.

Grade and record the results.

10. Confirm the validity of negative tests

by adding IgG-coated red cells.

Notes

1. The prewarming procedure described

above will not detect alloantibodies

that agglutinate at 37 C or lower and

are not reactive in the antiglobulin

phase. If detection of these antibod-

ies is desired, testing and centrifuga-

tion at 37 C are required. If time per-

mits, a tube containing a prewarmed

mixture of serum and cells can be

incubatedat37Cfor60to120min

utes, and the settled red cells exam-

ined for agglutination by resuspend-

ing the button without centrifugation.

2. Cold-reactive antibodies may not be

detectable when room-temperature

saline instead of 37 C saline is used

in the wash step.2The use of room-

temperature saline may avoid the

elution of clinically significant anti-

body(ies) from reagent red cells that

can occur with the use of 37 C saline.

Some strong cold-reactive autoanti-

bodies, however, may still react and

therefore require the use of 37 C sa-

line to avoid their detection.

References

1. Judd WJ. Controversies in transfusion medi-

cine. Prewarmed tests: Con. Transfusion 1995;

35:271-7.

2. Mallory D. Controversies in transfusion med-

icine. Prewarmed tests: Pro—why, when, and

how—not if. Transfusion 1995;35:268-70.

3. Leger RM, Garratty G. Weakening or loss of

antibody reactivity after prewarm technique.

Transfusion 2003;43:1611-14.

4. Silva MA, ed. Standards for blood banks and

transfusion services. 23rd ed. Bethesda, MD:

AABB, 2005.

Method 3.4. Saline

Replacement to Demonstrate

Alloantibody in the Presence

of Rouleaux

Principle

Rouleaux are aggregates of red cells that,

characteristically, adhere to one another

on their flat surface, giving a “stack of coins”

appearance when viewed microscopically.

Rouleaux formation is an in-vitro phe-

nomenon resulting from abnormalities of

serum protein concentrations. The pa-

tient is often found to have liver disease,

multiple myeloma, or another condition

associated with abnormal globulin levels.

It may be difficult to detect antibody-as-

sociated agglutination in a test system

containing rouleaux-promoting serum. In

the saline replacement technique, serum

and cells are incubated to allow antibody

Methods Section 3: Antibody Detection/Identification and Compatibility Testing 755

attachment, but the serum is removed

and saline is added as the resuspending

medium.

Reagents

Saline.

Procedure

After routine incubation and resuspension,

proceed with the following steps if the ap-

pearance of the resuspended cells suggests

rouleaux formation:

1. Recentrifuge the serum/cell mixture.

2. Remove the serum, leaving the cell

button undisturbed.

3. Replacetheserumwithanequal

volume of saline (2 drops).

4. Resuspend the cell button gently and

observe for agglutination. Rouleaux

will disperse when suspended in saline,

whereas true agglutination will remain.

Reference

Issitt PD, Anstee DJ. Applied blood group serology.

4th ed. Durham, NC: Montgomery Scientific Pub-

lications, 1998:1135.

Method 3.5. Enzyme

Techniques

For a discussion of the principles of en-

zyme testing, see Chapter 19.

Method 3.5.1. Preparation of Ficin

Enzyme Stock, 1% w/v

Principle

The enzyme preparations used in blood

banking differ from lot to lot; each time a

stock enzyme solution is prepared, its re-

activity should be tested and incubation

periods standardized for optimal effective-

ness. See Method 3.5.3.

Reagents

1. Dryenzymepowder,1g.

2. Phosphate-buffered saline (PBS), pH

7.3: see Method 1.7.

3. Phosphate buffer, pH 5.4.

Procedure

1. Place 1 g of powdered ficin in a 100-

mL volumetric flask. Handle the

ficin carefully; it is harmful if it gets

intheeyesorisinhaled.Itisdesir

able to wear gloves, mask, and apron,

or to work under a hood.

2. AddPBS,pH7.3to100mL,todis

solve the ficin. Agitate vigorously by

inversion, rotate for 15 minutes, or

mix with a magnetic stirrer until

mostly dissolved. The powder will not

dissolve completely.

3. Collect clear fluid, either by filtration

or centrifugation, and prepare small

aliquots. Store the aliquots at –20 C

or colder. Do not refreeze a thawed

solution.

Method 3.5.2. Preparation of Papain

Enzyme Stock, 1% w/v

Principle

The enzyme preparations used in blood

banking differ from lot to lot; each time a

stock enzyme solution is prepared, its re-

activity should be tested and incubation

periods standardized for optimal effective-

ness. See Method 3.5.3.

Reagents

1. L-cysteine hydrochloride 0.5 M, 0.88

g in 10 mL distilled water.

2. Dryenzymepowder,2g.

3. Phosphate buffer 0.067 M at pH 5.4,

prepared by combining 3.5 mL of

Na2HPO4and 96.5 mL of KH2PO4.

756 AABB Technical Manual

Procedure

1. Add 2 g of powdered papain to 100

mL of phosphate buffer (pH 5.4).

Handle papain carefully; it is harm-

ful to mucous membranes. Use ap-

propriate protective equipment.

2. Agitate enzyme solution for 15 min-

utes at room temperature.

3. Collect clear fluid by filtration or

centrifugation.

4. Add L-cysteine hydrochloride and

incubate solution at 37 C for 1 hour.

5. Add phosphate buffer (pH 5.4) to fi-

nal volume of 200 mL. Store aliquots

at –20 C or colder. Do not refreeze

aliquots.

Method 3.5.3. Standardization of Enzyme

Procedures

Principle

Foratwo-stageenzymeprocedure,the

optimal treatment time must be deter-

mined for each new lot of stock solution.

The technique given below for ficin can

be modified for use with other enzymes.

Reagents

1. 1% stock solution of ficin in PBS, pH

7.3.

2. Several sera known to lack unex-

pected antibodies.

3. Anti-D that agglutinates only en-

zyme-treated D+ red cells and does

not agglutinate untreated D+ cells.

4. Anti-Fyaof moderate or strong reac-

tivity.

5. D+ and Fy(a+b–) red cell samples.

6. Antihuman globulin (AHG) reagent.

Polyspecific or anti-IgG may be used

unless otherwise indicated.

7. IgG-coated red cells.

Procedure

1. Prepare 0.1% ficin by diluting one

volume of stock ficin solution with

nine volumes of PBS, pH 7.3.

2. Label three tubes: 5 minutes, 10

minutes, and 15 minutes.

3. Add equal volumes of washed red cells

and 0.1% ficin to each tube.

4. Mix and incubate at 37 C for the time

designated. Incubation times are

easily controlled if the 15-minute

tube is prepared first, followed by

the 10- and 5-minute tubes at

5-minute intervals. Incubation will

be complete for all three tubes at the

same time.

5. Immediately wash the red cells three

times with large volumes of saline.

6. Resuspend treated cells to 2% to 5%

in saline.

7. Label four tubes for each serum to

be tested: untreated, 5 minutes, 10

minutes, 15 minutes.

8. Add 2 drops of the appropriate se-

rum to each of the four tubes.

9. Add 1 drop of the appropriate red cell

suspension to each of the labeled

tubes.

10. Mix and incubate at 37 C for 15 min-

utes.

11. Centrifuge and examine for aggluti-

nation by gently resuspending the red

cell button.

12. Proceed with the AHG test described

in Method 3.2.1, steps 6 through 9.

Interpretation

Table 3.5.3-1 shows possible results with

D+, Fy(a+b–) cells and the sera indicated.

In this case, the optimal incubation time

would be 10 minutes. Incubation for only

5 minutes does not completely abolish Fya

activity or maximally enhance anti-D re-

activity. Incubation for 15 minutes causes

Methods Section 3: Antibody Detection/Identification and Compatibility Testing 757

false-positive antiglobulin reactivity with

inert serum.

If incubation for 5 minutes overtreats the

cells,itispreferabletouseamoredilute

working solution of enzyme than to reduce

incubation time because it is difficult to ac-

curately monitor very short incubation

times. Additional tests can evaluate a single

dilution at different incubation times, or a

single incubation time can be used for

different enzyme dilutions.

Method 3.5.4. Evaluating Enzyme-Treated

Red Cells

Principle

After optimal incubation conditions have

been determined for a lot of enzyme solu-

tion, treated red cells should be evaluated

before use to demonstrate that they are

adequately, but not excessively, modified.

Satisfactory treatment produces cells that

are agglutinated by an antibody that causes

only indirect antiglobulin test reactivity of

unmodified cells but are not agglutinated

or aggregated by inert serum.

Specimen

Enzyme-treated red cells.

Reagents

1. Sera known to contain antibody that

will agglutinate enzyme-treated cells.

2. Sera free of any unexpected antibod-

ies.

3. Antihuman globulin (AHG) reagent.

Polyspecific or anti-IgG may be used

unless otherwise indicated.

4. IgG-coated red cells.

Procedure

1. Select an antibody that agglutinates

enzyme-treated red cells positive for

the antigen but gives only AHG reac-

tions with unmodified cells. Many

examples of human source anti-D

behave in this way.

2. Add 2 drops of the selected antibody-

containing serum to a tube labeled

“positive.”

3. Add2dropsofaserumfreeofunex

pected antibodies to a tube labeled

“negative.”

4. Add 1 drop of 2% to 5% suspension

of enzyme-treated red cells to each

tube.

5. Mix and incubate 15 minutes at 37 C.

6. Centrifuge and resuspend the cells by

gentle shaking.

758 AABB Technical Manual

Table 3.5.3-1. Hypothetical Results with D+, Fy(a+b–) Red Cells

Cells and Enzyme Inert Serum Anti-D Anti-Fya

Untreated 37 C incubation 0 0 0

antihuman globulin test 0 1+ 3+

5 minutes 37 C incubation 0 1+ 0

antihuman globulin test 0 2+ 1+

10 minutes 37 C incubation 0 2+ 0

antihuman globulin test 0 2+ 0

15 minutes 37 C incubation 0 2+ 0

antihuman globulin test w+ 2+ w+

7. Examine macroscopically for the pre-

sence of agglutination.

8. Perform the AHG test described in

Method 3.2.1, steps 6 through 9, on

the tube labeled “negative.”

Interpretation

Thereshouldbeagglutinationinthe“pos

itive” tube and no agglutination in the

“negative” tube. If agglutination occurs in

the “negative” tube, the cells have been

overtreated; if agglutination does not oc-

cur in the “positive” tube, treatment has

been inadequate.

Method 3.5.5. One-Stage Enzyme

Technique

Specimen

Serum or plasma to be tested.

Reagent

1. Reagent red cells.

2. Antihuman globulin (AHG) reagent.

Polyspecific or anti-IgG may be used

unless otherwise indicated.

3. IgG-coated red cells.

Procedure

1. Add 2 drops of serum to an appro-

priately labeled tube.

2. Add 2 drops of a 2% to 5% saline sus-

pension of reagent red cells.

3. Add 2 drops of 0.1% papain solution

and mix well.

4. Incubate at 37 C for 15 minutes.

5. Centrifuge; gently resuspend the cells

and observe for agglutination. Grade

and record the results.

6. Proceed with the AHG test described

in Method 3.2.1, steps 6 through 9.

Method 3.5.6. Two-Stage Enzyme

Technique

Specimen

Serum or plasma to be tested.

Reagent

1. Reagent red cells.

2. Antihuman globulin (AHG) reagent.

Polyspecific or anti-IgG may be used

unless otherwise indicated.

3. IgG-coated red cells.

Procedure

1. Prepare a diluted enzyme solution

(papain or ficin) by adding 9 mL of

PBS, pH 7.3, to 1 mL of stock enzyme.

2. Add one volume of diluted enzyme

to one volume of packed, washed re-

agent red cells.

3. Incubate at 37 C for the time deter-

mined to be optimal for that enzyme

solution.

4. Wash treated cells at least three times

with large volumes of saline and re-

suspend the cells to a 2% to 5% con-

centration in saline.

5. Add 2 drops of serum or plasma to

be tested to an appropriately labeled

tube.

6. Add 1 drop of 2% to 5% suspension

of enzyme-treated cells.

7. Mix and incubate for 15 minutes at

37 C.

8. Centrifuge; gently resuspend the cells

and observe for agglutination. Grade

and record the results.

9. Proceed with the AHG test described

in Method 3.2.1, steps 6 through 9.

Notes

1. An alternative method for steps 4 and

5 (Method 3.5.5) or steps 7 and 8

(Method 3.5.6) is to incubate the se-

rum and enzyme-treated cells at 37

Methods Section 3: Antibody Detection/Identification and Compatibility Testing 759

C for 60 minutes and examine the

settled cells for agglutination with-

out centrifugation. This can be use-

ful for serum with strong cold-reac-

tive agglutinins and can sometimes

prevent the occurrence of false-posi-

tive results.

2. Microscopic examination is not rec-

ommended for routine use and is

particularly inappropriate with en-

zyme enhanced tests; false-positive

reactions will often be detected.

3. Either papain or ficin may be used in

a two-stage procedure.

4. Enzyme preparations are available

commercially. The manufacturer’s

directions should be followed for ap-

propriate use and quality control.

References

1. Issitt PD, Anstee DJ. Applied blood group se-

rology,4thed.Durham,NC:Montgomery

Scientific, 1998.

2. Judd WJ. Methods in immunohematology. 2nd

ed. Durham, NC: Montgomery Scientific, 1994.

Method 3.6. Direct

Antiglobulin Test (DAT)

Principle

See Chapter 20 for a discussion of the

principles of direct antiglobulin testing.

Specimen

Red cells from an anticoagulated blood

sample.

Reagents

1. Antihuman globulin (AHG) reagent:

polyspecific antiglobulin reagent,

anti-IgG, anti-complement antisera.

2. A control reagent (eg, PBS) is re-

quired when all antisera tested give

a positive result.

3. IgG-coated red cells.

Procedure

1. Dispense 1 drop of a 2% to 5% sus-

pension of red cells into each tube.

2. Wash each tube three or four times

with saline. Completely decant the fi-

nal wash.

3. Immediately add antisera and mix. For

the amount of antisera required, re-

fer to the manufacturer’s directions.

4. Centrifuge according to the manu-

facturer’s directions.

5. Examine the cells for agglutination.

Grade and record the reaction.

6. IfusingpolyspecificAHGoranti-

C3d, incubate nonreactive tests at

room temperature for 5 minutes, then

centrifuge, and read again.

7. Confirm the validity of negative tests

by adding IgG-coated red cells to tests

containing anti-IgG.

8. Centrifuge according to the manu-

facturer’s directions.

9. Examine the cells for agglutination and

record the reaction.

Interpretation

1. The DAT is positive when agglutina-

tion is observed either after im-

mediate centrifugation or after the

centrifugation that followed room-

temperature incubation. IgG-coated

red cells usually give immediate re-

actions, whereas complement coat-

ing may be more easily demonstra-

ble after incubation.1,2 Monospecific

AHG reagents are needed to confirm

which globulins are present.

2. The DAT is negative when no agglu-

tination is observed at either test phase

and the IgG-coated cells added in step

7 are agglutinated. If the IgG-coated

cells are not agglutinated, the nega-

tive DAT result is considered invalid

and the test must be repeated. A

negative DAT does not necessarily

760 AABB Technical Manual

mean that the red cells have no at-

tached globulin molecules. Poly-

specific and anti-IgG reagents detect

as few as 200 to 500 molecules of IgG

per cell,1but patients may experi-

ence autoimmune hemolytic ane-

mia when IgG coating is below this

level.2

3. No interpretation can be made if the

results with all antisera used to per-

form a DAT and the control are reac-

tive. This indicates spontaneous ag-

glutination, which must be resolved

before further testing is performed.

Notes

1. Steps 2 through 7 must be performed

without interruption.

2. Initial testing may be performed with

polyspecific reagent only. If the DAT

is negative with polyspecific reagent,

no further testing is necessary. If the

DAT is positive with polyspecific re-

agent, perform the DAT test with

monospecific reagents, anti-IgG, and

anticomplement, to determine which

globulins are present.

3. Verification of negative results with

anti-C3d is recommended. Refer to

the manufacturer’s instructions to

determine appropriate controls.

References

1. Mollison PL, Engelfriet CP, Contreras M, eds.

Blood transfusion in clinical medicine. 10th

ed. Oxford, England: Blackwell Scientific Pub-

lications, 1997.

2. Petz LD, Garratty G. Immune hemolytic ane-

mia. Philadelphia: Churchill-Livingstone, 2004.

Method 3.7. Antibody Titration

Principle

Titration is a semiquantitative method used

to determine the concentration of antibody

in a serum sample or to compare the

strength of antigen expression on differ-

ent red cell samples. The usual applica-

tions of titration studies are: 1) estimating

antibody activity in alloimmunized preg-

nant women to determine whether and

when to perform more complex invasive

investigation of the fetal condition (see

Chapter 23); 2) elucidating autoantibody

specificity (see Chapter 20); 3) character-

izing antibodies as high-titer, low-avidity,

traits common in antibodies to antigens

of the Knops and Chido/ Rodgers sys-

tems, Csa,andJMH(seeChapter15);and

4) observing the effect of sulfhydryl re-

agents on antibody behavior, to deter-

mine immunoglobulin class (IgG or IgM).

See Method 5.3 for titration studies spe-

cifically to assist in monitoring clinically

significant antibodies in the pregnant

woman.

Specimen

Serum or plasma antibody to be titrated.

Reagents

1. Red cells that express the antigen(s)

corresponding to the antibody spec-

ificity (ies), in a 2% to 5% saline sus-

pension. Uniformity of cell suspen-

sionsisveryimportanttoensure

comparability of results.

2. Saline.(Note:Dilutionsmaybemade

with albumin if desired.)

Procedure

The master dilution technique for titration

studiesisasfollows:

1. Label 10 test tubes according to the

serum dilution (eg, 1 in 1, 1 in 2, etc).

A 1 in 1 dilution means one volume

of serum undiluted; a 1 in 2 dilution

means one volume of serum in a fi-

Methods Section 3: Antibody Detection/Identification and Compatibility Testing 761

nal volume of two, or a 50% solution

of serum in the diluent. See Methods

1.4 and 1.5.

2. Deliver one volume of saline to all test

tubes except the first (undiluted 1 in

1) tube.

3. Add an equal volume of serum to

each of the first two tubes (undiluted

and 1 in 2).

4. Using a clean pipette, mix the con-

tents of the 1 in 2 dilution several

times and transfer one volume into

the next tube (the 1 in 4 dilution).

5. Continue the same process for all di-

lutions, using a clean pipette to mix

and transfer each dilution. Remove

one volume of diluted serum from

the final tube and save it for use if

further dilutions are required.

6. Label 10 tubes for the appropriate

dilutions.

7. Using separate pipettes for each di-

lution, transfer 2 drops of each di-

luted serum into the appropriately

labeled tubes and add 2 drops of a

2% red cell suspension. Alterna-

tively, for convenience, add 1 drop of

a 3%-4% suspension of red cells as

supplied by the reagent manufac-

turer, although this method is less

precise.

8. Mix well and test by a serologic tech-

nique appropriate to the antibody

(see Chapter 19).

9. Examine test results macroscopically;

grade and record the reactions. The

prozone phenomenon (see Chapter

12) may cause reactions to be weaker

in the more concentrated serum pre-

parations than in higher dilutions.

To avoid misinterpretation of results,

itmaybepreferabletoexaminefirst

the tube containing the most dilute

serum and proceed through the more

concentrated samples to the undiluted

specimen.

Interpretation

1. Observe the highest dilution that

produces 1+ macroscopic agglutina-

tion. The titer is reported as the re-

ciprocal of the dilution level, eg,

32—not 1in32or1:32(seeTable

3.7-1). If there is agglutination in the

tube containing the most dilute se-

rum, the endpoint has not been

reached, and additional dilutions

should be prepared and tested.

2. In comparative studies, a significant

difference in titer is three or more di-

lutions. Variations in technique and

inherent biologic variability can cause

duplicate tests to give results that

differ by one dilution in either direc-

tion. Serum containing antibody at a

true titer of 32 may show, on repli-

cate tests, the endpoint in the 1:32

tube, the 1:64 tube, or the 1:16 tube.

3. Titer values alone can be misleading

without also evaluating the strength

of agglutination. The observed strength

of agglutination can be assigned a

number and the sum of these num-

bers for all tubes in a titration study

represents the score, another semi-

quantitative measurement of antibody

reactivity. The arbitrarily assigned

threshold for significance in com-

paring scores is a difference of 10 or

more between different test samples.

See Table 3.7-1.

4. Antibodies with high-titer, low-avid-

ity characteristics generally have a ti-

ter greater than 64, with most tubes

showing consistently weak reactivity.

Table 3.7-1 shows the results obtained

with three sera, each of which shows no

more agglutination after 1:256. The differ-

ences in score, however, indicate consider-

able variation in strength of reactivity.

762 AABB Technical Manual

Notes

Titration is a semiquantitative technique.

Technical variables greatly affect the re-

sults and care should be taken to achieve

the most uniform possible practices.

1. Careful pipetting is essential. Pi-

pettes with disposable tips that can

be changed after each dilution are

recommended.

2. Optimal time and temperature of in-

cubation and time and force of centri-

fugation must be used consistently.

3. The age, phenotype, and concentra-

tion of the test red cells will influence

the results. When the titers of several

antibody-containing sera are to be

compared, all of them should be

tested against red cells (preferably

freshly collected) from the same do-

nor. If this is not possible, the tests

should use a pool of reagent red cells

from donors of the same phenotype.

When a single serum is to be tested

against different red cell samples, all

samples should be collected and pre-

servedinthesamemanneranddi

luted to the same concentration be-

fore use.

4. Completely reproducible results are

virtually impossible to achieve. Com-

parisons are valid only when speci-

mens are tested concurrently. In tests

with a single serum against different

red cell samples, material from the

master dilution must be used for all

the tests.

5. Measurements are more accurate with

large volumes than with small vol-

umes; a master dilution technique

(see above) gives more reliable re-

sults than individual dilutions for a

single set of tests. The volume needed

for all planned tests should be calcu-

Methods Section 3: Antibody Detection/Identification and Compatibility Testing 763

Table 3.7-1. Examples of Antibody Titers, Endpoints, and Scores

Reciprocal of Serum Dilution

1248163264128256512 Titer* Score

Strength: 3+ 3+ 3+ 2+ 2+ 2+ 1+ ± ± 0 64(256)

Sample 1

Score: 10101088853 20 64

Strength: 4+ 4+ 4+ 3+ 3+ 2+ 2+ 1+ ± 0 128(256)

Sample 2

Score: 12 12 12 10 10 8 8 5 3 0 80

Strength: 1+1+1+1+±±±±±0 8(256)

Sample 3

Score: 55553332 20 33

*The titer is often determined from the highest dilution of serum that gives a reaction ≥1+ (score 5). This may differ sig-

nificantly from the titration endpoint (shown in parentheses), as with the reactions of an antibody with high-titer,

low-avidity characteristics, manifested by Sample 3.

lated and an adequate quantity of

each dilution prepared.

6. When performing a titration for

anti-D for HDFN, see Method 5.3.

Method 3.8. Use of Sulfhydryl

Reagents to Distinguish IgM

from IgG Antibodies

Principle

Treating IgM antibodies with sulfhydryl

reagents abolishes both agglutinating and

complement-binding activities. Observa-

tions of antibody activity before and after

sulfhydryl treatment are useful in deter-

mining immunoglobulin class. Sulfhydryl

treatment can also be used to abolish IgM

antibody activity to permit detection of co-

existing IgG antibodies. For a discussion of

IgM and IgG structures, see Chapter 11.

Specimen

2 mL of serum or plasma to be treated.

Reagents

1. Phosphate-buffered saline (PBS) at

pH 7.3.

2. 0.01 M dithiothreitol (DTT), prepared

by dissolving 0.154 g of DTT in 100

mL of pH 7.3 PBS. Store at –18 C or

lower.

Procedure

1. Dispense 1 mL of serum or plasma

into each of two test tubes.

2. To one tube, labeled dilution control,

add 1 mL of pH 7.3 PBS.

3. To the other tube, labeled test, add 1

mL of 0.01 M DTT.

4. Mix and incubate at 37 C for 30 to 60

minutes.

5. Use the DTT-treated and dilution con-

trol samples in standard procedures.

Interpretation

1. Reactivity in the dilution control

serum and no reactivity in the DTT-

treated serum indicates an IgM anti-

body.

2. Reactivity in the dilution control se-

rum and the DTT-treated serum in-

dicates an IgG antibody or an IgG

and IgM mixture. Titration studies

may be necessary to distinguish be-

tween them. See Table 3.8-1.

764 AABB Technical Manual

Table 3.8-1. Effect of Dithiothreitol on Blood Group Antibodies

Dilution

Test Sample 1/2 1/4 1/8 1/16 1/32 Interpretation

Serum+DTT 3+2+2+1+ 0 IgG

Serum + PBS 3+ 2+ 2+ 1+ 0

Serum+DTT 0000 0 IgM

Serum + PBS 3+ 2+ 2+ 1+ 0

Serum+DTT 2+1+0 0 0 IgG + IgM*

Serum + PBS 3+ 2+ 2+ 1+ 0

*May also indicate only partial inactivation of IgM.

3. No reactivity in the dilution control

serum indicates dilution of weak an-

tibody reactivity and an invalid test.

Control

A serum or plasma sample known to con-

tain an IgM antibody should be treated and

tested in parallel.

Notes

1. 2-mercaptoethanol can also be used

for this purpose. See Method 2.11 for

preparation.

2. Sulfhydryl reagents used at low con-

centration may weaken antigens of

the Kell system. For investigation of

antibodies in the Kell system, it may

be necessary to use other methods.

3. Gelling of a serum or plasma sample

may be observed during treatment with

DTT. This can occur if the DTT has

been prepared incorrectly and has a

concentration above 0.01 M. Gelling

may also occur if serum and DTT are

incubated too long. An aliquot of the

sample undergoing treatment can be

tested after 30 minutes of incubation;

if the activity thought to be due to IgM

has disappeared, there is no need to

incubate further. Gelled samples can-

not be tested for antibody activity be-

cause overtreatment with DTT causes

the denaturation of all serum proteins.

Reference

Mollison PL, Engelfriet CP, Contreras M, eds. Blood

transfusion in clinical medicine. 10th ed. Oxford,

England: Blackwell Scientific Publications, 1997.

Method 3.9. Plasma Inhibition

to Distinguish Anti-Ch and

-Rg from Other Antibodies

with HTLA Characteristics

Principle

For a discussion of the principles of plasma

inhibition of anti-Ch and -Rg, see Chapter 19.

Specimen

Serum or plasma to be tested.

Reagents

1. Reactive red cell samples.

2. A pool of six or more normal plasma

samples.

3. 6% bovine albumin, see Method 1.5.

4. Anti-IgG.

5. IgG-coated red cells.

Procedure

1. Prepare serial twofold dilutions of test

serum in saline. The dilution range

should be from 1 in 2 to 1 in 512, or

toonetubebeyondtheknowntiter

as determined above (Method 3.7). The

volume prepared should be not less

than 0.3 mL for each red cell sample

to be tested.

2. For each red cell sample to be tested,

place 2 drops of each serum dilution

into each of two sets of appropriately

labeled 10 or 12 ×75-mm test tubes.

3. To one set, add 2 drops of pooled

plasma to each tube.

4. To the other set, add 2 drops of 6%

albumin to each tube.

5. Gently agitate the contents of each

tube and incubate the tubes at room

temperature for at least 30 minutes.

6. Add1dropofa2%to5%suspension

of red cells to each tube.

Methods Section 3: Antibody Detection/Identification and Compatibility Testing 765

7. Gently agitate the contents of each

tube and incubate the tubes at 37 C

for 1 hour.

8. Wash the cells four times in saline,

add anti-IgG, and centrifuge accord-

ing to the manufacturer’s directions.

9. Resuspend the cell buttons and ex-

amine for agglutination; confirm all

nonreactive tests microscopically.

Grade and record the results.

10. Confirm the validity of negative tests

by adding IgG-coated red cells.

Interpretation

1. Inhibition of antibody activity in the

tubes to which plasma has been added

suggests anti-Ch or anti-Rg specific-

ity; this inhibition is often complete.

2. Thepresenceofpartialinhibition

suggests the possibility of additional

alloantibodies. This can be tested by

preparingalargevolumeofinhibited

serum and testing it against a re-

agent red cell panel to see if the non-

neutralizable activity displays anti-

genic specificity.

3. Lack of reactivity in the control (6%

albumin) indicates dilution of weakly

reactive antibody and an invalid test.

Notes

1. Antibodies to other plasma antigens

may also be partially inhibited by

plasma.1

2. Adsorption with C4-coated red cells

is an alternative procedure that may

be used for identifying anti-Ch or

anti-Rg and for detecting underlying

alloantibodies.2

References

1. Reid ME, Lomas-Francis C. The blood group

antigenfactsbook.2nded.NewYork:Aca

demic Press, 2004.

2. Ellisor SS, Shoemaker MM, Reid ME. Adsorp-

tion of anti-Chido from serum using autolo-

gous red blood cells coated with homologous

C4. Transfusion 1982;22:243-5.

Method 3.10. Dithiothreitol

(DTT) Treatment of Red Cells

Principle

DTT is an efficient reducing agent that

can disrupt the tertiary structure of pro-

teins by irreversibly reducing disulfide

bonds to free sulfhydryl groups. Without

tertiary structure, protein-containing an-

tigens can no longer bind antibodies that

are specific for them. Red cells treated

with DTT will not react with antibodies in

the Kell blood group system, most anti-

bodies in the Knops system, or most ex-

amples of anti-LWa,-Yt

a,-Yt

b,-Do

a,-Do

-Gya,-Hy,and-Jo

a. This inhibition tech-

nique may be helpful in identifying some

of these antibodies or in determining if a

serum contains additional underlying allo-

antibodies.

Specimen

Red cells to be tested.

Reagents

1. Prepare 0.2 M DTT by dissolving 1 g

of DTT powder in 32 mL of phos-

phate-buffered saline (PBS), pH 8.0.

Divide it into 1-mL volumes and

freeze aliquots at –18 C or colder.

2. PBS at pH 7.3, see Method 1.7.

3. Red cells known to be positive for the

antigen in question and, as a control,

red cells known to be positive for K,

which is consistently disrupted by

DTT.

4. Anti-K, either in reagent form or

strongly reactive in a serum specimen.

766 AABB Technical Manual

Procedure

1. Wash one volume of the test cells and

the control cells with PBS. After de-

canting, add four volumes of 0.2 M

DTT, pH 8.0.

2. Incubate at 37 C for 30 to 45 min-

utes.

3. Wash four times with PBS. Slight

hemolysis may occur; if hemolysis is

excessive, repeat the procedure us-

ing fresh red cells and a smaller vol-

ume of DTT, eg, two or three volumes.

4. Resuspend the cells to a 2% to 5%

suspension in PBS.

5. Test DTT-treated cells with serum

containing the antibody in question.

Test K+ red cells with anti-K.

Interpretation

1. The control K+ red cells should give

negative reactions when tested with

anti-K; if not, the DTT treatment has

been inadequate. Other antigens in

the Kell system can also serve as the

control.

2. If reactivity of the test serum is elim-

inated, the suspected antibody spec-

ificity may be confirmed. Enough red

cell samples should be tested to ex-

clude most other clinically signifi-

cant alloantibodies.

Note

Treatment of red cells with 0.2 M DTT, pH

8.0, is optimal for denaturation of all anti-

gens of the Kell, Cartwright, LW, and

Dombrock systems, and most antigens of

the Knops system. Lower concentrations

of DTT may selectively denature particu-

lar blood group antigens (ie, 0.002 M DTT

will denature only Jsaand Jsbantigens). This

property may aid in certain antibody in-

vestigations.

Reference

BranchDR,MuenschHA,SySiokHianS,PetzLD.

DisulfidebondsarearequirementforKelland

Cartwright (Yta) blood group antigen integrity. Br J

Haematol 1983;54:573-8.

Method 3.11. Urine

Neutralization of Anti-Sda

Principle

For a discussion of anti-Sdaneutralization

by urine, see Chapter 15.

Specimen

Serum or plasma suspected of containing

anti-Sda.

Reagents

1. Urine from a known Sd(a+) individ-

ual, or from a pool of at least six in-

dividuals of unknown Sdatype, pre-

pared as follows: Collect urine and

immediately boil it for 10 minutes.

Dialyze it against phosphate-buf-

fered saline (PBS), pH 7.3, at 4 C for

48 hours. Change PBS several times.

Centrifuge. Dispense supernatant into

aliquots, which can be stored at –20

Cuntilthawedforuse.

2. PBS, pH 7.3. See Method 1.7.

Procedure

1. Mix equal volumes of thawed urine

and test serum.

2. Prepare a dilution control tube con-

taining equal volumes of serum and

PBS.

3. Prepare a urine control tube by mix-

ing equal volumes of thawed urine

and PBS.

4. Incubate all tubes at room tempera-

ture for 30 minutes.

5. Mix 1 drop of each test red cell sam-

plewith4dropsfromeachofthe

Methods Section 3: Antibody Detection/Identification and Compatibility Testing 767

tubes: neutralized serum, serum with

PBS, and urine with PBS. Test each

one using standard procedures.

Interpretation

1. Persistent agglutination in the serum

sample incubated with urine means

either that partial or no neutraliza-

tion was achieved or that underlying

antibodies are present. Microscopic

examination may be helpful; aggluti-

nation due to anti-Sdahas a refractile,

mixed-field appearance on micro-

scopic examination.

2. No agglutination in the neutralized

tube with persistent agglutination in

the dilution control tube and absence

of hemolysis and agglutination in

the urine control tube indicate that

the antibody has been neutralized

and is quite probably anti-Sda.

3. The absence of agglutination in the

dilution control tube means that the

dilution in the neutralization step

was too great for the antibody pres-

ent and the results of the test are in-

valid. The urine control tube provides

assurance that no substances in the

urine are agglutinating or damaging

the red cells.

Note

Urine may also contain ABO and Lewis

blood group substances, depending upon

the ABO, Lewis, and secretor status of the

donor.

Reference

Judd WJ. Methods in immunohematology. 2nd ed.

Durham, NC: Montgomery Scientific Publications,

1994.

Method 3.12. Adsorption

Procedure

Principle

See Chapter 19.

Specimen

Serum or plasma containing antibody to

be adsorbed.

Reagents

Red cells (eg, autologous or allogeneic) that

carry the antigen corresponding to the an-

tibody specificity to be adsorbed.

Procedure

1. Wash the selected red cells at least

three times with saline.

2. After the last wash, centrifuge the red

cells at 800 to 1000 ×gfor at least 5

minutes and remove as much of the

supernatant saline as possible. Addi-

tional saline may be removed by

touching the red cell mass with a

narrow piece of filter paper.

3. Mix appropriate volumes of the pack-

ed red cells and serum and incubate

at the desired temperature for 30 to

60 minutes.

4. Mix the serum/cell mixture periodi-

cally throughout the incubation

phase.

5. Centrifuge the red cells at 800 to

1000 ×gfor 5 minutes to pack cells

tightly. Centrifuge at the incubation

temperature, if possible, to avoid

dissociation of antibody from the red

cell membranes.

6. Transfer the supernatant fluid, which

is the adsorbed serum, to a clean test

tube.Ifaneluateistobeprepared,

save the red cells.

7. Test an aliquot of the adsorbed se-

rum, preferably against an addi-

768 AABB Technical Manual

tional aliquot of the cells used for

adsorption, to see if all antibody has

been removed.

Interpretation

If reactivity remains, the antibody has not

been completely removed. No reactivity

signifies that antibody has been com-

pletely adsorbed.

Notes

1. Adsorption is more effective if the

area of contact between the red cells

and serum is large; use of a large-

bore test tube (13 mm or larger) is

recommended.

2. Multiple adsorptions may be neces-

sary to completely remove an anti-

body, but each successive adsorp-

tion increases the likelihood that the

serum will be diluted and un-

adsorbed antibodies weakened.

3. Repeat adsorptions should use a

fresh aliquot of cells and not the

cells from the prior adsorption.

4. Enzyme pretreatment of adsorbing

cells can be performed to increase an-

tibody uptake for enzyme-resistant

antigens.

Reference

Judd WJ. Methods in immunohematology. 2nd ed.

Durham, NC: Montgomery Scientific Publications,

1994.

Method 3.13. Using the

American Rare Donor

Program

Principle

The American Rare Donor Program (ARDP)

helps to locate blood products for pa-

tients requiring rare or unusual blood.

The ARDP maintains a database of rare

donors submitted by immunohematology

reference laboratories that are accredited

by the AABB or the American Red Cross

(ARC). Donors are considered rare due to

the absence of a high-incidence antigen,

absence of multiple common antigens, or

IgA deficiency.

All requests to the ARDP must originate

from an AABB- or ARC-accredited im-

munohematology reference laboratory to

ensure that the patient in question has

been accurately evaluated and reported. All

shipping and rare unit fees are established

by the shipping institution.

Procedure

1. A hospital blood bank, transfusion

service, or blood center identifies a

patient who needs rare blood.

2. The institution contacts the nearest

AABB- or ARC-accredited immuno-

hematology reference laboratory to

supply the needed blood.

3. If the laboratory cannot supply the

blood, it contacts the ARDP. All re-

quests to the ARDP must come from

an AABB- or ARC-accredited labora-

tory (or another rare donor program).

Requests received directly from a non-

accredited facility will be referred to

the nearest accredited institution.

4. The institution contacting the ARDP

(requesting institution) must confirm

the identity of the antibody(ies) by

serologic investigation or by exam-

ining the serologic work performed

by another institution.

5. ARDP staff search their database for

centers that have identified donors

with the needed phenotype and con-

tact the centers for availability of

units. ARDP staff give the name(s) of

the shipping center(s) to the request-

ing institution.

Methods Section 3: Antibody Detection/Identification and Compatibility Testing 769

6. The requesting and shipping institu-

tions should discuss and agree on

charges and testing requirements

before units are shipped.

7. If an initial search does not result in

a sufficient number of units, the fol-

lowing mechanisms can be used by

ARDP staff to obtain needed units: 1)

communication to all ARDP partici-

pating centers alerting them to search

their inventories and/or recruit do-

nors matching the needed pheno-

type, 2) contacting other rare donor

filessuchasthoseadministeredby

the World Health Organization, Jap-

anese Red Cross, etc.

770 AABB Technical Manual

Methods Section 4: Investigation of a Positive DAT

Methods Section 4

Investigation of a Positive

Direct Antiglobulin Test

Elution Techniques

The objective of all elution techniques is

to interfere with the noncovalent binding

forces that hold antibody-antigen complexes

together on the red cell surface. The cell

membrane can be physically disrupted by

heat, ultrasound, freezing and thawing,

detergents, or organic solvents. The bind-

ing forces of antigen-antibody complexes

can be interrupted by alterations in pH or

salt concentration. For a comparison of the

advantages and disadvantages of various

elution methods, see Chapter 20. Selected

elution methods follow, including one ex-

ample of an organic solvent method. The

cold-acid elution method (Method 4.1) is

the basis of the commercially available acid

elution kits commonly used in the United

States. Because no single elution method

will result in the identification of all

antibodies, use of an alternative elution

method (eg, organic solvent) may be indi-

cated when a nonreactive eluate is not in

agreement with clinical data. The reader

should refer to Chapter 2 for the proper

handling of hazardous chemicals that are

sometimes used in these techniques. Ac-

cess to a chemical fume hood is desirable

when organic solvents are in use.

Very thorough washing of the red cells

before elution is essential to ensure that an-

tibody in the eluate is only red cell-bound

and does not represent free antibody, eg,

from plasma. A control to show that all free

antibody has been removed by washing can

be obtained by saving the saline from the

last wash and testing it in parallel with the

eluate. Additionally, transferring the red

cells into a clean test tube just before the

elution step eliminates the possibility of

dissociating antibody that may have non-

specifically bound to the glass test tube

during an adsorption or the initial eluate

preparation steps.

771

Section 4

Method 4.1. Cold-Acid Elution

Principle

Dissociation of antibodies from red cells

enables the identification of auto- or allo-

antibodies. Elution methods used in con-

junction with adsorption techniques are

also useful in detecting weak antigen ex-

pression on the adsorbing cells and in

separating mixtures of antibodies against

red cell antigens.

Specimen

Red cells positive by the direct antiglobulin

test (DAT) washed six times with large

volumes of saline (save the last wash).

Reagents

1. Glycine-HCl (0.1 M, pH 3.0), prepared

by dissolving 3.75 g of glycine and

2.922 g of sodium chloride in 500 mL

of deionized or distilled water. Adjust

the pH to 3.0 with 12 N HCl. Store at

4C.

2. Phosphate buffer (0.8 M, pH 8.2),

prepared by dissolving 109.6 g of

Na2HPO4and 3.8 g of KH2PO4in ap-

proximately 600 mL of deionized or

distilled water and adjusting the fi-

nalvolumeto1L.AdjustthepH,if

necessary,witheither1NNaOHor1

N HCl. Store at 4 C (see note 2).

3. NaCl, 0.9%, at 4 C.

4. Supernatant saline from the final

wash of red cells to be tested.

Procedure

1. Place 1 mL of red cells in a 13 ×

100-mm test tube and chill in an ice

water bath for 5 minutes before add-

ing the glycine-HCl.

2. Add 1 mL of chilled saline and 2 mL

of chilled glycine-HCl to the red cells.

3. Mix and incubate the tube in an ice

water bath (0 C) for 1 minute.

4. Quickly centrifuge the tube at 900 to

1000 ×gfor 2 to 3 minutes.

5. Transfer the supernatant eluate into

a clean test tube and add 0.1 mL of

pH 8.2 phosphate buffer for each 1

mL of eluate (see note 3).

6. Mix and centrifuge at 900 to 1000 ×g

for 2 to 3 minutes.

7. Transfer the supernatant eluate into

a clean test tube and test it in paral-

lel with the supernatant saline from

the final wash.

Notes

1. Keep glycine-HCl in an ice bath dur-

ing use, to maintain the correct pH.

2. Phosphate buffer will crystallize dur-

ing storage at 4 C. Redissolve it at 37

Cbeforeuse.

3. The addition of phosphate buffer re-

stores neutrality to the acidic eluate.

Acidity may cause hemolysis of the

reagent red cells used in testing the

eluate. The addition of 22% bovine

albumin (one part to four parts of

eluate) may reduce such hemolysis.

References

1. Judd WJ. Methods in immunohematology. 2nd

ed. Durham, NC: Montgomery Scientific Pub-

lications, 1994.

2. Rekvig OP, Hannestad K. Acid elution of blood

group antibodies from intact erythrocytes.

Vox Sang 1977;33:280-5.

Method 4.2.

Glycine-HCl/EDTA Elution

Principle

See Method 4.1.

Specimen

Red cells positive by the direct antiglobulin

test (DAT) washed six times with large

volumes of saline (save the last wash).

772 AABB Technical Manual

Reagents

1. Disodium EDTA (10% w/v): Na2EDTA,

10 g; distilled water to 100 mL.

2. Glycine-HCl (0.1 M at pH 1.5): 0.75 g

glycine diluted to 100 mL with 0.9%

NaCl;adjusttopH1.5with12NHCl.

3. TRIS-NaCl (1 M): Tris(hydroxymethyl)

aminomethane [TRIS] or TRIZMA

BASE, 12.1 g; 5.25 g NaCl; distilled

water to 100 mL.

4. Supernatant saline from the final wash

of the red cells to be tested.

Procedure

1. In a test tube, mix together 20 vol-

umes (eg, drops) of 0.1 M glycine-

HCl buffer and 5 volumes of 10%

EDTA. This is the eluting solution.

2. In a 12 ×75-mm tube, place 10 vol-

umes of packed red cells.

3. Add 20 volumes of the eluting solu-

tion to the red cells, mix well, and in-

cubate at room temperature for 2

minutes. Do not overincubate.

4. Add 1 volume of TRIS-NaCl, mix,

and immediately centrifuge the tube

at 900 to 1000 ×gfor 60 seconds.

5. Transfer the supernatant eluate into

a clean test tube and adjust it care-

fully dropwise to pH 7.0 to 7.4 with

1 M TRIS-NaCl. The pH can be

checked with pH paper.

6. Centrifuge at 900 to 1000 ×gfor 2 to

3 minutes to remove the precipitate.

7. Transfer the supernatant eluate into

a clean test tube and test it in paral-

lel with the supernatant saline from

the final wash.

Notes

1. Once the red cells have been rendered

DAT negative, they may be tested for

the presence of blood group anti-

gens, except those in the Kell system.

Treatment with glycine-HCl/EDTA

denatures Kell system antigens and

Era. Wash the red cells at least three

timesinsalinebeforeuse.

2. Red cells modified with glycine-

HCl/EDTA may be treated with a

protease and used in autologous ad-

sorption studies.

3. Overincubation with the eluting so-

lution (step 3) will irreversibly dam-

age the red cells.

4. TRIS-NaCl is very alkaline and only a

few drops should be required to at-

tain the desired pH (step 5).

5. Aliquots of the reagents can be stored

frozen and one tube of each can be

thawed just before use. The 10% EDTA

may precipitate when stored at 2 to 8 C.

6. Stored eluate (4 C or frozen) may be

more stable if albumin is added (1

volume of 30% bovine albumin for

every 10 volumes of eluate). If albu-

min is added to the eluate, it should

be added to the last wash.

Reference

ByrnePC.Useofamodifiedacid/EDTAelution

technique. Immunohematology 1991;7:46-7. [Cor-

rection note: Immunohematology 1991;7:106.]

Method 4.3. Heat Elution

Principle

Heatelutionusesanincreaseintempera

ture to dissociate antibodies from red cells.

This method is best suited for the investi-

gation of ABO hemolytic disease of the fe-

tus and newborn and for the elution of IgM

antibodies from red cells. It should not

routinely be used for the investigation of

abnormalities caused by IgG auto- or allo-

antibodies.

Specimen

Red cells positive by the direct antiglobulin

test (DAT) washed six times with large

Methods Section 4: Investigation of a Positive DAT 773

volumes of saline; save the last wash (see

note).

Reagents

1. 6% bovine albumin (see Method 1.5).

2. Supernatant saline from the final

wash of the red cells to be tested.

Procedure

1. Mix equal volumes of washed packed

cells and 6% bovine albumin in a

13 ×100-mm test tube.

2. Place the tube at 56 C for 10 min-

utes. Agitate the tube periodically

during this time.

3. Centrifuge the tube at 900 to 1000 ×

gfor 2 to 3 minutes, preferably in a

heated centrifuge.

4. Immediately transfer the supernatant

eluate into a clean test tube and test

in parallel with the supernatant sa-

line from the final wash.

Note

For optimal recovery of cold-reactive anti-

bodies, the red cells should be washed in

ice-cold saline to prevent dissociation of

bound antibody before elution.

References

1. Judd WJ. Methods in immunohematology. 2nd

ed. Durham, NC: Montgomery Scientific Pub-

lications, 1994.

2. Landsteiner K, Miller CPJr.Serologicalstud

ies on the blood of primates. II. The blood

groups in anthropoid apes. J Exp Med 1925;

42:853-62.

Method 4.4. Lui

Freeze-Thaw Elution

Principle

As red cells freeze, extracellular ice crys-

tals form that attract water from their sur-

roundings. This increases the osmolarity

of the remaining extracellular fluid, which

then extracts water from the red cells. The

red cells shrink, resulting in lysis. As the

membranes are disrupted, antibody is

dissociated. This method is used primar-

ily for the investigation of ABO hemolytic

disease of the fetus and newborn.

Specimen

1. Red cells washed six times with large

volumes of saline.

2. Supernatant saline from the final

wash of the red cells to be tested.

Procedure

1. Mix 0.5 mL of the red cells to be tested

with 3 drops of saline in a test tube.

2. Cap the tube, then rotate the tube to

coat the tube wall with cells.

3. Place the tube in a horizontal posi-

tioninafreezerat–6Cto–70Cfor

10 minutes.

4. Removethetubefromthefreezerand

thaw it quickly with warm, running

tap water.

5. Centrifuge for 2 minutes at 900 to

1000 ×g.

6. Transfer the supernatant to a clean

test tube and test it in parallel with

the supernatant saline from the final

wash.

References

1. Judd WJ. Methods in immunohematology. 2nd

ed. Durham, NC: Montgomery Scientific Pub-

lications, 1994.

2. FengCS,KirkleyKC,EicherCA,etal.TheLui

elution technique: A simple and efficient

method for eluting ABO antibodies. Transfu-

sion 1985;25:433-4.

774 AABB Technical Manual

Method 4.5. Methylene

Chloride Elution

Principle

Organic solvents can influence antigen-

antibody dissociation by several mecha-

nisms, including alteration of the tertiary

structure of antibody molecules and dis-

ruption of the red cell membrane. This

method is suitable for elution of IgG auto-

and alloantibodies.

Specimen

DAT-positive red cells washed six times with

large volumes of saline (save last wash).

Reagents

1. Methylene chloride (dichloromethane).

2. Supernatant saline from final wash

of the red cells to be tested.

Procedure

1. Mix 1 mL of red cells, 1 mL of saline,

and 2 mL of methylene chloride in a

test tube, eg, 13 ×100 mm.

2. Stopper the tube and mix by gentle

agitation for 1 minute.

3. Remove the stopper and centrifuge

the tube at 1000 ×gfor 10 minutes.

4. Remove the lower layer of methylene

chloride with a transfer pipette and

discard it.

5. Place the tube at 56 C for 10 min-

utes. Stir the eluate constantly with

wooden applicator sticks in the first

several minutes to avoid it boiling

over; thereafter, stir it periodically.

6. Centrifuge at 1000 ×gfor 10 min-

utes.

7. Transfer the supernatant eluate into

a clean test tube and test it in paral-

lel with the supernatant saline from

the final wash.

Reference

Judd WJ. Methods in immunohematology. 2nd ed.

Durham, NC: Montgomery Scientific Publications,

1994.

Immune Hemolytic Anemia

Serum/Plasma Methods

Included in this section are methods used

to remove warm or cold autoantibody re-

activity (eg, adsorptions) so that alloanti-

body detection tests and diagnostic tests

for differentiating the immune hemolytic

anemias can be performed. See Chapter

20 for a discussion of the immune hemoly-

tic anemias.

Method 4.6. Cold

Autoadsorption

Principle

Although most cold autoantibodies do not

cause a problem in serologic tests, some

potent cold-reactive autoantibodies may

mask the concomitant presence of clini-

cally significant alloantibodies. In these

cases, adsorbing the serum in the cold

with autologous red cells can remove the

autoantibody, permitting detection of un-

derlying alloantibodies. In the case of most

nonpathologic cold autoantibodies, a

simple quick adsorption of the patient’s

serum with enzyme-treated autologous

red cells will remove most cold antibody.

See Method 3.5.6. A more efficient method

of removing immunoglobulins is the use

of ZZAP reagent, a combination of pro-

teolytic enzyme and a powerful reducing

agent. ZZAP treatment removes IgM and

complement from autologous red cells

and uncovers antigen sites that can be

used to bind free autoantibody in the se-

rum.

Methods Section 4: Investigation of a Positive DAT 775

Specimen

1. 1 mL of serum or plasma to be ad-

sorbed.

2. Two 1-mL aliquots of autologous red

cells.

Reagents

1. 1% cysteine-activated papain or 1%

ficin (see Methods 3.5.1 and 3.5.2).

2. Phosphate-buffered saline (PBS), pH

7.3 (see Method 1.7).

3. 0.2 M dithiothreitol (DTT) prepared

by dissolving 1 g of DTT in 32.4 mL

of pH 7.3 PBS. Dispense into 3-mL

aliquots and store at –18 C or colder.

Procedure

1. Prepare ZZAP reagent by mixing 0.5

mL of 1% cysteine-activated papain

with2.5mLof0.2MDTTand2mL

of pH 7.3 PBS. Alternatively, use 1

mL of 1% ficin, 2.5 mL of 0.2 M DTT,

and 1.5 mL of pH 7.3 PBS. The pH

should be between 6.0 and 6.5.

2. Add 2 mL of ZZAP reagent to 1 mL of

autologous red cells. Mix and incu-

bate at 37 C for 30 minutes.

3. Wash the cells three times in saline.

Centrifuge the last wash for at least 5

minutes at 900 to 1000 ×gand re-

move as much of the supernatant sa-

lineaspossible(seenote1).

4. To the tube of ZZAP-treated red cells,

add 1 mL of the autologous serum.

Mixandincubateat4Cfor30min

utes.

5. Centrifuge at 900 to 1000 ×gfor 4 to

5 minutes and transfer the serum into

acleantube.

6. Steps 2 through 5 may be repeated if

the first autoadsorption does not sat-

isfactorily remove the autoantibody

activity.

7. After the final adsorption, test the

serum with reagent red cells for allo-

antibody activity.

Notes

1. To avoid dilution of the serum and

possible loss of weak alloantibody

activity, it is important in step 3 to

remove as much of the residual sa-

line as possible.

2. If the reactivity of the autoantibody

is not diminished, the target auto-

antigen may have been destroyed by

either the enzyme or the DTT. The

adsorption should be repeated against

untreated autologous red cells washed

several times in warm saline.

References

1. Branch DR. Blood transfusion in autoimmune

hemolytic anemias. Lab Med 1984;15:402-8.

2. Branch DR, Petz LD. A new reagent (ZZAP)

having multiple applications in immuno-

hematology. Am J Clin Pathol 1982;78:161-7.

Method 4.7. Determining the

Specificity of Cold-Reactive

Autoagglutinins

Principle

For a discussion of specificity of cold-re-

acting autoantibodies, see Chapter 20.

Specimen

1. Serum, separated at 37 C from a blood

sample allowed to clot at 37 C, or

plasma, separated from an anti-

coagulated sample after periodic in-

version at 37 C for approximately 15

minutes.

2. Autologous red cells.

Reagents

Test red cells of the following phenotypes:

776 AABB Technical Manual

1. A pool of two examples of adult

group O I adult red cells; they can be

the reagent cells routinely used for

alloantibody detection.

2. Group O i cord red cells.

3. The patient’s own (autologous) red

cells, washed at least three times

with 37 C saline.

4. Red cells of the same ABO group as

the patient, if the patient is not

group O. If the patient is group A or

AB, use both A1and A2cells.

5. Saline or phosphate-buffered saline

(PBS), pH 7.3 (see Method 1.7).

Procedure

1. Prepare serial twofold dilutions of

the serum or plasma in saline or

PBS. The dilution range should be

from 1 in 2 to 1 in 4096 (12 tubes),

andthevolumespreparedshouldbe

more than the total volume needed

to test all of the desired red cells. See

Method 3.7.

2. Label a set of 12 tubes with the dilu-

tion (eg, 2, 4, 8, etc) for each of the

red cells to be tested (eg, adult, cord,

autologous).

3. Dispense 2 drops of each dilution

into the appropriate tubes.

4. Add 1 drop of a 3% to 5% saline sus-

pension of each red cell sample to

the appropriate set of tubes.

5. Mix and incubate at room tempera-

turefor30to60minutes.

6. Centrifuge for 15 to 20 seconds at

900 to 1000 ×g. Examine the tubes

one by one macroscopically for agglu-

tination, starting with the set of tubes

at the highest dilution for each cell

tested (ie, read all the tubes for each

dilution as a set). Grade and record

the results.

7. Transfer the tubes to 4 C and incu-

bate them at this temperature for 1

to 2 hours.

8. Centrifuge for 15 to 20 seconds at

900 to 1000 ×g. Immediately place

thetubesinarackinanicewater

bath. Examine the tubes as in step 6.

Grade and record the results.

Interpretation

Table4.7-1summarizesthereactionsof

the commonly encountered cold-reactive

autoantibodies. In cold agglutinin syn-

Methods Section 4: Investigation of a Positive DAT 777

Table 4.7-1. Typical Relative Reactivity Patterns of Cold Autoantibodies

Red Cells

Antibody Specificity

Anti-I Anti-i Anti-ITAnti-IH Anti-Pr

O I adult + 0/↓0/↓++

Oicord 0/↓++↓+

O i adult 0/↓+0/↓↓ +

A1I adult + 0/↓0/↓↓ +

Autologous + 0/↓0/↓↓ +

O I enzyme-treated ↑↑↑↑0

+ = reactive; 0 = nonreactive; ↓= weaker reaction; ↑= stronger reaction.

drome, anti-I is seen most frequently, but

anti-i may also be encountered. When

cord cells react stronger than adult cells,

the specificity may be anti-i, but adult i

red cells need to be tested to confirm that

these reactions are due to anti-i and not

anti-IT. Some examples of anti-I react

more strongly with red cells that have a

strong expression of H antigen (eg, O and

A2cells); such antibodies are called anti-

IH. Rarely, the specificity may be anti-Pr,

which should be suspected if all the cells

tested react equally. Anti-Pr can be con-

firmed by testing enzyme-treated cells;

anti-Pr does not react with enzyme-

treated cells, whereas anti-I and anti-i

react better with enzyme-treated cells.

Anti-Pr reacts equally with untreated red

cells of I or i phenotypes.

Notes

1. It is important to use separate pi-

pettes or pipette tips for each tube

when preparing serum dilutions be-

cause the serum carried from one

tube to the next when a single pi-

pette is used throughout may cause

falsely high titration endpoints. The

difference can convert a true titer of

4000 to an apparent titer of 100,000,

when the use of separate pipettes is

compared with the use of a single pi-

pette.

2. Serum dilutions can be prepared

more accurately with large volumes

(eg, 0.5 mL) than with small vol-

umes.

3. Potent examples of cold-reactive

autoantibodies generally do not

show apparent specificity until titra-

tion studies are performed; this

specificity may not even be apparent

with dilutions at room temperature

or 4 C. In such circumstances, tests

can be incubated at 30 to 37 C. Dif-

ferential reactivity may be more

apparent if incubation times are

prolonged and agglutination is eval-

uated after settling, without centrifu-

gation. Settled readings are more ac-

curate after a 2-hour incubation.

4. This procedure can be used to deter-

mine both the titer and the specific-

ity. If incubations are started at 37 C

(set up prewarmed, and readings are

taken sequentially after incubation

at each temperature—eg, 37 C, 30 C,

room temperature, 4 C), the specific-

ity, titer, and thermal amplitude of

the autoantibody can be determined

with a single set of serum dilutions.

5. If testing will also be performed at

30 C and 37 C, include a test of the

neat (undiluted) serum.

Reference

Petz LD, Garratty G. Immune hemolytic anemias.

2nd ed. Philadelphia: Churchill Livingstone, 2004.

Method 4.8. Cold Agglutinin

Titer

Principle

Cold-reactive autoantibodies, if present at

very high titers, may suggest a pathologic

cold agglutinin disease. This may result in

overt hemolysis and systemic symptoms

and may indicate underlying B-cell hema-

tologic neoplasia.

Specimen

Serum, separated at 37 C from a sample

allowed to clot at 37 C, or plasma, sepa-

rated from an anticoagulated sample after

periodic inversion at 37 C for approxima-

tely 15 minutes.

778 AABB Technical Manual

Reagents

1. A pool of 2 examples of washed group

O I adult red cells, eg, antibody de-

tection cells.

2. Phosphate-buffered saline (PBS), pH

7.3 (see Method 1.7).

Procedure

1. Prepare serial twofold dilutions of the

patient’s serum or plasma in PBS.

Thedilutionrangeshouldbefrom1

in 2 to 1 in 4096 (12 tubes). See

Method 3.7.

2. Mix 2 drops of each dilution with 1

dropofa3%to5%cellsuspensionof

red cells.

3. Mix and incubate at 4 C for 1 to 2

hours.

4. Centrifuge the tubes for 15 to 20 sec-

onds at 900 to 1000 ×g,thenplace

thetubesinarackinanicewater

bath. Examine the tubes one by one

macroscopically for agglutination,

starting with the tube at the highest

dilution. Grade and record the re-

sults.

Interpretation

Thetiteristhereciprocalofthehighest

serum dilution at which macroscopic ag-

glutination is observed. Titers above 64

are considered elevated, but hemolytic

anemia resulting from cold-reactive auto-

agglutinins rarely occurs unless the titer is

>1000. Titers below 1000 may be obtained

when the autoantibody has a different

specificity (eg, anti-i), or if the cold agglu-

tinin is of the less common low-titer,

high-thermal-amplitude type. If the pa-

tient has a positive direct antiglobulin test

(DAT) because of complement only and

has clinical signs of hemolytic anemia,

specificity and thermal amplitude studies

should be performed (see Method 4.7).

Notes

1. It is important to use separate pi-

pettes for each tube when preparing

serum dilutions because the serum

carried from one tube to the next when

a single pipette is used throughout

may cause falsely high titration end-

points.

2. Serum dilutions can be prepared

more accurately with large volumes

(eg, 0.5 mL) than with small vol-

umes.

Reference

Petz LD, Garratty G. Immune hemolytic anemias.

2nd ed. Philadelphia: Churchill Livingstone, 2004.

Method 4.9. Autologous

Adsorption of

Warm-Reactive

Autoantibodies

Principle

Warm-reactive autoantibodies in serum

maymasktheconcomitantpresenceof

clinically significant alloantibodies. Ad-

sorption of the serum with autologous red

cells can remove autoantibody from the

serum, permitting detection of underly-

ing alloantibodies. However, autologous

red cells in the circulation are coated with

autoantibody. Autologous adsorption of

warm-reactive autoantibodies can be fa-

cilitated by dissociating autoantibody from

the red cell membrane, thereby uncover-

ing antigen sites that can bind free auto-

antibody to remove it from the serum.

Some autoantibody can be dissociated by

a gentle heat elution for 3 to 5 minutes at

56 C. Subsequent treatment of the cells

withenzymesenhancestheadsorption

process by removing membrane struc-

tures that otherwise hinder the associa-

Methods Section 4: Investigation of a Positive DAT 779

tion between antigen and antibody. The

most effective procedure involves the use

of ZZAP reagent, a mixture of a proteolytic

enzyme and a sulfhydryl reagent. ZZAP

removes immunoglobulins and comple-

ment from the red cells and enhances the

adsorption process. Red cells from pa-

tients transfused within the last 3 months

should not be used for autoadsorption

because transfused red cells present in

the circulation are likely to adsorb the

alloantibodies that are being sought (see

Chapter 20).

Specimen

1. 1 mL of serum or plasma (or eluate)

to be adsorbed.

2. 2 mL of autologous red cells.

Reagents

1. 1% cysteine-activated papain or 1%

ficin (see Methods 3.5.1 and 3.5.2).

2. Phosphate-buffered saline (PBS), pH

7.3 (see Method 1.7).

3. 0.2 M DTT prepared by dissolving 1

gofDTTin32.4mLofpH7.3PBS.

Dispense into 3-mL aliquots and

store at –18 C or colder.

Procedure

1. Prepare ZZAP reagent by mixing 0.5

mL of 1% cysteine-activated papain

with2.5mLof0.2MDTTand2mL

of pH 7.3 PBS. Alternatively, use 1

mL of 1% ficin, 2.5 mL of 0.2 M DTT,

and 1.5 mL of pH 7.3 PBS. The pH

should be between 6.0 and 6.5.

2. To each of two tubes containing 1

mL of red cells, add 2 mL of ZZAP re-

agent. Mix and incubate at 37 C for

30 minutes with periodic mixing.

3. Wash the red cells three times in sa-

line. Centrifuge the last wash for at

least 5 minutes at 900 to 1000 ×g

and remove as much supernatant

saline as possible.

4. Add serum to an equal volume of

ZZAP-treated red cells, mix, and in-

cubate at 37 C for approximately 30

to 45 minutes.

5. Centrifuge and carefully remove serum.

6. If the original serum reactivity was

only 1+, proceed to step 7; otherwise,

repeat steps 4 and 5 once more us-

ing the once-adsorbed patient’s se-

rum and the second aliquot of ZZAP-

treated cells.

7. Test the serum against a specimen of

group O reagent cells. If reactivity

persists, repeat steps 4 and 5.

Interpretation

One or two adsorptions ordinarily remove

sufficient autoantibody so that alloanti-

body reactivity, if present, is readily ap-

parent. If the twice-autoadsorbed serum

reacts with defined specificity, as shown

by testing against a small antibody identi-

fication panel, then the defined specificity

of the antibody is probably an alloantibody.

If the serum reacts with all cells on the

panel, either additional autoadsorptions

are necessary, the serum contains anti-

body to a high-incidence antigen, or the

serum contains an autoantibody (eg,

anti-Kpb) that does not react with ZZAP-

treated cells and thus will not be adsorbed

by this procedure. To check this latter

possibility, test the reactive autoadsorbed

serum against reagent cells that have

been pretreated with the ZZAP reagent.

Notes

1. ZZAP treatment destroys all Kell sys-

tem antigens and all other antigens

that are destroyed by proteases, eg,

M, N, Fya,andFy

b.ZZAPreagentalso

denatures the antigens of the LW,

Cartwright, Dombrock, and Knops

780 AABB Technical Manual

systems. If the autoantibody is sus-

pected to have specificity in any of

these latter blood groups, an alter-

native procedure is to perform auto-

adsorption with untreated autologous

cells or autologous cells treated only

with 1% ficin or 1% cysteine-activated

papain.

2. There is no need to wash packed red

cells before treatment with ZZAP.

3. Cold autoantibodies reactive at room

temperature can also be present in

the serum of about 30% of patients

with warm-reactive autoantibodies.

Removal of these cold antibodies can

be facilitated by placing the serum

and cell mixture at 4 C for about 15

minutes after incubation at 37 C.

4. As a guide, when the original serum

reactivity is 1+ in the low-ionic-

strength saline indirect antiglobulin

test (LISS-IAT), usually only one ad-

sorption would be required. Antibod-

ies with 2+ to 3+ reactivity will gen-

erally be removed in two to three

adsorptions. Performing greater

than four adsorptions increases the

risk of diluting alloantibody reactiv-

ity.

Reference

Branch DR, Petz LD. A new reagent (ZZAP) having

multiple applications in immunohematology. Am

J Clin Pathol 1982;78:161-7.

Method 4.10. Differential

Warm Adsorption Using

Enzyme- or ZZAP-Treated

Allogeneic Red Cells

Principle

Adsorption of serum with selected red cells

of known phenotypes will remove auto-

antibody and leave antibodies to most

blood group systems. The specificity of the

antibodies that remain after adsorption

can be confirmed by testing against a

panel of reagent red cells. This procedure

can be used to detect underlying alloanti-

bodies if the patient has been recently

transfused, or if insufficient autologous

red cells are available and the patient’s

phenotype is unknown.

Treating the adsorbing cells with enzyme

or ZZAP typically enhances the adsorption

process. In addition, the treated red cells will

lack the antigens destroyed by dithiothrei-

tol (DTT) and/or enzymes (see Chapter 19).

Specimen

Serum/plasma containing warm-reactive

autoantibodies or eluate from direct anti-

globulin test (DAT)-positive cells.

Reagents

1. 1% cysteine-activated papain or 1%

ficin (see Methods 3.5.1 and 3.5.2).

2. ZZAP reagent (papain or ficin plus

0.2 M DTT). See Method 4.9.

3. Phosphate-buffered saline (PBS), pH

7.3 (see Method 1.7).

4. Group O red cells of the phenotypes

R1R1,R

2R2, and rr; one of these cells

should be Jk(a–b+) and one should

be Jk(a+b–). Additionally, if the red

cells are to be only enzyme-treated,

at least one of the cells should also

be K–. They can be reagent cells or

from any blood specimen that will

yield a sufficient volume of red cells.

Procedure

1. Wash 1 mL of each red cell specimen

once in a large volume of saline,

centrifuge to pack the cells, and re-

move the supernatant saline.

2. To each volume of washed packed

cells,addonevolumeof1%enzyme

solution or two volumes of working

Methods Section 4: Investigation of a Positive DAT 781

ZZAP reagent. Invert several times to

mix them.

3. Incubate at 37 C: 15 minutes for en-

zyme or 30 minutes for ZZAP. Mix

periodically throughout incubation.

4. Wash the red cells three times with

large volumes of saline. Centrifuge

at 900 to 1000 ×gfor at least 5 min-

utes and remove the last wash as

completely as possible to prevent di-

lution of the serum.

5. For each of the three red cell speci-

mens, mix one volume of treated

cells with an equal volume of the pa-

tient’s serum and incubate at 37 C

for 30 minutes, mixing occasionally.

6. Centrifuge at 900 to 1000 ×gfor ap-

proximately 5 minutes and harvest

the supernatant serum.

7. Test the three samples of adsorbed

serum against the cells (untreated)

used for adsorption, respectively. If

reactivity is present, repeat steps 5

through 7 with a fresh aliquot of treat-

ed red cells until no reactivity re-

mains. The three samples of adsorbed

serum can then be tested against an-

tibody detection/panel cells and the

results compared for demonstration

of persisting and removed alloanti-

body activity. See section on allo-

geneic adsorption in Chapter 20.

Notes

1. If the autoantibody is very strong,

three or more aliquots of adsorbing

cells should be prepared. If the first

adsorption is unsuccessful, the use

of a higher proportion of cells to se-

rum/eluate may enhance effective-

ness.

2. The adsorbing red cells should be

tightly packed to remove residual sa-

line that might dilute the antibodies

remaining in the serum/eluate.

3. Agitate the serum/cell mixture dur-

ing incubation to provide maximum

surface contact.

4. A visible clue to the effectiveness of

adsorption is clumping of the en-

zyme- or ZZAP-treated cells when they

are mixed with the serum, especially

when strong antibodies are present.

5. As a guide, when the original serum

reactivity is 1+ in the low-ionic-

strength saline indirect antiglobulin

test (LISS-IAT), usually only one ad-

sorption would be required. Anti-

bodies with 2+ to 3+ reactivity will

generally be removed in two to three

adsorptions. Performing greater than

four adsorptions increases the risk of

diluting alloantibody reactivity.

6. If adsorption with enzyme- or ZZAP-

treated cells has no effect on the

autoantibody, adsorption with un-

treated red cells may be tried.

References

1. Branch DR, Petz LD. A new reagent (ZZAP)

having multiple applications in immuno-

hematology. Am J Clin Pathol 1982;78:161-7.

2. Judd WJ. Methods in immunohematology.

2nd ed. Durham, NC: Montgomery Scientific

Publications, 1994.

Method 4.11. One-Cell

Sample Enzyme or ZZAP

Allogeneic Adsorption

Principle

If the Rh and Kidd phenotypes of a re-

cently transfused patient are known or can

be determined, autoantibody activity can

be adsorbed from the serum onto a single

allogeneic red cell sample, leaving serum

that can be evaluated for the presence of

alloantibodies. The red cells used should

have the same Rh and Kidd phenotypes as

the patient; they can be treated with en-

782 AABB Technical Manual

zyme or ZZAP to denature antigens (see

Chapter 19). This method is a simplified

version of the previous adsorption proce-

dure, but it should be used only if the pa-

tient’s Rh and Kidd phenotypes are known

(see the note).

Specimen

Serum, plasma, or eluate to be tested.

Reagents

1. 1% cysteine-activated papain or 1%

ficin (see Methods 3.5.1 and 3.5.2).

2. ZZAP reagent (papain or ficin plus

0.2 M DTT). See Method 4.9.

3. ABO-compatible red cells of the pa-

tient’s Rh and Kidd phenotypes; they

can be reagent cells or cells from any

blood specimen that will yield suffi-

cient cells.

Procedure

1. Wash the selected allogeneic red cells

once in a large volume of saline and

centrifuge to pack them.

2. Add one volume of 1% enzyme solu-

tion or two volumes of ZZAP reagent

to one volume of these packed cells.

Invert several times to mix them.

3. Incubate at 37 C: 15 minutes for en-

zyme or 30 minutes for ZZAP. Mix

periodically throughout incubation.

4. Wash the cells three times with sa-

line. Centrifuge at 900 to 1000 ×gfor

at least 5 minutes and remove the

last wash as completely as possible

to prevent dilution of the serum.

5. To one volume of treated cells, add

an equal volume of the patient’s se-

rum, mix, and incubate at 37 C for 30

minutes, mixing occasionally.

6. Centrifuge at 900 to 1000 ×gfor ap-

proximately 5 minutes and harvest

the supernatant serum.

7. Test the adsorbed serum against the

cells (untreated) used for adsorption.

If reactivity persists, repeat steps 5

through 7 with a fresh aliquot of

treated cells until the serum is no

longer reactive.

Note

The s antigen may not be denatured by a

particular enzyme or ZZAP solution. The s

antigen status of the adsorbing red cells

may need to be considered.

Method 4.12. Polyethylene

Glycol Adsorption

Principle

Polyethylene glycol (PEG) enhances the

adsorption of antibody by untreated red

cells. Testing the adsorbed aliquot against

a panel of red cells can identify the speci-

ficity of antibodies that remain after ad-

sorption. This method can be used for both

autologous and allogeneic adsorption.

Specimen

Serum or plasma to be tested.

Reagents

1. PEG, 20% (20 g PEG, 3350 MW, in

100 mL of PBS, pH 7.3) or commer-

cial PEG enhancement reagent.

2. Autologous red cells or ABO-com-

patible allogeneic red cells of known

phenotype.

Procedure

1. Wash aliquots of red cells in large

volumes of saline three times and

centrifuge for 5 to 10 minutes at

1000 ×g. Remove all residual saline.

2. To 1 volume (eg, 1 mL) of red cells,

add 1 volume of serum and 1 vol-

Methods Section 4: Investigation of a Positive DAT 783

ume of PEG. Mix well and incubate

at 37 C for 15 minutes.

3. Centrifuge the serum/PEG/cell mix-

ture for 5 minutes and harvest the

adsorbed serum/PEG mixture.

4. To test the adsorbed serum, add 4

drops of serum to 1 drop of test red

cells, incubate for 15 minutes at 37

C, and proceed to the antiglobulin

test with anti-IgG. The larger volume

of serum tested (4 drops) is required

to account for the dilution of the se-

rum by the PEG. See the notes.

5. To check for completeness of ad-

sorption, test the adsorbed serum

against the red cells used for the ad-

sorption. If positive, repeat the ad-

sorption by adding the adsorbed se-

rum to a fresh aliquot of red cells but

do not add additional PEG. If the test

was negative, test the adsorbed se-

rum with a panel of cells.

Notes

1. Red cells for adsorption may be chem-

ically modified (eg, with enzymes or

ZZAP) before adsorption if denatur-

ation of antigens is desired.

2. The adsorbing cells should be thor-

oughly packed to remove any resid-

ual saline that could result in dilu-

tion of the antibodies remaining in

the serum.

3. Test the adsorbed serum on the day

it was adsorbed. Weak antibody re-

activity may be lost upon storage of

PEG-adsorbed sera, possibly due to

precipitation of the protein noticeable

after 4 C storage.

4. Although many laboratories suc-

cessfully use the PEG adsorption

method, some serologists have re-

ported a weakening or loss of anti-

body reactivity in some samples

when compared with results ob-

tained using a different technique.

To accommodate this potential

weakening of antibody reactivity,

some serologists test 6 drops of the

PEG-adsorbed serum.

5. Agglutination of the adsorbing red

cells does not occur when PEG is

used; therefore, there is no visible

clue to the efficiency of the adsorp-

tion process. As a guide, when the

original serum reactivity is 1+ in

low-ionic-strength saline indirect

antiglobulin test (LISS-IAT), usually

only one adsorption would be re-

quired. Antibodies with 2 to 3+ reac-

tivity will generally be removed in

two adsorptions.

References

1. Leger RM, Garratty G. Evaluation of methods

for detecting alloantibodies underlying warm

autoantibodies. Transfusion 1999;39:11-6.

2. Leger RM, Ciesielski D, Garratty G. Effect of

storage on antibody reactivity after adsorp-

tion in the presence of polyethylene glycol.

Transfusion 1999;39:1272-3.

Method 4.13. The

Donath-Landsteiner Test

Principle

IgG autoantibodies that cause paroxysmal

cold hemoglobinuria (PCH) act as bi-

phasic hemolysins in vitro. The IgG auto-

antibodies bind to the red cells at cold

temperatures, and, as the test is warmed

to 37 C, complement is activated and lysis

of the red cells occurs. The patient for whom

this procedure should be considered is

one with a positive direct antiglobulin test

(DAT) resulting from C3; demonstrable

hemoglobinemia, hemoglobinuria, or

both; and no evidence of autoantibody

activity in the serum or the eluate made

from the DAT-positive cells. For a discus-

sion of PCH, see Chapter 20.

784 AABB Technical Manual

Specimen

Serum separated from a freshly collected

blood sample maintained at 37 C.

Reagents

1. Freshly collected pooled normal sera

known to lack unexpected antibod-

ies, to use as a source of complement.

2. 50% suspension of washed group O

red cells that express the P antigen,

eg, antibody detection cells.

Procedure

1. Label three sets of three 10 ×75-mm

test tubes as follows: A1-A2-A3;

B1-B2-B3; C1-C2-C3.

2. To tubes 1 and 2 of each set, add 10

volumes (eg, drops) of the patient’s

serum.

3. To tubes 2 and 3 of each set, add 10

volumes of fresh normal serum.

4. To all tubes, add one volume of the

50% suspension of washed P-posi-

tive red cells and mix well.

5. Place the three “A” tubes in a bath of

melting ice for 30 minutes and then

at 37 C for 1 hour.

6. Place the three “B” tubes in a bath of

melting ice and keep them in melt-

ing ice for 90 minutes.

7. Placethethree“C”tubesat37Cand

keep them at 37 C for 90 minutes.

8. Centrifuge all tubes and examine the

supernatant fluid for hemolysis.

Interpretation

The Donath-Landsteiner test is considered

positive when the patient’s serum, with or

without added complement, causes

hemolysis in the tubes that were incu-

bated first in melting ice and then at 37 C

(ie, tubes A1 and A2), and there is no

hemolysis in any of the tubes maintained

throughout at 37 C (ie, tubes C1, C2) or in

melting ice (ie, tubes B1, B2). The A3, B3,

and C3 tubes serve as a control of the nor-

mal sera complement source and should

notmanifesthemolysis.

Notes

1. Thebiphasicnatureofthehemoly

sin associated with PCH requires

that serum be incubated with cells at

a cold temperature first (eg, melting

ice bath) and then at 37 C.

2. Active complement is essential for

demonstration of the antibody. Be-

cause patients with PCH may have

low levels of serum complement,

fresh normal serum should be in-

cluded in the reaction medium as a

source of complement.

3. To avoid loss of antibody by cold

autoadsorption before testing, the

patient’s blood should be allowed to

clot at 37 C, and the serum sepa-

rated from the clot at this tempera-

ture.

4. If a limited amount of blood is avail-

able (eg, from young children), set

up tubes A-1, A-2, A-3 and C-1, C-2;

if there is only enough serum for two

tests (ie, 20 drops), set up tubes A-2,

A-3, and C-2.

5. To demonstrate the P specificity of

the Donath-Landsteiner antibody,

ABO-compatible p red cells should

be tested in a second set of tubes

A-1, A-2, and A-3. No lysis should

develop in these tubes, confirming

the P specificity of the antibody.

References

1. Judd WJ. Methods in immunohematology.

2nd ed. Durham, NC: Montgomery Scientific

Publications, 1994.

2. Dacie JV, Lewis SM. Practical hematology. 7th

ed. New York: Churchill Livingstone, 1991:

500-1.

Methods Section 4: Investigation of a Positive DAT 785

Method 4.14. Detection of

Antibodies to Penicillin or

Cephalosporins by Testing

Drug-Treated Red Cells

Principle

See Chapter 20 for a discussion of the

mechanisms by which drugs cause a posi-

tive direct antiglobulin test (DAT). The

preparations of drugs used should, to the

extent possible, be the same as those

given to the patient. For drugs other than

penicillin and the cephalosporins, refer to

published reports for the method used to

treat the red cells.

Specimen

Serum or plasma and eluate (and last

wash) to be studied.

Reagents

1. 0.1 M sodium barbital-buffer (BB) at

pH 9.6 to 9.8, prepared by dissolving

2.06 g of sodium barbital in 80 mL of

distilled or deionized H20. Adjust the

pH to between 9.6 and 9.8 with 0.1 N

HCl. Bring total volume to 100 mL.

Store at 2 to 8 C.

2. Phosphate-buffered saline (PBS), pH

7.3 (see Method 1.7).

3. Drug, eg, penicillin, cephalosporin.

4. Washed, packed, group O red cells.

5. Normal sera/plasma.

6. IgG-coated red cells.

Procedure

1. For penicillin-treated cells, dissolve

600 mg of penicillin in 15 mL of BB.

This high pH is optimal, but if the

buffer is unavailable, PBS, pH 7.3,

can be used. Add 1 mL of red cells. In

a separate tube, prepare control cells

by adding 1 mL of untreated red

cells (without the drug) to 15 mL of

the same buffer. Incubate both tubes

for 1 hour at room temperature with

occasional mixing. Wash three times

in saline and store in PBS at 2 to 8 C

for up to 1 week. See note 1.

2. For cephalosporin-treated cells, dis-

solve400mgofthedrugin10mLof

PBS, pH 7.3. Add 1 mL of red cells. In

a separate tube, prepare control cells

by adding 1 mL of untreated red cells

(without the drug) to 10 mL of PBS.

Incubate both tubes for 1 hour at 37

C with occasional mixing. Wash three

timesinsalineandstoreinPBSfor

up to 1 week at 2 to 8 C. See notes 1

and 2.

3. Mix 2 or 3 drops of each specimen

(serum, eluate, and last wash) and

controls with 1 drop of 5% saline

suspension of drug-treated red cells.

4. In parallel, test each specimen and

control with the untreated red cells.

See notes 3 and 4.

5. Incubate the tests at 37 C for 60 min-

utes. Centrifuge and examine for

hemolysis and agglutination. Record

the results.

6. Wash the cells four times in saline

and test by an indirect antiglobulin

technique using polyspecific anti-

human globulin or anti-IgG reagent.

Centrifuge and examine for aggluti-

nation. Record the results.

7. Confirm the validity of negative tests

by adding IgG-coated red cells.

Interpretation

Reactivity (hemolysis, agglutination, and/

or positive indirect antiglobulin test) with

drug-treated cells, but not with untreated

cells, indicates that drug antibodies are

present (see notes 3 and 4). No hemolysis

786 AABB Technical Manual

will be seen in tests with plasma or the

eluate. Antibodies to either penicillin or

cephalothin may cross-react with cells

treated with the other drug (ie, penicillin

antibodies may attach to cephalothin-

treated cells and vice versa). Antibodies to

other cephalosporins may react with

cephalothin-treated cells. It is best to treat

cells with the drug that is suspect.

Negative results without a positive con-

trol can only be interpreted to mean that

drug antibodies were not detected. The

drug may or may not be bound to the test

red cells.

Notes

1. The volume of drug-treated red cells

can be scaled down as long as the ra-

tio of the 40 mg/mL drug solution to

red cells is constant; eg, 120 mg pen-

icillin in 3 mL BB plus 0.2 mL red

cells or 100 mg cephalosporin in 2.5

mL PBS plus 0.25 mL red cells.1

2. Cephalosporins do not require a

high pH for optimal coating of red

cells. In fact, a lower pH, ie, pH 6 to

7, decreases nonspecific protein ad-

sorption seen when a high pH buffer

is used. The least amount of nonspe-

cific protein adsorption by drug-

treated red cells will occur if a pH 6.0

buffer is used, but this leads to a

slight decrease in coating by the

drug.

3. Test normal pooled serum and PBS

as negative controls and, when avail-

able, a specimen known to contain

an antibody to the drug being inves-

tigated as a positive control.

4. To control for nonspecific protein

adsorption and nonspecific aggluti-

nation of normal sera observed with

some cephalosporins (eg, cephalo-

thin), test the normal serum and the

test serum at a 1 in 20 dilution in PBS.

Normal sera diluted 1 in 20 generally

do not react nonspecifically. Thus,

reactivity of the diluted serum with

the drug-treated cells but not with

the untreated cells indicates that

drug antibody is present.

5. When testing cefotetan-treated red

cells, test the serum at a 1 in 100 di-

lutioninPBStopreventafalse-posi

tive test result. In addition to the

nonspecific uptake of protein onto

cefotetan-treated red cells, some

normal sera appear to have a “natu-

rally occurring” anticefotetan,2afew

of which react weakly at a 1 in 20 di-

lution. Cases of cefotetan-induced

immune hemolytic anemia are asso-

ciated with very high antibody titers

(eg, mean antiglobulin test titer =

16,000).3

6. About 30% of patients with antice-

fotetan also have a drug-independ-

ent antibody3; in these cases, the

serum and/or eluate, when tested

undilute, may react with both the

cefotetan-treated and untreated red

cells.

7. The last wash control can some-

times react with cefotetan-treated

red cells when antibodies to cefo-

tetan are present, regardless of the

wash solution used (commercial

acid eluate kit wash solution, 4 C

LISS, or 4 C PBS) or the number of

washes performed.3

8. Prepare drug solutions just before

use.

9. Drug-treated red cells may be kept

in PBS at 4 C for up to 1 week; how-

ever, there may be some weakening

of drug coating upon storage. Drug-

treated and untreated red cells may

also be stored frozen.

10. When antibodies are not detected

with drug-treated red cells, test by

Methods Section 4: Investigation of a Positive DAT 787

the immune complex method (Method

4.15). Antibodies to some third-gen-

eration cephalosporins (eg, ceftriaxone)

do not react with drug-treated red cells.

References

1. Petz LD, Garratty G. Immune hemolytic

anemias. 2nd ed. Philadelphia: Churchill

Livingstone, 2004.

2. Arndt P, Garratty G. Is severe immune

hemolytic anemia, following a single dose of

cefotetan, associated with the presence of

“naturally occurring” anti-cefotetan? (ab-

stract) Transfusion 2001;41(Suppl):24S.

3. Arndt PA, Leger RM, Garratty G. Serology of

antibodies to second- and third-generation

cephalosporins associated with immune

hemolytic anemia and/or positive direct

antiglobulin tests. Transfusion 1999;39:1239-

46.

Method 4.15. Demonstration

of Immune-Complex

Formation Involving Drugs

Principle

For a discussion of the mechanism of

drug-induced immune-complex forma-

tion, see Chapter 20.

Specimen

The patient’s serum.

Reagents

1. The drug under investigation, in the

same form (powder, tablet, capsules)

that the patient is receiving.

2. Phosphate-buffered saline (PBS) at

pH 7.3 (see Method 1.7).

3. Fresh, normal serum known to lack

unexpected antibodies, as a source

of complement.

4. Pooled group O reagent red cells, 5%

suspension, one aliquot treated with

a proteolytic enzyme (see Method

3.5.6) and one untreated.

5. Polyspecific antihuman globulin re-

agent.

6. IgG-coated red cells.

Procedure

1. Prepare a 1 mg/mL solution of the

drug in PBS. Centrifuge to remove

any particulate matter and adjust

the pH of the supernatant fluid to

approximately 7 with either 1 N

NaOH or 1 N HCl, as required, if the

pH is below 5 or above 8.

2. Label two sets of tubes for the fol-

lowing test mixtures:

a. Patient’s serum + drug.

b. Patient’s serum + PBS.

c. Patient’s serum + complement

(normal serum) + drug.

d. Patient’s serum + complement

(normal serum) + PBS.

e. Normal serum + drug.

f. Normal serum + PBS.

3. Add 2 volumes (eg, 2 drops) of each

component in the appropriate tubes

(eg,2dropsofserum+2dropsof

drug).

4. Add 1 drop of a 5% saline suspen-

sion of untreated group O reagent

red cells to one set of tubes. Add 1

drop of a 5% saline suspension of

enzyme-treated group O reagent red

cells to the second set of tubes.

5. Mix and incubate at 37 C for 1 to 2

hours, with periodic gentle mixing.

6. Centrifuge, examine for hemolysis

and agglutination, and record the re-

sults.

7. Wash the cells four times in saline

and test with a polyspecific antiglo-

bulin reagent.

8. Centrifuge, examine for agglutina-

tion, and record the results.

9. Confirm the validity of negative tests

by adding IgG-coated red cells.

788 AABB Technical Manual

Interpretation

Hemolysis, direct agglutination, or posi-

tive indirect antiglobulin tests can occur

together or separately. Reactivity in any of

the tests containing the patient’s serum to

which the drug was added, and absence

of reactivity in the corresponding control

tests containing PBS instead of the drug,

indicate that antibody to the drug is pres-

ent.Seenote4.

Notes

1. Thedrugmaybemoreeasilydis

solved by incubation at 37 C and vig-

orous shaking of the solution. If the

drug is in tablet form, crush it with a

mortar and pestle and remove any

visible outer tablet coating material

before adding PBS.

2. Not all drugs will dissolve com-

pletely in PBS. Consult the manufac-

turer or a reference such as the Merck

Index for the solubility of the drug in

question. A previous report of drug-

induced immune hemolytic anemia

resulting from the drug in question

mayprovideinformationonthe

drug solution preparation.

3. When available, a serum/plasma

known to contain antibody with the

drug specificity being evaluated

should be included as a positive

control.

4. Tests without the drug added may be

positive if autoantibodies or circu-

lating drug-antibody immune com-

plexes are present in the patient’s

sample. Autoantibody reactivity would

be persistent over time, whereas cir-

culating immune complexes are

transient.

5. Testing with enzyme-treated red

cells and the addition of fresh nor-

mal serum as a source of comple-

ment may increase the sensitivity of

the test.

6. If tests for drug antibodies by the

immune-complex method and drug

adsorption method are noninforma-

tive, consider testing with ex-vivo

antigen (see Method 4.16).

Reference

Petz LD, Garratty G. Immune hemolytic anemias.

2nd ed. Philadelphia: Churchill Livingstone, 2004.

Method 4.16. Ex-Vivo

Demonstration of

Drug/Anti-Drug Complexes

Principle

Immune drug/anti-drug complexes can

activate complement and cause hemolysis

in vivo. These immune complexes may be

demonstrable by serologic testing in the

presence of the drug, but with some drugs

(notably nomifensine), antibodies are di-

rected against metabolites of the drug,

rather than the native drug. Serum and/or

urine from volunteers who have ingested

therapeutic levels of the drug can be used

as a source of these metabolites. See note

This procedure is used to investigate

drug-associated immune hemolysis, partic-

ularly when use of the preceding methods

has been noninformative.

Specimen

Patient’s serum.

Reagents

1. Polyspecific antihuman globulin

(AHG) reagent.

2. Drug metabolites from volunteer

drug recipients. See note 2.

Methods Section 4: Investigation of a Positive DAT 789

a. Volunteer serum (VS) obtained

immediately before (VS0), at 1

hour (VS1), and 6 hours (VS6)

after drug administration. Di-

vide serum into 1-mL aliquots

and store them at 2 to 8 C for a

few hours or at –20 C or colder

until use.

b. Volunteer urine (VU) obtained

immediately before (VU0), at 1

hour (VU1), 3.5 hours (VU3.5), 7

hours (VU7), and 16 hours

(VU16)afterdrugadministra

tion. Divide into 1-mL aliquots

and store them at 2 to 8 C for a

few hours or at –20 C or colder

until use.

3. Fresh normal serum, known to lack

unexpected antibodies, as a source of

complement.

4. Phosphate-buffered saline (PBS), pH

7.3 (see Method 1.7).

5. Pooled group O reagent red cells

washed three times with saline and

resuspended to a 5% concentration

with PBS.

6. Pooled, enzyme-treated, group O red

cells, 5% suspension in PBS.

7. IgG-coated red cells.

Procedure

1. For each volunteer serum and/or

volunteer urine sample collected, la-

bel two sets of the following test

mixtures:

a. Patient’s serum + VS (or VU).

b. Patient’s serum + PBS.

c. Patient’s serum + complement

+VS(orVU).

d. Patient’s serum + complement

+PBS.

e. Complement + VS (or VU).

f. Complement + PBS.

2. Add 0.1 mL of each component to the

appropriate tubes.

3. Add 1 drop of a 5% saline suspen-

sion of the untreated group O re-

agent red cells to one set of tubes.

Add 1 drop of a 5% saline suspen-

sion of enzyme-treated reagent red

cells to the second set of tubes.

4. Mix the contents of each tube and

incubate at 37 C for 1 to 2 hours, with

periodic mixing.

5. Centrifuge, examine for agglutina-

tion and/or hemolysis, and record

the results.

6. Wash the cells four times with saline

and test with a polyspecific antiglo-

bulin reagent.

7. Centrifuge, examine for agglutina-

tion, and record the results.

8. Confirm the validity of negative tests

by adding IgG-coated red cells.

Interpretation

Hemolysis, direct agglutination, or reac-

tivity with AHG in any of the tubes con-

taining test serum and VS or VU, and ab-

sence of reactivity in all the control tubes,

indicate antibody against a metabolite of

thedruginquestion.

Notes

1. Approval of the institutional ethics

committeeshouldbeobtainedfor

the use of volunteers for obtaining

drug metabolites.

2. The urine sample collection times

given are those optimal for antibod-

ies to nomifensine metabolites; dif-

ferent collection times may be re-

quired for other drugs.

3. Complement may be omitted from

step 1 if the VS samples have been

keptoniceandareusedfortesting

within 8 hours of collection.

790 AABB Technical Manual

4. Testing with enzyme-treated red

cells and the addition of fresh nor-

mal serum as a source of complement

may increase the sensitivity of the

test.

References

1. Judd WJ. Methods in immunohematology.

2nd ed. Durham, NC: Montgomery Scientific

Publications, 1994.

2. Salama A, Mueller-Eckhardt C. The role of me-

tabolite-specific antibodies in nomifensine-

dependent immune hemolytic anemia. N

Engl J Med 1985;313:469-74.

Methods Section 4: Investigation of a Positive DAT 791

Methods Section 5: Hemolytic Disease of the Fetus and Newborn

Methods Section 5

Hemolytic Disease of the

Fetus and Newborn

Method 5.1. Indicator Cell

Rosette Test for

Fetomaternal Hemorrhage

Principle

This test detects D+ red cells in the blood

of a D– woman whose fetus or recently

delivered infant is D+. When reagent

anti-D is added to maternal blood con-

taining D+ fetal cells, fetal red cells be-

come coated with anti-D. When D+ re-

agent cells are subsequently added, easily

visible rosettes are formed, with several

redcellsclusteredaroundeachanti

body-coated D+ red cell.

Although the number of rosettes is

roughly proportional to the number of D+

red cells present in the original mixture,

this test provides only qualitative informa-

tion about fetal-maternal admixture. Speci-

mens giving a positive result should be sub-

jected to further testing to quantify the

number of fetal cells. The acid-elution pro-

cedure given below and flow cytometry are

acceptable choices. If a commercial test is

available, the directions in the package in-

sert should be followed.

Specimen

A 2% to 5% saline suspension of washed

red cells from a maternal blood sample.

Reagents

Prepared reagents are commercially avail-

able. The steps below can be used for in-

house preparation.

1. Negative control: a 2% to 5% saline

suspension of washed red cells known

to be D–.

2. Positivecontrol:a2%to5%saline

suspension of a mixture containing

approximately 0.6% D+ red cells and

99.4% D– red cells. The positive con-

trol can be prepared by adding 1 drop

793

Section 5

ofa2%to5%suspensionofD+con

trol cells to 15 drops of a 2% to 5%

suspension of washed D– control cells.

Mix well, then add 1 drop of this cell

suspension to 9 drops of the 2% to

5% suspension of D– red cells. Mix well.

3. Indicator red cells: a 2% to 5% saline

suspension of group O, R2R2red cells.

Either enzyme-treated or untreated

cells in an enhancing medium can be

used.

4. Chemically modified or high-protein

reagent anti-D serum. Some mono-

clonal/polyclonal blended reagents

areunsuitableforuseinthismethod.

The antisera selected for use should

be evaluated for suitability before in-

corporation into the test procedure.

Procedure

1. To each of three test tubes, add 1 drop

(or volume specified in the manufac-

turer’s instructions) of reagent anti-D.

2. Add 1 drop of maternal cells, nega-

tive control cells, and positive control

cells to the appropriately labeled tubes.

3. Incubate at 37 C for 15 to 30 min-

utes, or as specified by the manufac-

turer’s instructions.

4. Wash cell suspensions at least four

times with large volumes of saline, to

remove all unbound reagent anti-D.

Decant saline completely after last

wash.

5. To the dry cell button, add 1 drop of

indicator cells and mix thoroughly to

resuspend them.

6. Centrifuge the tubes for 15 seconds

at 900 to 1000 ×g.

7. Resuspend cell button and examine

the red cell suspension microscopi-

cally at 100 to 150 ×magnification.

8. Examine at least 10 fields and count

the number of red cell rosettes in each

field.

Interpretation

The absence of rosettes is a negative re-

sult. With enzyme-treated indicator cells,

up to one rosette per three fields may oc-

cur in a negative specimen. With un-

treated indicator cells and an enhancing

medium, there may be up to six rosettes

per five fields in a negative test. The pres-

ence of more rosettes than these allow-

able maxima constitutes a positive result,

and the specimen should be examined

using a test that quantifies the amount of

fetal blood present.

Thepresenceofrosettesoragglutination

in the negative control tube indicates inad-

equate washing after incubation, allowing

residual anti-D to agglutinate the D+ indi-

cator cells. A strongly positive result is seen

with red cells from a woman whose Rh

phenotype is weak D rather than D–; mas-

sive fetomaternal hemorrhage may pro-

duce an appearance difficult to distinguish

from that caused by a weak D phenotype,

and a quantitative test for fetal cells should

be performed. If the infant’s cells are shown

to be weak D, a negative result on the

mother’s specimen should be interpreted

with caution. In this situation, a quantita-

tive test that does not rely on D antigen

expression should be performed.

Reference

Sebring ES, Polesky HF. Detection of fetal mater-

nal hemorrhage in Rh immune globulin candi-

dates. Transfusion 1982;22:468-71.

Method 5.2. Acid-Elution Stain

(Modified Kleihauer-Betke)

Principle

Fetal hemoglobin resists elution from red

cells under acid conditions, whereas adult

hemoglobin is eluted. When a thin blood

smear is exposed to an acid buffer, hemo-

794 AABB Technical Manual

globin from adult red cells is leached into

the buffer so that only the stroma remains;

fetal cells retain their hemoglobin and

can be identified by a positive staining

pattern. The approximate volume of feto-

maternal hemorrhage can be calculated

from the percentage of fetal red cells in

the maternal blood film.

Specimen

Maternal anticoagulated whole blood

sample.

Reagents

Prepared reagents are commercially avail-

able in kits. The steps below can be used

for in-house preparations.

1. Stock solution A (0.1 M of citric acid).

C6H8O7•H2O, 21.0 g, diluted to 1 liter

with distilled water. Keep it in the re-

frigerator.

2. Stock solution B (0.2 M of sodium

phosphate). Na2HPO4•7H2O, 53.6 g,

diluted to 1 liter with distilled water.

Keep it in the refrigerator.

3. McIlvaine’s buffer, pH 3.2. Add 75

mL of stock solution A to 21 mL of

stock solution B. Prepare fresh mix-

ture for each test. This buffer mix-

ture should be brought to room tem-

perature or used at 37 C.

4. Erythrosin B, 0.5% in water.

5. Harris hematoxylin (filtered).

6. 80% ethyl alcohol.

7. Positive control specimen. Ten parts

of anticoagulated adult blood, mixed

with one part of anticoagulated ABO-

compatible cord blood.

8. Negative control specimen. Anticoa-

gulated adult blood.

Procedure

1. Prepare very thin blood smears, di-

luting blood with an equal volume of

saline. Air dry.

2. Fix the smears in 80% ethyl alcohol

for 5 minutes.

3. Wash the smears with distilled wa-

ter.

4. Immerse the smears in McIlvaine’s

buffer, pH 3.2, for 11 minutes at room

temperature or 5 minutes at 37 C. This

reaction is temperature-sensitive.

5. Wash the smears in distilled water.

6. Immerse the smears in erythrosin B

for 5 minutes.

7. Wash the smears completely in dis-

tilled water.

8. Immerse the smears in Harris hema-

toxylin for 5 minutes.

9. Wash the smears in running tap wa-

ter for 1 minute.

10. Examine dry using 40×magnifica-

tion, count a total of 2000 red cells,

and record the number of fetal cells

observed.

11. Calculate the percent of fetal red cells

in the total counted.

Interpretation

1. Fetal cells are bright pink and re-

fractile; normal adult red cells ap-

pear as very pale ghosts.

2. The conversion factor used to indi-

cate the volume (as mL of whole

blood) of fetomaternal hemorrhage

is the percent of fetal red cells ob-

served times 50.

Note

The accuracy and precision of this proce-

dure are poor, and decisions regarding Rh

Immune Globulin (RhIG) dosage in mas-

sive fetomaternal hemorrhage should be

made accordingly. If there is a question

regarding the need for additional RhIG, it

is preferable to administer another dose

to prevent the risks of undertreatment. (See

Table 23-1 for dosage.)

Methods Section 5: Hemolytic Disease of the Fetus and Newborn 795

Reference

Sebring ES. Fetomaternal hemorrhage—incidence

and methods of detection and quantitation. In:

Garratty G, ed. Hemolytic disease of the newborn.

Arlington, VA: AABB, 1984:87-118.

Method 5.3. Antibody

Titration Studies to Assist in

Early Detection of Hemolytic

Disease of the Fetus and

Newborn

Principle

Antibody titration is a semiquantitative

method of determining antibody concen-

tration. Serial twofold dilutions of serum

are prepared and tested for antibody ac-

tivity. The reciprocal of the highest dilu-

tion of plasma or serum that gives a 1+ re-

action is referred to as the titer (ie, 1 in

128 dilution; titer = 128).

In pregnancy, antibody titration is per-

formed to identify women with significant

levels of antibodies that may lead to

hemolytic disease of the fetus and newborn

(HDFN) and, for low-titer antibodies, to es-

tablish a baseline for comparison with

titers found later in pregnancy. Titration of

non-Rh antibodies should be undertaken

only after discussion with the obstetrician

about how the data will be used in the clini-

cal management of the pregnancy. The sig-

nificance of titers has been sufficiently es-

tablished only for anti-D (using a saline

technique).

Specimen

Serum for titration (containing potentially

significant unexpected antibodies to red

cell antigens, 1 mL). If possible, test the

current sample in parallel with the most

recent previously submitted (preceding)

sample from the current pregnancy.

Materials

1. Antihuman IgG: need not be heavy-

chain-specific.

2. Isotonic saline.

3. Volumetric pipettes, or equivalent:

0.1- to 0.5-mL delivery, with dispos-

able tips.

4. Red cells: group O reagent red cells,

2% suspension. (See note 1 regard-

ing the selection of red cells for test-

ing.) Avoid using Bg+ red cells be-

cause they may result in falsely high

values, especially with sera from

multiparous women.

5. IgG-coated red cells.

Quality Control

1. Test the preceding sample in parallel

with the most recent sample.

2. Prepare the dilutions using a sepa-

rate pipette for each tube. Failure to

do so will result in falsely high titers

because of carryover.

3. Confirm all negative reactions with

IgG-coated red cells (see step 9 be-

low).

Procedure

1. Using 0.5-mL volumes, prepare serial

twofold dilutions of serum in saline.

The initial tube should contain un-

diluted serum and the doubling di-

lutionrangeshouldbefrom1in2to

1 in 2048 (total of 12 tubes). (See

Method 3.7.)

2. Place 0.1 mL of each dilution into

appropriately labeled test tubes.

3. Add 0.1 mL of the 2% suspension of

red cells to each dilution. Alterna-

tively, for convenience, add 1 drop of

a solution of a 3% to 4% suspension

of red cells as supplied by the re-

agent manufacturer, although this

method is less precise.

796 AABB Technical Manual

4. Gently agitate the contents of each

tube; incubate at 37 C for 1 hour.

5. Wash the red cells four times with

saline; completely decant the final

wash supernatant.

6. To the dry red cell buttons thus ob-

tained, add anti-IgG according to the

manufacturer’s directions.

7. Centrifuge as for hemagglutination

tests.

8. Examine the red cells macroscopically;

grade and record the reactions.

9. Add IgG-coated red cells to all nega-

tive tests; recentrifuge and examine

the tests for macroscopic agglutina-

tion; repeat the testing if the tests with

IgG-coated red cells are nonreactive.

Interpretation

The titer is reported as the reciprocal of

the highest dilution of serum at which 1+

agglutination is observed. A titer ≥16 (this

value may vary according to the labora-

tory) is considered significant and may

warrant further monitoring for HDFN.

Notes

1. The selection of the most suitable

phenotype of red cells to use when

performing titration studies for HDFN

is controversial. Some workers select

red cells that have the strongest ex-

pression of antigen, such as R2R2for

anti-D. Others select red cells with the

phenotype that would be expected

in fetal circulation—ie, red cells that

express a single dose of the antigen,

such as R1r for testing for anti-D.

Whichever viewpoint is followed, it

is important that the laboratory be

consistent and use red cells of the

same phenotype for future titrations

to test the same patient’s serum.

2. Titration studies should be performed

upon initial detection of the anti-

body; save an appropriately labeled

aliquot of the serum (frozen at –20 C

or colder) for comparative studies

with the next submitted sample.

3. When the titer (eg, >16) and the anti-

body specificity have been associ-

ated with HDFN, it is recommended

that repeat titration studies be per-

formed every 2 to 4 weeks, beginning

at 18 weeks’ gestation; save an ali-

quot of the serum (frozen at –20 C or

colder) for comparative studies with

the next submitted sample.

4. When invasive procedures (eg, am-

niocentesis) have demonstrated fetal

compromise and are being used to

monitor the pregnancy, use the opti-

mal method for follow-up of fetal

well-being. However, if initial studies

do not show fetal compromise or the

Liley curve result is borderline, addi-

tional titrations may be helpful as a

means of following the pregnancy in

a less invasive manner.

5. Each institution should develop a

policy to ensure a degree of unifor-

mity in reporting and interpreting

antibody titers.

6. For antibodies to low-incidence an-

tigens, consider using putative pa-

ternal red cells, having established

that they express the antigen in ques-

tion.

7. Do not use enhancement techniques

[albumin, polyethylene glycol, low-

ionic-strength saline (LISS)] or en-

zyme-treated red cells because falsely

elevated titers may be obtained. Gel

testing is not recommended.

8. LISS should not be used as a diluent

in titration studies; nonspecific up-

take of globulins may occur in serum-

LISS dilutions.

9. Failure to obtain the correct results

may be caused by 1) incorrect tech-

nique, notably, failure to use sepa-

Methods Section 5: Hemolytic Disease of the Fetus and Newborn 797

rate pipette tips for each dilution or

2) failure to adequately mix thawed

frozen serum.

References

1. Issitt PD, Anstee DJ. Applied blood group se-

rology. 4th ed. Durham, NC: Montgomery

Scientific Publications, 1998:1067-9.

2. Judd WJ, Luban NLC, Ness PM, et al. Prenatal

and perinatal immunohematology: Recom-

mendations for serologic management of the

fetus, newborn infant, and obstetric patient.

Transfusion 1990;30:175-83.

3. Judd WJ. Methods in immunohematology.

2nd ed. Durham, NC: Montgomery Scientific

Publications, 1994.

4. Judd WJ. Practice guidelines for prenatal and

perinatal immunhematology, revisited. Trans-

fusion 2001;41:1445-52.

798 AABB Technical Manual

Methods Section 6: Blood Collection, Storage, and Component Preparation

Methods Section 6

Blood Collection, Storage,

and Component Preparation

Method 6.1. Copper Sulfate

Method for Screening Donors

for Anemia

Principle

This method estimates the hemoglobin

content of blood from its specific gravity.

A drop of blood in contact with copper

sulfate solution of specific gravity 1.053

becomes encased in a sac of copper pro-

teinate, which prevents dispersion of the

fluid or any change in specific gravity for

about 15 seconds. If the specific gravity of

the blood is higher than that of the solu-

tion, the drop will sink within 15 seconds;

if not, the drop will hesitate and remain

suspended or rise to the top of the solu-

tion. A specific gravity of 1.053 corre-

sponds to a hemoglobin concentration of

12.5 g/dL.

This is not a quantitative test; it shows

only whether the prospective donor’s he-

moglobin is below or above the acceptable

level of 12.5 g/dL. False-positive reactions

are rare; donors whose drop of blood sinks

nearly always have an acceptable hemoglo-

bin level. False-negative reactions occur

fairly commonly and can cause inappropri-

ate deferral.1,2 Measuring hemoglobin by

another method or determining hematocrit

sometimes reveals that the prospective do-

nor is acceptable.

Reagents and Materials

1. Copper sulfate solution at specific

gravity 1.053, available commer-

cially. Store it in tightly capped con-

tainers to prevent evaporation. The

solution should be kept at room tem-

perature or brought to room temper-

ature before it is used.

2. Sterile gauze, antiseptic wipes, and

sterile lancets.

3. Containers for the disposal of sharps

and other biohazardous materials.

4. Capillary tubes and dropper bulbs or

a device to collect capillary blood

without contact.

799

Section 6

Procedure

1. Into a labeled, clean, dry tube or

bottle, dispense a sufficient amount

(atleast30mL)ofcoppersulfateso

lution to allow the drop to fall ap-

proximately 3 inches. Change the so-

lution daily or after 25 tests. Be sure

that the solution is adequately mixed

before beginning each day’s deter-

minations.

2. Clean the site of the skin puncture

thoroughly with antiseptic solution

and wipe it dry with sterile gauze.

3. Puncture the finger firmly, near the

end but slightly to the side, with a

sterile, disposable lancet or spring-

loaded, disposable needle system. A

good free flow of blood is important.

Donotsqueezethepuncturesitere-

peatedly because this may dilute the

drop of blood with tissue fluid and

lower the specific gravity.

4. Collect the blood in a capillary tube

without allowing air to enter the

tube.

5. Let one drop of blood fall gently

from the tube at a height about 1 cm

above the surface of the copper sul-

fate solution.

6. Observe for 15 seconds.

7. Dispose of lancets and capillary tubes

in appropriate biohazard containers.

Dispose of gauze appropriately;

gauze contaminated with droplets of

blood that subsequently dry such

that the item is stained but not

soaked or caked may be considered

nonhazardous.

Interpretation

1. If the drop of blood sinks, the do-

nor’s hemoglobin is at an acceptable

level for blood donation.

2. If the drop of blood does not sink,

the donor’s hemoglobin may not be

at an acceptable level for blood do-

nation. If time and equipment permit,

it is desirable to perform a quantita-

tive measurement of hemoglobin or

hematocrit.

Notes

1. A certificate of analysis from the man-

ufacturer should be obtained with

each new lot of copper sulfate solu-

tion.

2. Used solution should be disposed of

as biohazardous or chemical mate-

rial because of the blood in the con-

tainer. Refer to local and state laws

regarding disposal.

3. Usecaretopreventbloodfromcon-

taminating work surfaces, the donor’s

clothing, or other persons or equip-

ment.

4. Cover the container between uses to

prevent evaporation.

References

1. Lloyd H, Collins A, Walker W, et al. Volunteer

blood donors who fail the copper sulfate

screening test: What does failure mean, and

what should be done? Transfusion 1988;28:

467-9.

2. Morris MW, Davey FR. Basic examination of

blood. In: Henry JB, ed. Clinical diagnosis

and management by laboratory methods.

20th ed. Philadelphia: WB Saunders, 2001:

479-519.

Method 6.2. Arm Preparation

for Blood Collection

Detailed instructions are specific to each

manufacturerandshouldbefollowedas

indicated. The following procedure is

writteningeneraltermsasanexample.

800 AABB Technical Manual

Principle

Iodophor compounds, or other sterilizing

compounds, are used to sterilize the veni-

puncture site before blood collection.

Materials

1. Scrub solution: Disposable povidone-

iodine scrub 0.75% or disposable

povidone-iodine swabstick 10%; avail-

able in prepackaged single-use form.

2. Preparation solution: 10% povidone-

iodine; available in prepackaged sin-

gle-use form.

3. Sterile gauze.

Procedure

1. Apply tourniquet or blood pressure

cuff; identify venipuncture site, then

release tourniquet or cuff.

2. Scrub area at least 4 cm (1.5 inches)

in all directions from the intended

site of venipuncture (ie, 8 cm or 3

inches in diameter) for a minimum

of 30 seconds with 0.7% aqueous so-

lution of iodophor compound. Excess

foam may be removed, but the arm

need not be dry before the next step.

3. Starting at the intended site of veni-

puncture and moving outward in a

concentric spiral, apply “prep” solu-

tion; let stand for 30 seconds or as

indicated by manufacturer.

4. Cover the area with dry, sterile gauze

until the time of venipuncture. After

the skin has been prepared, it must

not be touched again. Do not repal-

pate the vein at the intended veni-

puncture site.

Notes

1. For donors sensitive to iodine (tinc-

ture or povidone preparations), an-

other method (eg, ChloraPrep 2%

chlorhexidine and 70% isopropyl al-

cohol) should be designated by the

blood bank physician. Green soap

should not be used.

2. For donors sensitive to both iodine

and chlorhexidine, a method using

only isopropyl alcohol could be con-

sidered. The preferred procedure is

the use of a 30-second up-and-down

scrub, followed by enough time for

the skin to dry. A second scrub is

then applied. This method may re-

quire a variance from the Food and

Drug Administration.

3. Arm preparation methods approved

bytheFoodandDrugAdministra

tion are available at http://www.fda.

gov/cber/infosheets/armprep.htm.

Reference

Goldman M, Roy G, Frechette N, et al. Evaluation

of donor skin disinfection methods. Transfusion

1997;37:309-12.

Method 6.3. Phlebotomy and

Collection of Samples for

Processing and Compatibility

Tests

Principle

Blood for transfusion and accompanying

samples is obtained from prominent veins

on the donor’s arm, usually in the area of

the antecubital fossa.

Materials

1. Sterile collection bag containing an-

ticoagulant, with integrally attached

tubing and needle.

2. Metal clips and hand sealers.

3. Balance system to monitor volume

of blood drawn.

4. Sterile gauze and clean instruments

(scissors, hemostats, forceps).

Methods Section 6: Blood Collection, Storage, and Component Preparation 801

5. Test tubes for sample collection.

6. Device for stripping blood in the

tubing.

7. Dielectric sealer (optional).

Procedure

1. Ask donor to confirm his or her iden-

tification.

2. Ensure that all labeling on blood con-

tainer, processing tubes, retention

segment, and donor records is cor-

rect.

3. Prepare donor’s arm as described in

Method 6.2.

4. Inspect bag for any defects and dis-

coloration. The anticoagulant and

additive solutions should be in-

spected for particulate contaminants.

5. Position bag below the level of the

donor’s arm.

a. If a balance system is used, be

sure the counterbalance is level

andadjustedfortheamountof

blood to be drawn. Unless metal

clips and a hand sealer are used,

make a very loose overhand knot

in the tubing. Hang the bag and

route the tubing through the

pinch clamp. A hemostat

should be applied to the tubing

before the needle is uncapped

to prevent air from entering the

line.

b. If a balance system is not used,

be sure to monitor the volume

of blood drawn.

6. Reapply tourniquet or inflate blood

pressure cuff. Ask the donor to open

and close hand until previously se-

lected vein is again prominent.

7. Uncover sterile needle and perform

the venipuncture immediately. A

clean, skillful venipuncture is essen-

tial for collection of a full, clot-free

unit. Once the bevel has penetrated

the skin, palpation of the skin above

the needle stem may be performed

with a gloved finger, provided the

needle is not touched. When the

needle position is acceptable, tape

the tubing to the donor’s arm to hold

the needle in place and cover the

site with sterile gauze.

8. Release the hemostat. Open the tem-

porary closure between the interior

of the bag and the tubing.

9. Ask the donor to open and close hand

slowly every 10 to 12 seconds during

collection.

10. Keep the donor under observation

throughout the donation process.

The donor should never be left unat-

tended during or immediately after

donation.

11. Mix blood and anticoagulant gently

and periodically (approximately ev-

ery 45 seconds) during collection.

Mixingmaybedonebyhandorby

continuous mechanical mixing.

12. Be sure blood flow remains fairly

brisk, so that coagulation activity is

not triggered. If there is continuous,

adequate blood flow and constant

agitation, rigid time limits are not

necessary. However, units requiring

more than 15 minutes to draw may

notbesuitableforpreparationof

Platelets, Fresh Frozen Plasma, or

Cryoprecipitated AHF. The time re-

quired for collection can be moni-

tored by indicating the time of phle-

botomy or the maximal allowable

time (start time plus 15 minutes) on

the donor record.

13. Monitor volume of blood being drawn.

If a balance is used, the device will

interrupt blood flow after the proper

amount has been collected. One mL

of blood weighs at least 1.053 g, indi-

cated by the minimum allowable

specific gravity for donors. A conve-

802 AABB Technical Manual

nient figure to use is 1.06 g/mL; a unit

containing 405 to 550 mL should

weigh 429 to 583 g plus the weight of

the container and anticoagulant. For

a 500-mL bag, this is 565 to 671 g.

14. Clamp the tubing near the veni-

puncture using a hemostat, metal

clip, or other temporary clamp. Re-

lease the blood pressure cuff/tourni-

quet to 20 mm Hg or less and fill the

tube(s) for blood processing sam-

ple(s) by a method that prevents

contamination of the contents of the

bag. This can be done in several ways.

a. If the blood collection bag con-

tains an inline needle, make an

additional seal with a hemo-

stat, metal clip, hand sealer, or

a tight knot made from previ-

ously prepared loose knot just

distal to the inline needle. Open

the connector by separating

the needles. Insert the proximal

needle into a processing test

tube, remove the hemostat, al-

low the tube to fill, and re-

clamp the tubing. The donor

needle is now ready for removal.

b. If the blood collection bag con-

tains an inline processing tube,

be certain that the processing

tube,orpouch,isfullwhenthe

collection is complete and the

original clamp is placed near

the donor needle. The entire

assembly may now be removed

from the donor.

c. If a straight-tubing assembly

set is used, the following proce-

dure should be followed. Place

a hemostat on the tubing, al-

lowing about four segments be-

tween the hemostat and the

needle. Pull tight the loose

overhand knot made in step 3.

Release the hemostat and strip

asegmentofthetubingfreeof

blood between the knot and

the needle (about 1 inch in

length). Reapply the hemostat

and cut the tubing in the strip-

ped area between the knot and

the hemostat. Fill the required

tube(s) by releasing the hemo-

stat and then reclamp the tub-

ing with the hemostat. Because

this system is open, Biosafety

Level 2 precautions should be

followed.

15. Deflate the cuff and remove the

tourniquet. Remove the needle from

the donor’s arm, if not already re-

moved. Apply pressure over the gauze

and ask the donor to raise his or her

arm (elbow straight) and hold the

gauze firmly over the phlebotomy

site with the other hand.

16. Discard the needle assembly into a

biohazard container designed to pre-

vent accidental injury to, and con-

tamination of, personnel.

17. Strip donor tubing as completely as

possible into the bag, starting at the

seal.Workquickly,topreventthe

blood from clotting in the tubing. In-

vert the bag several times to mix the

contents thoroughly; then allow the

tubing to refill with anticoagulated

blood from the bag. Repeat this pro-

cedure a second time.

18. Seal the tubing attached to the col-

lection bag into segments, leaving a

segment number clearly and com-

pletely readable. Attach a unit iden-

tification number to one segment to

be stored as a retention segment.

Knots, metal clips, or a dielectric

sealer may be used to make seg-

ments suitable for compatibility

testing. It must be possible to sepa-

rate segments from the unit without

breaking sterility of the bag. If a di-

Methods Section 6: Blood Collection, Storage, and Component Preparation 803

electric sealer is used, the knot or

clip should be removed from the dis-

tal end of the tubing after creating a

hermetic seal.

19. Reinspect the container for defects.

20. Recheck numbers on the container,

processing tubes, donation record,

and retention segment.

21. Place blood at appropriate tempera-

ture. Unless platelets are to be re-

moved, whole blood should be placed

at 1 to 6 C immediately after collec-

tion. If platelets are to be prepared,

blood should not be chilled but

should be stored in a manner in-

tended to reach a temperature of 20

to 24 C until platelets are separated.

Platelets must be separated within 8

hours after collection of the unit of

Whole Blood.

Notes

1. If the needle is withdrawn and veni-

puncture is attempted again, prepa-

rationofthesitemustberepeatedas

in Method 6.2.

2. In addition to routine blood donor

phlebotomy, this procedure may be

adapted for use in therapeutic phle-

botomy.

References

1. Silva MA, ed. Standards for blood banks and

transfusion services. 23rd ed. Bethesda, MD:

AABB, 2005.

2. Smith LG. Blood collection. In: Green TS,

Steckler D, eds. Donor room policies and pro-

cedures. Arlington, VA: AABB, 1985:25-45.

3. Huh YO, Lightiger B, Giacco GG, et al. Effect

of donation time on platelet concentrates

and fresh frozen plasma. Vox Sang 1989;56:

21-4.

4. Sataro P. Blood collection. In: Kasprisin CA,

Laird-Fryer B, eds. Blood donor collection

practices. Bethesda, MD: AABB, 1993:89-103.

Method 6.4. Preparation of

Red Blood Cells

Principle

Red Blood Cells (RBCs) are obtained by

removal of supernatant plasma from cen-

trifuged Whole Blood. The volume of

plasma removed determines the hema-

tocrit of the component. When RBCs are

preserved in CPDA-1, maximal viability

during storage requires an appropriate ra-

tio of cells to preservative. A hematocrit of

80% or lower ensures the presence of ade-

quate glucose for red cell metabolism for

up to 35 days of storage.

Materials

1. Freshly collected Whole Blood, ob-

tained by phlebotomy as described

in Method 6.3. Collect blood in a col-

lection unit with integrally attached

transfer container(s).

2. Plasma extractor.

3. Metal clips and hand sealer.

4. Clean instruments (scissors, hemo-

stats).

5. Dielectric sealer (optional).

6. Refrigerated centrifuge.

7. Scale.

Procedure

1. Centrifuge whole blood using a “heavy”

spin (see Method 7.4), with a tem-

perature setting of 4 C.

2. Place the primary bag containing

centrifuged blood on a plasma ex-

pressor, and release the spring, al-

lowing the plate of the expressor to

contact the bag.

3. Temporarily clamp the tubing be-

tween the primary and satellite bags

with a hemostat or, if a mechanical

804 AABB Technical Manual

sealer will not be used, make a loose

overhand knot in the tubing.

4. If two or more satellite bags are at-

tached, apply the hemostat to allow

plasma to flow into only one of the

satellite bags. Penetrate the closure

of the primary bag. A scale, such as a

dietary scale, may be used to mea-

sure the expressed plasma. Remove

the appropriate amount of plasma

to obtain the desired hematocrit.

5. Reapply the hemostat when the de-

sired amount of supernatant plasma

has entered the satellite bag. Seal the

tubing between the primary bag and

the satellite bag in two places.

6. Check that the satellite bag has the

same donor number as that on the

primary bag and cut the tubing be-

tween the two seals.

Notes

1. If blood was collected in a single bag,

modify the above directions as fol-

lows: after placing the bag on the

expressor, apply a hemostat to the

tubing of a sterile transfer bag, asep-

tically insert the cannula of the trans-

fer bag into the outlet port of the bag

of blood, release the hemostat, and

continue as outlined above. The ex-

piration date will change, however.

2. Collection of blood in an additive so-

lution allows removal of a greater

volume of plasma in step 4. After the

plasma has been removed, the addi-

tive solution is allowed to flow from

the attached satellite bag into the red

cells. This will result in a hematocrit

of 55% to 65%. Be sure that an ap-

propriate label and dating period are

used.

3. The removal of 230 to 256 g (225 to

250 mL) of plasma and preservation

of the red cells in the anticoagulant-

preservative solution will generally

result in a red cell component with a

hematocrit between 70% and 80%.

4. See Table 6.4-1 to prepare Red Blood

Cells with a specific (desired) hema-

tocrit.

Method 6.5. Preparation of

Prestorage Red Blood Cells

Leukocytes Reduced

Principle

The general principle and materials of

Method 6.4 apply, except that the red cells

are filtered using a special leukocyte re-

duction filter. All red cell leukocyte reduc-

tion filters licensed in the United States

remove platelets to some degree. Anti-

coagulated whole blood may be filtered,

from which only platelet-poor plasma

Methods Section 6: Blood Collection, Storage, and Component Preparation 805

Table 6.4-1. Removing Plasma from

Units of Whole Blood (To Prepare RBCs

in Anticoagulant-Preservative with a

Known Hematocrit)

Hematocrit of

Segment from

Whole Blood

Unit

Volume of

Plasma

to Be

Removed

Final

Hematocrit of

Red Blood Cell

Unit

40% 150 mL 56%

39% 150 mL 55%

38% 160 mL 55%

37% 165 mL 54%

36% 170 mL 54%

35% 180 mL 54%

34% 195 mL 55%

33% 200 mL 55%

(leukocyte reduced) and red cells may be

made. Alternatively, the red cells may be

filtered in additive solution, potentially

allowing the preparation of platelets,

plasma, and red cells. Nonleukocyte-re-

duced red cells may also undergo leuko-

cyte reduction after preparation by at-

taching a leukocyte reduction filter con-

nected to a storage container using a ster-

ile connection device.

Procedure

1. Before centrifugation, the antico-

agulated Whole Blood may be fil-

tered by hanging the container up-

side down and allowing the blood to

flow through an in-line filter by grav-

ity into a secondary container. The

steps in Method 6.4 are then fol-

lowed (see note 2, for addition of ad-

ditive solution).

2. The anticoagulated Whole Blood

may be centrifuged with the in-line

filter attached. After centrifugation,

the plasma is expressed. The addi-

tive solution is added, and the red

cells in the additive solution are fil-

tered by gravity, as in step 1 above.

3. A red cell component prepared us-

ing Method 6.4 either in residual

anticoagulated plasma or in additive

solution (AS-1, AS-3, AS-5) may have

a secondary container with an in-

line filter attached using a sterile

connection device. Filtration can

proceed according to the manufac-

turer’s directions using gravity, as in

step 1. The timing of this filtration is

often within 24 hours of collection

but can be up to 5 days.

4. Red cells that are leukocyte reduced

are labeled “Red Blood Cells Leuko-

cytes Reduced.” There is no specific

label for prestorage leukocyte reduc-

tion.

Notes

1. If the collection system does not in-

clude an in-line filter, a sterile con-

nection device can be used to attach

a leukocyte reduction filter to the

collection system. The filter should

be used according to the manufac-

turer’s directions.

2. Whole-blood-derived platelets can

be manufactured only before leuko-

cyte reduction (see Method 6.14).

Method 6.6. Rejuvenation of

Red Blood Cells

Principle

Rejuvenation is a process to restore de-

pleted metabolites and improve the func-

tion and posttransfusion survival of stored

red cells. The rejuvenating solution is not

intended for intravenous administration;

after warm incubation with the solution,

the red cells are washed and either gly-

cerolized for frozen storage or kept at 1 to

6 C for transfusion within 24 hours.

The rejuvenating solution approved by

the Food and Drug Administration contains

pyruvate, inosine, phosphate, and adenine.

Its use is permitted only with RBCs pre-

pared from Whole Blood collected into

CPD, CP2D, or CPDA-1, and it may be

added at any time between 3 days after col-

lection of the blood and 3 days after the ex-

piration of the unit. However, the use of the

rejuvenation solution with RBC units be-

fore 14 days of storage is not routinely ac-

cepted because the treated cells may develop

supranormal levels of 2,3-diphospho-

glycerate, which impairs oxygen uptake.

Reagents and Materials

1. RBCs stored at 1 to 6 C and prepared

from Whole Blood collected in CPD

or CPDA-1. After collection, RBCs

806 AABB Technical Manual

suspended in CPD from day 3 to day

24(orinCPDA-1fromday3today

38)maybeused.Thesolutionisnot

approved for use with cells stored in

additive solutions.

2. Red Blood Cell rejuvenation solu-

tion, in 50-mL sterile vial (Rejuvesol,

Cytosol Laboratories, Braintree, MA);

also called rejuvenating solution.

3. Waterproof plastic bag.

4. Metal clips and hand sealer.

5. Sterile airway.

Procedure

1. Connect the container of rejuvenat-

ing solution to the RBCs, using a

transfer set and aseptic technique.

2. Allow 50 mL of rejuvenating solution

to flow by gravity into the container

of red cells. Gently agitate the cell/

solution mixture during this addition.

Note: A sterile airway is required if

the solution is in a bottle.

3. Sealthetubingnearthebloodbag

and incubate the mixture for 1 hour

at 37 C. Either a dry incubator or cir-

culating waterbath can be used. If

placed in a waterbath, the container

should be completely immersed; use

of a waterproof overwrap is essential

to prevent contamination.

4. For use within 24 hours, wash the re-

juvenated cells with saline (2 L un-

buffered 0.9% NaCl) by the use of an

approved protocol. Storage of the

washed cells from the start of the

wash procedure should be at 1 to 6 C

fornolongerthan24hours.

5. If the rejuvenated cells are to be cryo-

preserved, the standard glyceroli-

zation protocol adequately removes

the rejuvenation solution from the

processed cells. Expiration date re-

mains 10 years from the date of col-

lection.

6. Be sure that units are appropriately

labeled and that all applicable re-

cords are complete.

Reference

Valeri CR, Zaroules CG. Rejuvenation and freezing

of outdated stored human red cells. N Engl J Med

1972;287:1307-13.

Method 6.7. Red Cell

Cryopreservation Using

High-Concentration

Glycerol—Meryman Method

Principle

Cryoprotectiveagentsmakepossiblethe

long-term (10 or more years) preservation

of red cells in the frozen state. High-con-

centration glycerol is particularly suitable

for this purpose. A practical method for

RBCs collected in a 450-mL bag is de-

scribed below.

Materials

(See Chapter 8 for additional information

on frozen cellular components.)

1. Donor blood, collected into CPD,

CD2D, CPDA-1, or AS.

a. Complete all blood processing

on units intended for freezing.

b. RBCs preserved in CPD or CPDA-1

may be stored at 1 to 6 C for up

to 6 days before freezing.

c. RBCs preserved in AS-1 and AS-3

may be stored at 1 to 6 C for up

to 42 days before freezing.

d. RBCs that have undergone re-

juvenation (see Method 6.6) may

be processed for freezing up to

3 days after their original expi-

ration.

e. RBCs in any preservative solu-

tion that have been entered for

Methods Section 6: Blood Collection, Storage, and Component Preparation 807

processing must be frozen with-

in 24 hours of puncturing the

seal.

2. Storage containers, either polyvinyl

chloride or polyolefin bags.

3. 6.2 M of glycerol lactate solution

(400 mL).

4. Cardboard or metal canisters for

freezing.

5. Hypertonic (12%) sodium chloride

solution.

6. 1.6% NaCl, 1 liter for batch wash.

7. Isotonic (0.9%) NaCl with 0.2% dex-

trose solution.

8. 37 C waterbath or 37 C dry warmer.

9. Equipment for batch or continuous-

flow washing, to deglycerolize cells

frozen in high-concentration glyc-

erol.

10. Freezer tape.

11. Freezer (–65 C or colder).

Procedure

Preparing RBCs for Glycerolization

1. Prepare RBCs from Whole Blood

units by removal of supernatant an-

ticoagulant-preservative or additive

solution. Weigh the RBC unit to be

frozen and obtain the net weight of

the RBCs. The combined weight of

the cells and the collection bag

should be between 260 g and 400 g.

2. Underweight units can be adjusted

to approximately 300 g either by the

addition of 0.9% NaCl or by the re-

moval of less plasma than usual. Re-

cord the weight and, if applicable,

document the amount of NaCl added.

3. Record the Whole Blood number,

ABO group and Rh type, anticoagu-

lant, date of collection, date frozen,

expiration time, and the identifica-

tion of the person performing the

procedure. If applicable, document

the lot number of the transfer bag.

4. Warm the red cells and the glycerol

to at least 25 C by placing them in a

dry warming chamber for 10 to 15

minutes or by allowing them to re-

main at room temperature for 1 to 2

hours. The temperature must not

exceed 42 C.

5. Apply a “Red Blood Cells Frozen” la-

bel to the freezing bag in which the

unit will be frozen. The label must

also include: name of the facility

freezing the unit; Whole Blood num-

ber; ABO group and Rh type; date

collected; date frozen; the cryopro-

tective agent used; and the expira-

tion date.

Glycerolization

1. Document the lot numbers of the

glycerol, the freezing bags, and, if

used, the 0.9% NaCl.

2. Place the container of red cells on a

shaker and add approximately 100

mL of glycerol as the red cells are

gently agitated.

3. Turn off the shaker and allow the

cells to equilibrate, without agita-

tion, for 5 to 30 minutes.

4. Allow the partially glycerolized cells

to flow by gravity into the freezing bag.

5. Add the remaining 300 mL of glyc-

erol slowly in a stepwise fashion,

with gentle mixing. Add smaller vol-

umes of glycerol for smaller volumes

of red cells. The final glycerol con-

centration is 40% w/v. Remove any

air from the bag.

6. Allow some glycerolized cells to flow

back into the tubing so that seg-

ments can be prepared.

7. Maintain the glycerolized cells at

temperatures between 25 and 32 C

until freezing. The recommended in-

808 AABB Technical Manual

terval between removing the RBC

unit from refrigeration and placing

the glycerolized cells in the freezer

should not exceed 4hours.

Freezing and Storage

1. Place the glycerolized unit in a card-

board or metal canister and place it

in a freezer at –65 C or colder.

2. Label the top edge of the canister

with freezer tape marked with the

Whole Blood number, ABO group

and Rh type, the date frozen, and the

expiration date.

3. Do not bump or handle the frozen

cells roughly.

4. Thefreezingrateshouldbelessthan

10 mL/minute.

5. Store the frozen RBCs at –65 C or

colder for up to 10 years. For blood

of rare phenotypes, a facility’s medi-

cal director may wish to extend the

storage period. The unusual nature

of such units and the reason for re-

taining them past the routine 10-

year storage period must be docu-

mented.

Thawing and Deglycerolizing

1. Putanoverwrapontheprotective

canister containing the frozen cells

and place it in either a 37 C water-

bath or 37 C dry warmer.

2. Agitate it gently to speed thawing.

The thawing process takes at least 10

minutes. Thawed cells should be at

37 C.

3. After the cells have thawed, use a

commercial instrument for batch or

continuous-flow washing to degly-

cerolize cells. Follow the manufac-

turer’s instructions.

4. Record the lot numbers and manu-

facturer of all the solutions and soft-

ware used. Apply a “Red Blood Cells

Deglycerolized” label to the transfer

packandbesurethatthelabelin

cludes identification of the collect-

ing facility, the facility preparing the

deglycerolized cells, the ABO group

and Rh type of the cells, the Whole

Blood number, and the expiration

date and time.

5. Dilute the unit with a quantity of

hypertonic (12%) NaCl solution ap-

propriate for the size of the unit. Al-

low it to equilibrate for approxi-

mately5minutes.

6. Wash the cells with 1.6% NaCl until

deglycerolization is complete. Ap-

proximately 2 liters of wash solution

are required. To check for residual

glycerol, see Method 6.8.

7. Suspend the deglycerolized cells in

isotonic (0.9%) saline with 0.2% dex-

trose.

8. Fill the integrally attached tubing

with an aliquot of cells sealed in

such a manner that it will be avail-

able for subsequent compatibility

testing.

9. Deglycerolized RBCs must be stored

at 1 to 6 C for no longer than 24 hours.

(A closed system has been licensed

that allows storage of deglycerolized

RBCsat1to6Cfor2weeks.)

Notes

1. An aliquot of the donor’s serum or

plasma should be frozen and stored

at –65 C or colder for possible future

use if new diagnostic tests are imple-

mented.

2. When new diagnostic tests have

been implemented and stored units

do not have aliquots available for

testing, the units may have to be is-

sued with a label stating that the test

has not been performed. The reason

for distributing an untested compo-

Methods Section 6: Blood Collection, Storage, and Component Preparation 809

nent should be documented. If a

specimen from the donor is obtained

and tested after the unit was stored,

the date of testing should be noted

ontheunitwhenitisissued.

Reference

Meryman HT, Hornblower M. A method for freez-

ing and washing RBCs using a high glycerol con-

centration. Transfusion 1972;12:145-56.

Method 6.8. Red Cell

Cryopreservation Using

High-Concentration

Glycerol—Valeri Method

Principle

RBCs collected in an 800-mL primary col-

lection bag in CPDA-1 and stored at 1 to 6

C for 3 to 38 days can be biochemically re-

juvenated and frozen with 40% w/v glyc-

erol in the 800-mL primary container. See

Method 6.6 for additional information.

Materials

1. Quadruple plastic bag collection

system with 800-mL primary bag.

2. Hand sealer clips.

3. Empty 600-mL polyethylene cryo-

genic vials (eg, Corning 25702 or

Fisher 033746).

4. Sterile connection device with wa-

fers.

5. Freezer tape.

6. 600-mL transfer bag.

7. 50 mL of Red Blood Cell Processing

Solution (Rejuvesol, Cytosol Labora-

tories, Braintree, MA).

8. Heat-sealable 8" × 12" plastic bags.

9. Rejuvenation harness (Fenwal 4C1921

or Cutter 98052).

10. Sterile filtered airway needle (BD

5200), for Fenwal rejuvenation har-

ness only.

11. 500 mL of glycerolyte 57 solution

(Fenwal 4A7833) or 500 mL of 6.2 M

glycerolization solution (Cytosol

PN5500).

12. Labels—Red Blood Cells Frozen Re-

juvenated.

13. Corrugated cardboard storage box

(7" × 5.5" × 2" outside dimensions).

14. Heat sealing device.

15. Plastic bag for overwrapping.

Procedure

Preparing RBCs for Glycerolization

1. Collect 450 mL of Whole Blood in

the primary bag. Invert the bag, fold

it about 2 inches from the base, se-

cure the fold with tape, and place

the bag upright in a centrifuge. Cen-

trifuge and remove all visible super-

natant plasma. The hematocrit of

the RBC unit must be 75% ± 5%.

2. Store RBCs at 1 to 6 C in the 800-mL

primary bag, along with the adapter

port on the tubing that connects the

primary bag and transfer pack.

3. Centrifuge the stored cells to remove

all visible plasma before undertaking

rejuvenation. The gross and net

weights of the RBCs should not ex-

ceed 352 g and 280 g, respectively.

4. Transfer the plasma to the integrally

connected transfer pack, fold the in-

tegral tubing, and replace the hand

sealer clip (not crimped).

5. Attach an empty 600-mL transfer

pack to the integral tubing of the pri-

mary collection bag, using a sterile

connection device.

6. Transfer 1 mL of plasma to each of

threecryogenicvialstobeusedfor

future testing.

810 AABB Technical Manual

Biochemical Modification of the Cells

1. Using the Fenwal Rejuvenation Har-

ness: Aseptically insert the needle of

the Y-type Fenwal Harness into the

rubberstopperofa50-mLRed

Blood Cell Processing Solution bot-

tle and the coupler of the set into the

adapter port of the primary collec-

tion bag. Insert the filtered airway

needle into the rubber stopper of the

Red Blood Cell Processing Solution

bottle.

2. Using the Cutter Rejuvenation Har-

ness: Aseptically insert the vented

white spike with the drip chamber

into the rubber stopper of the Red

Blood Cell Processing Solution bot-

tle and the nonvented spike into the

special adapter port on the primary

collection bag.

3. With gentle manual agitation, allow

50 mL of Red Blood Cell Processing

Solution to flow directly into the red

cells.

4. Heat-seal the tubing of the harness

setthatconnectstheRedBloodCell

Processing Solution to the adapter

port. The second tubing of the har-

ness Y-set is used to add glycerol

(see below).

5. Completely overwrap the 800-mL

primary bag, the integrally con-

nected empty transfer pack, and the

coupler of the Y-type harness and

incubate them in a 37 C waterbath

for 1 hour.

Glycerolization

1. Remove the numbered crossmatch

segments, leaving the initial seg-

ment and number attached to the

collection bag. Weigh the unit.

2. Determine the amount of glycerol to

be added, based on the gross or net

weight of the unit, from the values

showninTable6.8-1.

3. Aseptically insert the coupler of the

rejuvenation harness into the outlet

port of the rubber stopper on the

glycerol solution bottle. For the

Fenwal harness only, insert a filtered

airway needle into the vent portion

of the glycerol bottle stopper.

4. Placethebagonashaker.Addthe

amount of glycerol shown in Table

6.8-1 for the first volume while the

bag is shaking at low speed (180 os-

cillations/minute).

5. Equilibrate the mixture for 5 min-

utes without shaking and add the

second volume. Equilibrate it for 2

minutes. Add the third volume of

glycerol, using vigorous manual

shaking.

Methods Section 6: Blood Collection, Storage, and Component Preparation 811

Table 6.8-1. Amount of Glycerol Needed for Different Weights of Red Cell Units

Gross Weight

of Unit

(grams)*

Net Weight

of Unit

(grams)

Initial

Addition of

Glycerol (mL)

Second

Addition of

Glycerol (mL)

Third

Addition of

Glycerol (mL)

Total

Glycerol

Added (mL)

222-272 150-200 50 50 250 350

273-312 201-240 50 50 350 450

313-402 241-330 50 50 400 500

*Weight of the empty 800-mL primary bag with the integrally attached transfer pack and the adapter port is 72 grams

(average).

6. Heat-seal the tubing between the

empty bottle of glycerol and the tub-

ing proximal to the adapter port. En-

sure that the transfer pack remains

integrally attached to the primary

collection bag.

7. Centrifuge the mixture of red cells

and glycerol and transfer all visible

supernatant glycerol to the transfer

pack, resuspend, and mix. Note: This

step differs from Method 6.7.

8. Sealthetubing4"fromtheprimary

collection bag, detach the transfer

pack containing the supernatant fluid,

and discard it.

9. Affix an overlay blood component

label, the facility label, and an ABO/

Rh label. Record the expiration date

on the label.

10. Weigh the unit just before freezing

and record the weight.

11. Fold over the top portion of the pri-

mary bag (approximately 2"). Place

theprimarybagintoaplasticbag

overwrap and heat-seal the outer

bag across the top so that there is as

littleairaspossiblebetweenthebags.

12. Place one vial of plasma and the

plastic bag containing the glyceroli-

zed red cells in the cardboard box.

Store the other two vials, suitably

identified, at –65 C or colder for fu-

ture testing, if needed.

13. Affix a “Red Blood Cells Frozen Reju-

venated” label, an ABO/Rh label, a

facility label, and the original unit

number on the outside of the box.

Record separately or affix on the

cardboard box the collection, freez-

ing, and expiration dates.

14. Freeze the unit in a –80 C freezer. No

more than 4 hours should be al-

lowed to elapse between the time

the unit was removed from the 4 C

refrigerator and the time the cells

are placed in the –80 C freezer.

Thawing and Deglycerolization

See Method 6.7. Note, however, that the

supernatant glycerol is removed before

freezing. Therefore, only two salt solu-

tions (the hypertonic 12% saline and the

0.9% saline-0.2% dextrose solution) are

used in the deglycerolization process.

References

1. Rejuvesol Package insert. Braintree, MA:

Cytosol Laboratories, 2002.

2. Valeri CR, Ragno G, Pivacek LE, et al. A

multicenter study of in vitro and in vivo val-

ues in human RBCs frozen with 40% (wt/vol)

glycerol and stored after deglycerolization for

15 days at 4 C in AS-3: Assessment of RBC

processing in the ACP 215. Transfusion 2001;

41:933-9.

Method 6.9. Checking the

Adequacy of

Deglycerolization of Red

Blood Cells

Principle

Glycerolization of red cells for frozen stor-

age creates a hyperosmolar intracellular

fluid, which must be restored to physio-

logically compatible levels before the cells

are transfused. Inadequately deglycerolized

red cells will be hemolyzed by contact

with normal saline, or with serum or

plasma if subjected to crossmatching.

During deglycerolization, the last solution

in contact with the cells is normal saline.

The easiest way to determine adequacy of

glycerol removal is to determine the level

of free hemoglobin (mg/dL) in the final

wash. An adequate estimate of hemolysis

can be achieved by comparing the color

of the final wash fluid with the blocks in a

commercially available color comparator.

Alternatively, normal saline can be added

to an aliquot of deglycerolized cells and

812 AABB Technical Manual

the color of the supernatant fluid evalua-

ted against the color comparator.

Materials and Equipment

1. Semiautomated instrument for de-

glycerolizing cryopreserved RBCs.

2. Transparent tubing, as part of dis-

posable material used to deglycerol-

ize individual unit.

3. Color comparator, available com-

mercially.

Procedure

1. Interrupt the last wash cycle at a

point when wash fluid is visible in

the tubing leading to the disposal bag.

2. Hold the comparator block next to

an accessible segment of tubing,

against a well-lighted white back-

ground.

3. Note coloration of the wash fluid,

whichshouldbenostrongerthan

the block, indicating 3% hemolysis

(3% of the red cells are hemolyzed).

4. If the level of hemolysis is excessive,

continue the wash process until the

color is within acceptable limits.

5. Record observation for the individ-

ual unit and for the quality assurance

program.

6. If unacceptable hemolysis occurs re-

peatedly, document corrective action.

Method 6.10. Preparation of

Fresh Frozen Plasma from

Whole Blood

Principle

Plasma is separated from cellular blood

elements and frozen to preserve the activ-

ity of labile coagulation factors. Plasma

must be placed in the freezer within the

time frame required for the anticoagulant

or collection process.

Materials

1. Freshly collected Whole Blood, ob-

tained by phlebotomy as described

in Method 6.3, in a collection unit

with integrally attached transfer con-

tainer(s).

2. Metal clips and hand sealer.

3. Clean instruments (scissors, hemo-

stats).

4. Dielectric sealer (optional).

5. Plasma extractor.

6. Freezing apparatus.

7. Refrigerated centrifuge.

8. Scale.

Procedure

1. Centrifuge blood soon after collection,

usinga“heavy”spin(seeMethod7.4).

Use a refrigerated centrifuge at 1 to 6

C unless also preparing platelets (see

Method 6.13).

2. Place the primary bag containing

centrifuged blood on a plasma ex-

tractor and place the attached satel-

lite bag on a scale adjusted to zero.

Express the plasma into the satellite

bag and weigh the plasma.

3. Sealthetransfertubingwithadi

electric sealer or metal clips but do

not obliterate the segment numbers

ofthetubing.Placeanotherseal

nearer the transfer bag.

4. Label the transfer bag with the unit

number before it is separated from

the original container. Record the

volume of plasma on the label.

5. Cut the tubing between the two seals.

The tubing may be coiled and taped

against the plasma container, leaving

thesegmentsavailableforanytest

ing desired.

Methods Section 6: Blood Collection, Storage, and Component Preparation 813

6. Place the plasma at –18 C or colder

within the time frame required for the

anticoagulant or collection process.

Method 6.11. Preparation of

Cryoprecipitated AHF from

Whole Blood

Principle

Coagulation Factor VIII (antihemophilic

factor, AHF) can be concentrated from

freshly collected plasma by cryoprecipita-

tion. Cryoprecipitation is accomplished

by slow thawing, at 1 to 6 C, plasma that

has been prepared for freezing within the

time frame required for the anticoagulant

or collection process.

Materials

1. Freshly collected Whole Blood, ob-

tained by phlebotomy as described

in Method 6.3, in a collection unit

with at least two integrally attached

transfer containers.

2. Metal clips and hand sealer.

3. Clean instruments (scissors, hemo-

stats).

4. Dielectric sealer (optional).

5. Plasma extractor.

6. Refrigerated centrifuge.

7. Freezing apparatus: suitable freezing

devices include blast freezers or me-

chanical freezers capable of main-

taining temperatures of –18 C or

colder; dry ice; or an ethanol dry ice

bath. In a bath of 95% ethanol and

chipped dry ice, freezing will be

complete in about 15 minutes.

8. 1 to 6 C circulating waterbath or re-

frigerator.

9. Scale.

Procedure

1. Collect blood in a collection unit

with two integrally attached transfer

containers.

2. Centrifuge blood shortly after collec-

tion at 1 to 6 C, using a “heavy” spin

(see Method 7.4). Collect at least 200

mL (205 g) of cell-free plasma for

processing into cryoprecipitate.

3. Promptly place plasma in a freezing

device so that freezing is started

within the time frame required for

the anticoagulant or collection pro-

cess. Plasma containers immersed in

liquid must be protected with a plas-

tic overwrap.

4. Allow the frozen plasma to thaw at 1

to6Cbyplacingthebagina1to6C

circulating waterbath or in a refrig-

erator. If thawed in a waterbath, use

a plastic overwrap (or other means)

to keep container ports dry.

5. Whentheplasmahasaslushycon-

sistency, separate liquid plasma from

the cryoprecipitate by one of the

procedures below:

a. Centrifuge the plasma at 1 to 6

C using a “heavy” spin. Hang the

baginaninvertedpositionand

allow the separated plasma to

flow rapidly into the transfer

bag, leaving the cryoprecipitate

adhering to the sides of the pri-

mary bag. Separate the cryo-

precipitate from the plasma

promptly, to prevent the cryo-

precipitate from dissolving and

flowingoutofthebag.Tento

15 mL of supernatant plasma

maybeleftinthebagforresus

pension of the cryoprecipitate

after thawing. Refreeze the cryo-

precipitate immediately.

b. Place the thawing plasma in a

plasma expressor when appro-

814 AABB Technical Manual

ximately one-tenth of the con-

tents is still frozen. With the

baginanuprightposition,al

low the supernatant plasma to

flow slowly into the transfer

bag, using the ice crystals at

the top as a filter. The cryo-

precipitate paste will adhere to

the sides of the bag or to the ice.

Seal the bag when about 90% of

the cryoprecipitate-reduced

plasma has been removed and

refreeze the cryoprecipitate im-

mediately.

6. The cryoprecipitate should be re-

frozen within 1 hour of thawing.

Store at –18 C or colder, preferably

–30 C or colder, for up to 12 months

from the date of blood collection.

Note

Cryoprecipitated AHF may be prepared

fromFreshFrozenPlasmaatanytime

within 12 months of collection. The expi-

ration date of Cryoprecipitated AHF is 12

months from the date of phlebotomy, not

from the date it was prepared.

Method 6.12. Thawing and

Pooling Cryoprecipitated

AHF

Principle

Cryoprecipitated AHF should be rapidly

thawed at 30 to 37 C but should not re-

main at this temperature once thawing is

complete. The following method permits

rapid thawing and pooling of this product.

Materials

1. Circulating waterbath at 37 C (water-

baths designed for thawing plasma

are available commercially, as are

specially designed dry heat devices).

2. Medication injection ports.

3. Sterile 0.9% sodium chloride for in-

jection.

4. Syringes and needles.

Procedure

1. Cover the container with a plastic

overwrap to prevent contamination

of the ports with unsterile water, or

use a device to keep the containers

upright with the ports above water.

2. Resuspend the thawed precipitate

carefully and completely, either by

kneading it into the residual 10 to 15

mL of plasma or by adding approxi-

mately 10 mL of 0.9% sodium chlo-

ride and gently resuspending.

3. Pool by inserting a medication injec-

tion site into a port of each bag. As-

pirate contents of one bag into a sy-

ringe and inject into the next bag. Use

the ever-increasing volume to flush

each subsequent bag of as much dis-

solved cryoprecipitate as possible,

until all contents are in the final bag.

4. Thawed Cryoprecipitated AHF must

be stored at room temperature. If

pooled,itmustbeadministered

within 4 hours. Thawed single units,

if not entered, must be administered

within 6 hours of thawing if intend-

ed for replacement of Factor VIII.

Pools of thawed individual units may

not be refrozen.

Method 6.13. Preparation of

Platelets from Whole Blood

Principle

Platelet-rich plasma is separated from

Whole Blood by “light-spin” centrifugation

and the platelets are concentrated by

Methods Section 6: Blood Collection, Storage, and Component Preparation 815

“heavy-spin” centrifugation, with subse-

quent removal of supernatant plasma (see

Method 7.4).

Materials

1. Freshly collected Whole Blood, ob-

tained by phlebotomy as described

in Method 6.3, in a collection unit

with two integrally attached transfer

containers. The final container must

be a plastic approved for platelet

storage. Keep blood at room temper-

ature (20 to 24 C) before separating

platelet-rich plasma from the red cells.

This separation must take place

within 8 hours of phlebotomy.

2. Metal clips and hand sealer.

3. Scissors, hemostats.

4. Plasma extractor.

5. Dielectric sealer (optional).

6. Centrifuge, calibrated as in Method

7.4.

7. Scale.

8. Rotator.

Procedure

1. Do not chill the blood at any time

before or during platelet separation.

If the temperature of the centrifuge

is 1 to 6 C, set the temperature con-

trol of the refrigerated centrifuge at

20 C and allow the temperature to

risetoapproximately20C.Centri

fuge the blood using a “light” spin

(see Method 7.4).

2. Express the platelet-rich plasma into

the transfer bag intended for platelet

storage. Seal the tubing twice be-

tween the primary bag and Y con-

nector of the two satellite bags and

cut between the two seals. Place the

red cells at 1 to 6 C.

3. Centrifuge the platelet-rich plasma

at 20 C using a “heavy” spin (see

Method 7.4).

4. Express the platelet-poor plasma

into the second transfer bag and seal

the tubing. Some plasma should re-

main on the platelet button for stor-

age, but no exact volume can be des-

ignated. AABB Standards for Blood

Banks and Transfusion Services re-

quires that sufficient plasma remain

with the platelet concentrate to main-

tain the pH at 6.2 or higher for the

entire storage period. This usually

requires a minimum of 35 mL of

plasma when storage is at 20 to 24 C,

but50to70mLispreferable.

5. The platelet concentrate container

should be left stationary, with the la-

bel side down, at room temperature

for approximately 1 hour.

6. Resuspend the platelets in either of

the following ways:

a. Manipulate the platelet con-

tainer gently by hand to achieve

uniform resuspension.

b. Place the container on a rotator

at room temperature. The slow,

gentle agitation should achieve

uniform resuspension within 2

hours.

7. Maintain the platelet suspensions at

20 to 24 C with continuous gentle

agitation.

8. Platelets should be inspected before

issue to ensure that no platelet ag-

gregates are visible.

Notes

The platelet-reduced plasma may be frozen

promptlyandstoredasFreshFrozen

Plasma (FFP), if the separation and freez-

ing are completed within the time frame

required for the anticoagulant or collec-

tion process. The volume of FFP prepared

after platelet preparation will be substan-

tially less than that prepared directly from

Whole Blood.

816 AABB Technical Manual

Reference

Silva MA, ed. Standards for blood banks and trans-

fusion services. 23rd ed. Bethesda, MD: AABB,

2005:30.

Method 6.14. Preparation of

Prestorage Platelets

Leukocytes Reduced from

Whole Blood

Principle

Prestorage leukocyte-reduced platelets

may be prepared from whole blood using

in-line filtration of the platelet-rich plasma

(PRP). The resulting intermediate product

is a filtered PRP, from which a leukocyte-

reduced platelet concentrate and leuko-

cyte-reduced plasma may be manufac-

tured. Materials and procedures are the

same as for Method 6.13, except that the

PRP is expressed through an in-line filter.

References

1. Sweeney JD, Holme S, Heaton WAL, Nelson E.

Leukodepleted platelet concentrates pre-

pared by in-line filtration of platelet rich

plasma. Transfusion 1995;35:131-6.

2. Sweeney JD, Kouttab N, Penn LC, et al. A

comparison of prestorage leukoreduced

whole blood derived platelets with bedside

filtered whole blood derived platelets in

autologous stem cell transplant. Transfusion

2000;40:794-800.

Method 6.15. Removing

Plasma from Platelet

Concentrates (Volume

Reduction)

Principle

Although optimal storage of platelets re-

quires an adequate volume of plasma, a

few patients may not tolerate large-vol-

ume infusion. Stored platelets may be

centrifuged and much of the plasma re-

moved shortly before transfusion, but

appropriate resuspension is essential.

The platelets must remain at room tem-

perature, without agitation, for 20 to 60

minutes before resuspension into the re-

maining plasma. Transfusion must take

place within 4 hours of the time the

platelet bag was entered. Volume reduc-

tion can be performed on individual or

pooled units.

No consensus exists regarding the opti-

mal centrifugation rate. One study1found

35% to 55% platelet loss in several units

centrifuged at 500 × gfor 6 minutes, com-

pared with 5% to 20% loss in units centri-

fuged at 5000 × gfor 6 minutes or 2000 × g

for 10 minutes. The authors recommend

2000 × gfor 10 minutes, to avoid any risk

that a higher centrifugal force might inflict

on the plastic container. A study by Moroff

et al2found mean platelet loss to be less

than 15% in 42 units centrifuged at 580 × g

for 20 minutes. High gforces are of theoret-

ical concern because they may damage the

platelets when they are forced against the

wall of the container and also increase the

possibility of container breakage.

Materials

1. Platelet concentrate(s), prepared as

described in Method 6.13.

2. Metal clips and hand sealer.

3. Scissors, hemostats.

4. Dielectric sealer (optional).

5. Centrifuge, calibrated as in Method

7.4.

6. Plasma extractor.

Procedure

1. Pool platelets, if desired, into a trans-

fer pack, using standard technique.

Single platelet concentrates may

need volume reduction for pediatric

Methods Section 6: Blood Collection, Storage, and Component Preparation 817

recipients. Apheresis components may

be processed directly.

2. Centrifuge at 20 to 24 C, using one of

the following protocols:

a. 580 ×gfor 20 minutes.

b. 2000 ×gfor 10 minutes.

c. 5000 ×gfor 6 minutes.

3. Without disturbing the contents,

transfer the bag to a plasma extrac-

tor. Remove all but 10 to 15 mL plasma

from single units, or somewhat more

volume, proportionately, from a pool

or from a component prepared by

apheresis.

4. Mark expiration time on bag as 4

hoursafterthetimetheunitwasen

tered.

5. Leave bag at 20 to 24 C without agi-

tation for 20 minutes if centrifuged

at 580 ×g, or for 1 hour if centrifuged

at 2000 or 5000 ×g.

6. Resuspend platelets as described in

Method 6.13.

Notes

1. If a sterile connection device is used

for removing plasma from a hema-

pheresis component or individual

platelet concentrate, the unit can be

considered sterile and it is not nec-

essary to impose the 4-hour expira-

tion interval required for entered

Platelets. However, no data exist to

support storage of reduced volume

platelet concentrates; therefore, it is

preferable to transfuse them as soon

as possible.

2. Reduced-volume platelet concentrates

may not be distributed as a licensed

product.

3. Platelets that have been pooled must

be used within 4 hours of entering

the units, whether or not they have

been volume-reduced. Pooled plate-

lets may not be distributed as a li-

censed product.

References

1. Simon TL, Sierra ER. Concentration of plate-

let units into small volumes. Transfusion

1984;24:173-5.

2. Moroff G, Friedman A, Robkin-Kline L, et al.

Reduction of the volume of stored platelet

concentrates for use in neonatal patients.

Transfusion 1984;24:144-6.

818 AABB Technical Manual

Methods Section 7: Quality Control

Methods Section 7

Quality Control

Method 7.1. Quality Control

for Copper Sulfate Solution

Either of the two methods presented be-

low is acceptable for quality control of cop-

per sulfate solution.

Method 7.1.1. Functional Validation of

Copper Sulfate Solution

Principle

Copper sulfate solution can be checked for

suitability in donor screening by observing

the behavior (sinking or floating) of drops

of blood of known hemoglobin concentra-

tion.

Materials

1. Copper sulfate—specific gravity 1.053.

2. Capillary tubes.

3. Worksheet for recording results.

Procedure

1. Obtain several (three to six, if possi-

ble) blood samples with known he-

moglobin levels. Samples should in-

clude hemoglobin levels slightly

above and below 12.5 g/dL.

2. Gently place a drop of each blood

sample into a vial of copper sulfate

solution of stated specific gravity of

1.053.

3. Drops of all blood samples with he-

moglobin at or above 12.5 g/dL must

sink and those with hemoglobin lev-

els below 12.5 g/dL must float.

4. Record the date of testing; the man-

ufacturer, lot number, and expiration

date of the copper sulfate; sample

identity; the results; and the identity

of the person performing the test.

5. Document the corrective action taken

if the results are outside acceptable

limits.

819

Section 7

Method 7.1.2. Use of Measurement

Instruments for Specific Gravity, Density,

or Refractive Index of Copper Sulfate

Solution

Principle

The specific gravity, density, or refractive

index of the copper sulfate solution can

be measured directly and the result com-

pared with the value stated by the manu-

facturer. An error in the specific gravity

reading of ±0.0001 corresponds to ±0.06

g/dL hemoglobin in whole blood.1There-

fore, a copper sulfate solution with a spe-

cific gravity of 1.053 ± 0.0003 would result

in a corresponding hemoglobin range of

12.5 ± 0.18 g/dL.

Materials

1. Copper sulfate—specific gravity 1.053.

2. Measurement instruments

•For specific gravity—high-preci-

sion hydrometer, with gradations

of 0.0005 or smaller.

•For refractive index—refracto-

meter.

3. Pipette.

4. For specific gravity method—gradu-

ated cylinder or other container suit-

able for use with a hydrometer.

5. Alcohol for cleaning hydrometer.

6. Worksheet for recording results.

Procedure

Hydrometer method:

1. Wipe the hydrometer clean with al-

cohol and dry it.

2. Gently lower the hydrometer into the

solution until it floats on its own.

Drops of solution on the stem will

cause inaccurate readings.

3. To read, observe a point just below

the plane of the liquid surface and

then raise the line of vision until the

surface is seen as a straight line in-

stead of an ellipse. Read at the point

where the line intersects the scale on

the instrument.

Refractometer method:

1. Follow manufacturer’s instructions for

operation and maintenance of the in-

strument.

2. Add a drop of copper sulfate to the

refractometer.

3. Observe refractive index.

Note: Some refractometers designed

for urine analysis provide both a re-

fractive index scale and a specific

gravity scale. Do not read specific

gravity directly from this type of

refractometer.

4. A copper sulfate solution with a spe-

cific gravity of 1.053 will have a re-

fractive index of about 1.3425 at room

temperature.2

Both methods:

1. Record the results; the date of test-

ing; the manufacturer, lot number,

and expiration date of the copper

sulfate solution; the identity of the

person performing the test; and the

identification of the instrument used

for measurement.

2. Document the corrective action taken

if the results are outside the accept-

able limits.

Note

A liquid densitometer may be used to

measure density directly. At 4 C, the den-

sity of the solution will be exactly the

same as the specific gravity (specific

gravity = density of solution/density of

water; density of water at 4 C = 1 g/mL).

When density is measured at room tem-

perature, a conversion factor of 0.9970

(the density of water at 25 C) is used to

calculate specific gravity.2Specific gravity

of copper sulfate solution at 25 C = the

820 AABB Technical Manual

observed density in g/mL divided by

0.9970 g/mL.

References

1. Phillips RA, Van Slyke DD, Hamilton PB, et al.

Measurement of specific gravities of whole

blood and plasma by standard copper sulfate

solutions. J Biol Chem 1950;183:305-30.

2. Blood hemoglobin screening (specific gravity

method). Technical Reference Document 12.

Arlington, TX: Ricca Chemical Company,

2002.

Method 7.2. Standardization

and Calibration of

Thermometers

Principle

Thermometers used during laboratory

testing and in the collection (donor suit-

ability), processing, and storage of blood

components and reagents should be cali-

brated and standardized to ensure accu-

rate indication of temperatures. Calibra-

tion should be performed at temperatures

close to the temperature at which the

thermometers will be used. Over time,

liquid-in-glass thermometers may give a

different reading at a given temperature

because of permanent changes in the vol-

ume of the bulb related to relaxation of

the glass.1Each thermometer should be

calibrated before initial use and periodi-

cally thereafter, as well as any time there

is reason to suspect change or damage.

Calibration must be verified for all elec-

tronic thermometers, even those describ-

ed as “self-calibrating.”

Materials

1. National Institute of Standards and

Technology (NIST)-certified thermo-

meterorthermometerwithNIST-

traceable calibration certificate.

2. Thermometer to be calibrated.

3. Suitable container, eg, 250-500 mL

beaker.

4. Water.

5. Crushed ice.

6. 37 C waterbath.

7. Worksheet for recording results.

Method 7.2.1. Liquid-in-Glass Laboratory

Thermometers

Procedure

1. Before choosing a thermometer for a

particular application, consider all

the governing factors; be sure that

the thermometer will be used at its

proper immersion; and follow the

manufacturer’s instructions for its

proper use. When using a certified

thermometer, read and follow the

applicable notes. Be sure to include

any correction factors noted on the

certificate for the NIST-traceable

thermometer and apply them in

calculations.

2. Categorize the thermometers by key

factors, such as immersion, incre-

ments, and temperature of intended

use. Test them in groups, comparing

similar thermometers. Do not attempt

to compare dissimilar thermometers

in a single procedure.

3. Number each thermometer being

tested (eg, place a numbered piece

of tape around the top of each ther-

mometer or use the manufacturer’s

serial number).

4. Perform calibration with water at a

temperature close to that which the

thermometer will monitor.

a. To calibrate at 37 C, place the

thermometers to be tested and

the NIST thermometer at a uni-

form depth in a standard 37 C

waterbath, making sure that the

tips of all devices are at the same

level in the liquid.

Methods Section 7: Quality Control 821

b. To calibrate at 1 to 6 C, fill a

suitable container with water.

Add crushed ice until the ap-

proximate desired temperature

is reached. Place the thermom-

eters to be tested and the NIST

thermometer at a uniform depth

in the water/ice mixture, mak-

ingsurethatthetipsofallde

vices are at the same level and

are in the liquid, not the upper

ice.

5. Stir constantly in a random motion

until the temperature equilibrates,

approximately 3 to 5 minutes.

6. Observe temperatures. Record each

thermometer’s identification and re-

sults. Acceptance criteria depend on

the level of precision required, but,

for most blood banking applications,

agreement within 1 C between the two

thermometers may be considered

acceptable. If the reading varies by

more than one degree from the stan-

dard, the thermometer may be re-

turned to the distributor (if newly

purchased), labeled with the correc-

tion factor (degrees different from

theNISTthermometer)thatmustbe

applied to each reading, or dis-

carded.

7. Complete the calibration record

with the date of testing and identity

of the person who performed the

test.

Notes

1. If a thermometer is to be used for

temperatures over a range greater

than a few degrees (eg, 10 degrees), a

three-point calibration should be

performed. Use water of appropriate

temperature. Test at temperatures

just below, just above, and at the

midway point of intended use. All

results should be within 1 C of the

NIST thermometer to be considered

acceptable.

2. Thermometers should be observed

routinely for any split in the column

because this will cause inaccurate

readings. The methods for reuniting

the separation can be found in NCCLS

Standard I2-A2.2When this occurs,

document corrective action and

recalibrate the thermometer.

References

1. WiseJA.Aprocedurefortheeffectiverecali

bration of liquid-in-glass thermometers.

NIST Special Publication 819. Gaithersburg,

MD: National Institute of Standards and

Technology, 1991.

2. Temperature calibration of water baths, in-

struments, and temperature sensors. 2nd ed;

approved standard I2-A2 Vol. 10 No. 3. Wayne,

PA: National Committee for Clinical Labora-

tory Standards, 1990.

Method 7.2.2. Electronic Oral

Thermometers

Procedure

1. Useanyofthefollowingmethodsto

verify calibration:

a. Follow manufacturer’s instruc-

tions for verifying calibration.

b. Use a commercially available

calibration device by following

the instructions provided by

the device’s manufacturer.

c. Calibrate the thermometer by

inserting the probe in a 37 C

waterbath with a NIST-certified

thermometer.

2. A result is acceptable if the reading

on the thermometer agrees with the

NIST thermometer within 0.1 C. If

expected results are not achieved,

unsatisfactory thermometers should

be returned to the distributor. Docu-

ment corrective actions.

822 AABB Technical Manual

3. Record the date of testing, thermom-

eter identification numbers, temper-

ature readings, and the identity of the

person performing the test.

Method 7.3. Testing Blood

Storage Equipment Alarms

Blood storage refrigerators and freezers

must be equipped with a system for con-

tinuous temperature monitoring and an

audible alarm. If a storage unit goes into

alarm, it is essential that personnel know

the appropriate actions to take. Direc-

tions for such events should be available

in a conspicuous location, and personnel

should be trained to initiate these actions

if the temperature cannot be corrected

rapidly. The alarm on each storage unit

should be checked periodically for proper

functioning. Monthly checks are appro-

priate until consistent behavior of a par-

ticular storage unit has been demon-

strated. Thereafter, alarms should be

tested regularly and frequently enough to

achieve and maintain personnel compe-

tency as well as to detect malfunctions.

For equipment in good condition, quar-

terly checks are usually sufficient. Be-

cause alarms may be disconnected or si-

lenced during repairs, it is also prudent to

verify alarm functioning after repairs.

The high and low temperatures of activa-

tion must be checked and the results re-

corded. AABB Standards for Blood Banks

and Transfusion Services1(p5) requires that

thealarmbesettoactivateatatempera

ture that will allow appropriate interven-

tion before blood or components reach an

undesirable temperature. Because of the di-

versity of equipment available, it is not pos-

sibletogivespecificinstructions for all ap-

plicable alarm systems. If the equipment

user’s manual does not provide suitable di-

rections for testing the alarm, consult the

manufacturer or other equipment storage

expert. The facility procedures manual

must include a detailed description of the

method(s) in local use. (See Appendix 10 for

quality control testing intervals.)

Method 7.3.1. Testing Refrigerator Alarms

Principle

Refrigerator temperatures may increase

beyond acceptable limits for several rea-

sons, including:

1. Improperly closed door.

2. Insufficient refrigerant.

3. Compressor failure.

4. Dirty or blocked heat exchanger.

5. Loss of electrical power.

Materials

1. Calibrated thermometer.

2. Pan large enough to hold the ther-

mocouple container.

3. Water.

4. Crushed ice.

5. Table salt.

6. Worksheet for recording results.

Procedure

1. Verify that the alarm circuits are op-

erating, the alarm is switched on, and

the starting temperature is 1 to 6 C.

Immerse an easy-to-read calibrated

thermometer in the container with

the alarm thermocouple.

For low activation:

2. Place the container with the thermo-

couple and thermometer in a pan

containing an ice and water slush at

a temperature of –4 C or colder. To

achieve this temperature, add several

spoonfuls of table salt to the slush.

3. Close the refrigerator door to avoid

changing the temperature of the

storage compartment. Keep the con-

tainer in the pan of cold slush, and

Methods Section 7: Quality Control 823

gently agitate it periodically until the

alarm sounds.

4. Record this temperature as the low-

activation temperature.

For high activation:

5. Place the container with thermocou-

ple and thermometer in a pan con-

taining cool water (eg, 12 to 15 C).

6. Close the refrigerator door. Allow the

fluid in the container to warm slowly,

with occasional agitation.

7. Record the temperature at which the

alarm sounds as the high-activation

temperature.

8. Record the date of testing, the refrig-

erator identification, the thermome-

ter identification, and the identity of

the person performing the test.

9. If temperatures of activation are too

low or too high, take appropriate

corrective actions such as those sug-

gested by the manufacturer, record

the nature of the corrections, and re-

peat the alarm check to document

that the corrections were effective.

Notes

1. The thermocouple for the alarm should

be easily accessible and equipped

with a cord long enough so that it

can be manipulated easily.

2. Thethermocoupleforthecontinu

ous temperature monitor need not

be in the same container as that of

the alarm. If it is in the same con-

tainer, a notation should be made in

the records that explains any out-

of-range temperature registered as a

result of the alarm check.

3. When the temperatures of alarm ac-

tivation are checked, the temperature

change should occur slowly enough

so that the measurements and re-

cording are accurate. Too rapid a

change in temperature may give the

false impression that the alarm does

not sound until an inappropriate

temperature is registered.

4. Thelowtemperatureofactivation

should be greater than 1 C (eg, 1.5 C);

the high temperature of activation

should be less than 6 C (eg, 5.5 C).

5. Alarms should sound simultaneou-

sly at the site of the refrigerator and

at the location of remote alarms,

when employed. If remote alarms

are used, the alarm check should in-

clude a verification that the alarm

sounded at the remote location.

6. The amount of fluid in which the

thermocouple is immersed must be

no larger than the volume of the

smallest component stored in that

refrigerator. The thermocouple may

be immersed in a smaller volume,

but this means that the alarm will

go off with smaller temperature

changes than those registered in a

larger volume of fluid. Excessive sen-

sitivity may create a nuisance.

7. With the one-time assistance of a

qualified electrician, the required re-

frigerator alarm checks of units with

virtually inaccessible temperature

probes can be performed with an

electrical modification cited by Wenz

and Owens.2

References

1. Silva MA, ed. Standards for blood banks and

transfusion services. 23rd ed. Bethesda, MD:

AABB, 2005.

2. Wenz B, Owens RT. A simplified method for

monitoring and calibrating refrigerator alarm

systems. Transfusion 1980;20:75-8.

Method 7.3.2. Testing Freezer Alarms

Principle

Freezer temperatures may rise to unac-

ceptable levels for a variety of reasons.

824 AABB Technical Manual

Common causes of rising temperatures

include:

1. Improperly closed freezer door or lid.

2. Low level of refrigerant.

3. Compressor failure.

4. Dirty or blocked heat exchanger.

5. Loss of electrical power.

Materials

1. Protection for the freezer contents,

eg, a blanket.

2. Calibrated thermometer or thermo-

couple independent from that built

into the system.

3. Warm water or an oven mitt.

4. Worksheet for recording results.

Procedure

1. Protect frozen components from ex-

posure to elevated temperatures dur-

ing the test.

2. Use a thermometer or thermocou-

ple, independent from that built into

the system, that will accurately indi-

cate the temperature of alarm acti-

vation. Compare these readings with

the temperatures registered on the

recorder.

3. Warm the alarm probe and thermo-

meter slowly (eg, in warm water, by

an oven-mitt-covered hand, expo-

sure to air). The specific temperature

of activation cannot be determined

accurately during rapid warming,

and the apparent temperature of ac-

tivation will be too high.

4. Record the temperature at which the

alarm sounds, the date of testing,

the identity of the person perform-

ing the test, the identity of the

freezer and calibrating instrument,

and any observations that might

suggest impaired activity.

5. Return the freezer and the alarm sys-

tem to their normal conditions.

6. If the alarm sounds at too high a

temperature, take appropriate cor-

rective actions such as those sug-

gested by the manufacturer, record

the nature of the correction, and re-

peat the alarm check to document

that the corrections were effective.

Notes

1. Alarms should sound simultaneou-

sly at the site of the freezer and at

the location of the remote alarms,

when employed. If remote alarms

are used, the alarm check should in-

clude a verification that the alarm

sounded at the remote location.

2. Test battery function, electrical cir-

cuits, and power-off alarms more

frequently than the activation tem-

perature. Record function, freezer

identification, date, and identity of

person performing the testing.

3. For units with the sensor installed in

the wall or in air, apply local warmth

to the site or allow the temperature

of the entire compartment to rise to

the point at which the alarm sounds.

Remove the frozen contents or pro-

tect them with insulation while the

temperature rises.

4. For units with the thermocouple lo-

cated in antifreeze solution, pull the

container and the cables outside the

freezer chest for testing, leaving the

door shut and the contents protected.

5. For units with a tracking alarm that

sounds whenever the temperature

reaches a constant interval above

the setting on the temperature con-

troller, set the controller to a warmer

setting and note the temperature in-

terval at which the alarm sounds.

Methods Section 7: Quality Control 825

6. Liquid nitrogen freezers must have

alarm systems that activate at an un-

safe level of contained liquid nitro-

gen.

Method 7.4. Functional

Calibration of Centrifuges for

Platelet Separation

Principle

Successful preparation of platelet concen-

trates requires adequate but not excessive

centrifugation; the equipment used must

perform in a consistent and dependable

manner. Each centrifuge used to prepare

platelets should be calibrated upon re-

ceipt and after adjustment or repair.

Functional calibration of the centrifuge

for both the preparation of platelet-rich

plasma (PRP) from whole blood and sub-

sequent preparation of platelet concen-

trate from PRP can be performed during

thesameprocedure.

Materials

1. Freshly collected whole blood, ob-

tained by phlebotomy into a bag with

two integrally attached transfer con-

tainers.

2. A specimen of blood from the donor,

anticoagulated with EDTA and col-

lected in addition to the specimens

drawn for routine processing.

3. Metal clips and hand sealer or di-

electric sealer.

4. Clean instruments (scissors, hemo-

stats, tubing stripper).

5. Plasma extractor.

6. Centrifuge suitable for preparation

of platelet concentrates.

7. Worksheet for recording results.

Procedure

Preparation of Platelet-Rich Plasma

1. Perform a platelet count on the anti-

coagulated specimen. If the platelet

count is below 133,000/µL, do not

use this donor’s blood for calibration.

2. Calculateandrecordthenumberof

platelets in the Whole Blood unit: pla-

telets/µL×1000 ×mL of whole blood

= number of platelets in whole blood.

3. Prepare PRP at a selected speed and

time. (See “light spin” in Table 7.4-1

or guidance provided by the centri-

fuge manufacturer.)

4. Place a temporary clamp on the tub-

ing so that one satellite bag is closed

off. Express the PRP into the other

satellite bag. Seal the tubing close to

the primary bag, leaving a long sec-

tion of tubing, the “tail.” Disconnect

the two satellite bags from the pri-

mary bag. Do not remove the tem-

porary clamp between the satellite

bags until the platelets are prepared

(see next section).

5. Strip the tubing and “tail” several

times so that they contain a repre-

sentative sample of PRP.

6. Seal off a segment of the “tail” and

disconnect it, so that the bag of PRP

remains sterile.

7. Perform a platelet count on the sam-

ple of PRP in the sealed segment.

Calculateandrecordthenumberof

platelets in the bag of PRP: platelets/

µL×1000 ×mL of PRP = number of

platelets in PRP.

8. Calculate and record percent yield:

(number of platelets in PRP ×100)

divided by (number of platelets in

whole blood) = % yield.

9. Repeat the above process three or four

times with different donors, using

different speeds and times of centri-

fugation, and compare the yields

826 AABB Technical Manual

achieved under each set of test con-

ditions.

10. Select the shortest time/lowest speed

combination that results in the high-

est percent of platelet yield without

unacceptable levels of red cell con-

tent in the PRP.

11. Record the centrifuge identification,

the calibration settings selected, the

date, and the identity of the person

performing the calibration.

Preparation of Platelets

1. Centrifuge the PRP (as prepared

above) at a selected time and speed

to prepare platelets. (See “heavy spin”

in Table 7.4-1 or guidance provided

by the centrifuge manufacturer.)

2. Remove the temporary clamp be-

tween the two satellite bags and ex-

press the platelet-reduced plasma

into the second attached satellite

bag, leaving approximately 55 to 60

mL volume in the platelet bag. Seal

the tubing, leaving a long section of

tubing attached to the platelet bag.

3. Allow the platelets to rest for approx-

imately 1 hour.

4. Place the platelets on an agitator for

at least 1 hour to ensure that they are

evenly resuspended. Platelet counts

performed immediately after centri-

fugation will not be accurate.

5. Strip the tubing several times, mix-

ing tubing contents well with the

contents of the platelet bag. Seal off

asegmentofthetubinganddiscon

nect it, so that the platelet bag re-

mains sterile.

6. Perform a platelet count on the con-

tents of the segment.

7. Calculateandrecordthenumberof

platelets in the concentrate: platelets/

µL×1000 ×mL of platelets = number

of platelets in platelet concentrate.

8. Calculate and record percent yield.

9. Repeat the above process with PRP

from different donors, using differ-

ent speeds and times of centrifuga-

tion, and compare the yields achieved

under each set of test conditions.

Methods Section 7: Quality Control 827

Table 7.4-1. Centrifugation for Component Preparation

Heavy Spin

}

Red cells

Platelets from whole blood 55000 ×

, 5 minutes*

}

Cell- free plasma

Cryoprecipitate 55000 ×

, 7 minutes*

Light Spin

Platelet-rich plasma 2000 ×

, 3 minutes*

To calculate relative centrifugal force in

rcf (in

) = 28.38 ×R†×(rpm/1000)2

*Times include acceleration but not deceleration times. Times given are approximations only. Each individual centrifuge

must be evaluated for the preparation of the various components.

†R = radius of centrifuge rotor in inches.

10. Select the shortest time/lowest speed

combination that results in the high-

est percent of platelet yield in the

platelet concentrate.

11. Record the centrifuge identification,

the calibration settings selected, the

date, and the identity of the person

performing the calibration.

Notes

1. It is not necessary to perform func-

tional recalibration of a centrifuge

unless the instrument has under-

gone adjustments or repairs, or

component quality control indicates

that platelet counts have fallen be-

low acceptable levels. However,

timer, speed, and temperature cali-

brations of the centrifuge should oc-

cur on a regularly scheduled basis

(see Appendix 10).

2. Each centrifuge used for preparing

platelets must be calibrated individ-

ually. Use the conditions determined

to be optimal for each instrument.

3. When counting platelet samples on

an instrument intended for whole

blood, it may be necessary to use a

correction factor to obtain accurate

results.

4. When determining the appropriate

time and speed of centrifugation,

consideration should also be given to

other products that will be prepared

from the whole blood. Final size and

hematocrit of red cell and plasma

volume made available for further

processing are important factors to

consider.

References

1. Kahn R, Cossette I, Friedman L. Optimum

centrifugation conditions for the preparation

of platelet and plasma products. Transfusion

1976;16:162-5.

2. McShine R, Das P, Smit Sibinga C, Brozovic B.

Effect of EDTA on platelet parameters in

blood and blood components collected with

CPDA-1. Vox Sang 1991;61:84-9.

Method 7.5. Functional

Calibration of a Serologic

Centrifuge

Principle

Each centrifuge should be calibrated

upon receipt, after adjustments or repairs,

and periodically. Calibration evaluates the

behavior of red cells in solutions of differ-

ent viscosities, not the reactivity of differ-

ent antibodies.

For Immediate Agglutination

Materials

1. Test tubes, 10 ×75 mm or 12 ×75

mm (whichever size is routinely

used in the laboratory)

2. Worksheet for recording results.

3. For saline-active antibodies:

■SerumfromagroupAperson

(anti-B) diluted with 6% albu-

min to give 1+ macroscopic ag-

glutination (3 mL of 22% bovine

albumin + 8 mL of normal sa-

line = 6% bovine albumin). See

Method 1.5.

■Positive control: Group B red cells

in a 2% to 5% saline suspension.

■Negative control: Group A red cells

in a 2% to 5% saline suspension.

4. For high-protein antibodies:

■Anti-D diluted with 22% or 30%

albumin to give 1+ macro-

scopic agglutination.

■Positive control: D+ red cells in

a2%to5%salinesuspension.

■Negative control: D– red cells in

a2%to5%salinesuspension.

828 AABB Technical Manual

Procedure

1. For each set of tests (saline and high-

protein antibodies), label five test

tubes for positive reactions and five

for negative reactions.

2. In quantities that correspond to rou-

tine use, add diluted anti-B to each

of 10 tubes for the saline test and

add diluted anti-D to each of 10 tubes

for the high-protein test. Add serum

and reagents in quantities that cor-

respond to routine use.

3. Add the appropriate control cell sus-

pension to one set of tubes (one pos-

itiveandonenegativetubeforthe

saline test, and one positive and one

negative tube for the high-protein

antibody test). Centrifuge immedi-

ately for the desired time interval

(eg, 10 seconds).

4. Observe each tube for agglutination

and record observations. (See exam-

ple in Table 7.5-1.)

5. Repeat steps 2 and 3 for each time

interval (eg, 15, 20, 30, and 45 sec-

onds). Do not allow cells and sera to

incubate before centrifugation.

6. Select the optimal time of centrifu-

gation, which is the shortest time re-

quired to fulfill the following criteria:

a. Agglutination in the positive

tubesisasstrongasdetermined

in preparing reagents.

b. There is no agglutination or

ambiguity in the negative tubes.

c. The cell button is clearly delin-

eated and the periphery is

sharply defined, not fuzzy.

d. The supernatant fluid is clear.

e. The cell button is easily resus-

pended.

IntheexampleshowninTa

ble 7.5-1, these criteria are met

by the 30-second and the 45-

second spins; the optimal time

for these tests in this centrifuge

is 30 seconds.

7. Record centrifuge identification, the

times selected, the date, and the

identity of the person performing

the calibration.

For Washing and Antiglobulin Testing

Tests in which antihuman globulin (AHG)

serum is added to red cells may require

Methods Section 7: Quality Control 829

Table 7.5-1. Example of Serologic Centrifuge Test Results*

Time in Seconds

Criteria 10152030 45

Supernatant fluid is clear No No Yes Yes Yes

Cell button is clearly delineated No No No Yes Yes

Cells are easily resuspended Yes Yes Yes Yes Yes

Agglutination is observed ± ± 1+ 1+ 1+

Negative tube is negative Yes Yes Yes Yes Resuspends

roughly

*The optimal time for centrifugation in this example is 30 seconds.

centrifugation conditions different from

those for immediate agglutination. Cen-

trifugation conditions appropriate for

both washing and AHG reactions can be

determined in one procedure. Note that

this procedure does not monitor the com-

pleteness of washing; use of IgG-coated cells

to control negative AHG reactions provides

this check. The following procedure ad-

dresses only the mechanics of centrifu-

gation.

Materials

1. AHG reagent, unmodified.

2. Saline, large volumes.

3. Test tubes, 10 ×75 mm or 12 ×75

mm (whichever size is routinely used

in the laboratory).

4. Worksheet for recording results.

5. Positive control: a2%to5%saline

suspension of D+ red cells incubated

for 15 minutes at 37 C with anti-D

diluted to give 1+ macroscopic ag-

glutination after addition of AHG.

6. Negative control: a2%to5%suspen-

sion of D+ red cells incubated for 15

minutes at 37 C with 6% albumin.

[Note: D– red cells incubated with

diluted anti-D may also be used as a

negative control.]

Procedure

1. Prepare five test tubes containing 1

drop of positive cells and five tubes

containing 1 drop of negative con-

trol cells.

2. Fill tubes with saline and centrifuge

them in pairs, one positive and one

negative, for different times (eg, 30,

45, 60, 90, and 120 seconds). The red

cells should form a clearly delin-

eated button, with minimal cells

trailing up the side of the tube. After

the saline has been decanted, the

cell button should be easily resus-

pended in the residual fluid. The

optimal time for washing is the

shortest time that accomplishes

these goals.

3. Repeat washing process on all pairs

three more times, using time deter-

mined to be optimal.

4. Decant supernatant saline thoroughly.

5. Add AHG to one positive control test

tube and one negative control test tube.

Centrifuge immediately for the de-

sired interval (eg, 10 seconds).

6. Observe each tube for agglutination

and record observations.

7. Repeat steps 5 and 6 for each inter-

val (eg, 15, 20, 30, and 45 seconds).

Do not allow cells and AHG to incu-

bate before centrifugation.

8. Select optimal time as in immediate

agglutination procedure.

9. Record centrifuge identification, the

times selected, the date, and the

identity of the person performing

the calibration.

Notes

Periodic recalibration is performed to ver-

ify that the timing in use continues to be

the optimal timing. This may be accom-

plished by using a shortened version of

the procedures outlined above. For exam-

ple, use the current timing for a particular

centrifuge and each medium and those

times just above and just below the cur-

rent timing.

Method 7.6. Performance

Testing of Automatic Cell

Washers

Principle

Antihuman globulin (AHG) is inactivated

readily by unbound immunoglobulin. The

red cells to which AHG will be added

830 AABB Technical Manual

must be washed free of all proteins and

suspended in a protein-free medium. A

properly functioning cell washer must add

large volumes of saline to each tube, re-

suspend the cells, centrifuge them ade-

quately to avoid excessive red cell loss, and

decant the saline to leave a dry cell button.

Materials

1. Test tubes routinely used in the lab-

oratory, 10 ×75 mm or 12 ×75 mm.

2. Additive routinely used to potentiate

antigen-antibody reactions.

3. Human serum, from patient or do-

nor.

4. IgG-coated red cells, known to give a

1 to 2+ reaction in antiglobulin test-

ing.

5. Normal saline.

6. AHG reagent, anti-IgG or polyspeci-

fic.

7. Worksheet for recording results.

Procedure

1. To each of 12 tubes, add potentiator

and human serum in quantities that

correspond to routine use and 1 drop

of IgG-coated red cells.

2. Place the tubes in a centrifuge car-

rier, seat the carrier in the cell washer,

and start the wash cycle.

3. After addition of saline in the second

cycle, stop the cell washer. Inspect

the contents of all tubes. There

should be an approximately equal

volume of saline in all tubes; some

variation is acceptable. Tubes should

be approximately 80% full, to avoid

splashing and cross-contamination.

(Refer to manufacturer’s instructions

for specific requirements.) Record

observations.

4. Observe all tubes to see that the red

cells have been completely resus-

pended. Record observations.

5. Continue the washing cycle.

6. After addition of saline in the third

cycle, stop the cell washer and in-

spect tubes as above. Record obser-

vations.

7. Complete the wash cycle.

8. At the end of the wash cycle, inspect

all tubes to see that saline has been

completely decanted and that each

tube contains a dry cell button. Re-

cord observations.

9. Add AHG according to the manufac-

turer’s directions, centrifuge, and ex-

amine all tubes for agglutination. If

the cell washer is functioning prop-

erly, the size of the cell button

shouldbethesameinalltubes.All

tubes should show the same degree

of agglutination. Record observa-

tions.

10. Record identity of centrifuge, the date

of testing, and the identity of the

person performing the testing.

Notes

1. Further investigation is needed if:

a. The amount of saline varies

significantly from tube to tube

or cycle to cycle.

b. The cell button is not resus-

pended completely after being

filled with saline.

c. Any tube has weak or absent

agglutination in the antiglobu-

lin phase.

d. Any tube has a significant de-

crease in the size of the cell

button.

2. Cell washers that automatically add

AHG should also be checked for uni-

form addition of AHG. In step 9

above, AHG would be added auto-

matically, and failure of addition

would be apparent by absence of ag-

glutination. The volume of AHG

Methods Section 7: Quality Control 831

should be inspected and found to be

equal in all tubes. The volume of

AHG delivered automatically by cell

washers should be checked monthly

to ensure that it is as specified in the

manufacturer’s directions and that

delivery is uniform in all tubes.

3. Some manufacturers market AHG

colored with green dye for use in au-

tomated cell washers so that it will

be immediately obvious if no re-

agent has been added.

Method 7.7. Monitoring Cell

Counts of Apheresis

Components

Principle

When cellular components are prepared

by apheresis, it is essential to determine

cell yields without compromising the ste-

rility of the component.

Materials

1. Component collected by apheresis.

2. Metal clips and hand sealer or di-

electric sealer.

3. Tubing stripper.

4. Clean instruments (scissors, hemo-

stats).

5. Test tubes.

6. Cell-counting equipment.

7. Worksheet for recording results.

Procedure

1. Ensure that the contents of the aphere-

sis component bag are well mixed.

2. Strip the attached tubing at least four

times, mixing the contents of the

tubing with the contents of the bag,

to ensure that the contents of the

tubing accurately represent the en-

tire contents of the bag.

3. Seal a 5- to 8-cm (2- to 3-inch) seg-

ment distal to the collection bag.

There should be approximately 2 mL

of fluid in the segment. Double-seal

theendofthetubingnexttothe

component bag and detach the seg-

ment.

4. Empty the contents of the segment

into a suitably labeled tube.

5. Determine and record cell counts in

cells/mL.

a. For results reported as cells/µL,

change values to cells/mL by

multiplying by 1000 (or 103).

b. For results reported as cells/L,

change values to cells/mL by

dividing by 1000 (or 103).

6. Multiply cells/mL by the volume of

the component, in mL, to obtain to-

tal cell count in the component.

7. Record component’s identity, the date,

and the identity of the person per-

forming the testing.

Note

Refer to manufacturer’s directions for any

additional requirements.

Method 7.8. Manual Method

for Counting Residual White

Cells in Leukocyte-Reduced

Blood and Components

Principle

The residual white cell content of leuko-

cyte-reduced whole blood and components

can be determined using a large-volume

hemocytometer. For red-cell-containing

components, the red cells in the aliquot to

be counted are first lysed. Crystal violet is

used to stain the leukocyte nuclei. The

Nageotte counting chamber has a volume

56 times that of the standard hemocyto-

832 AABB Technical Manual

meter. Accuracy of counting is improved

by examining a larger volume of mini-

mally diluted specimen, compared with

standard counting techniques.

Materials

1. Hemocytometer chamber with 50 µL

counting volume (eg, Nageotte Brite

Line Chamber).

2. Crystal violet stain: 0.01% w/v crys-

tal violet in 1% v/v acetic acid (eg,

Turks solution).

3. Red cell lysing agent (eg, Zapoglobin,

Coulter Electronics, Hialeah, FL), for

red-cell-containing components

only.

4. Pipettor (40 µL and 100 µL) with dis-

posable tips.

5. Talc-free gloves, clean plastic test

tubes, plastic petri dish, filter paper.

6. Light microscope with 10×ocular

lens and 20×objective.

7. Worksheet for recording results.

Procedure

1. Dilute and stain leukocyte-reduced

blood and component samples as

follows:

a. For Red-Cell-Containing Com-

ponents

1) Pipette 40 µLoflysing

agent into a clean test tube.

2) Place a representative

sample of the component

to be tested in a clean test

tube. The hematocrit of the

sample to be tested should

not exceed 60%.

3) Pipette 100 µLofthesam

ple into the tube contain-

ing 40 µLoflysingagent.

Rinse the pipette several

times to mix the two flu-

ids, until the pipette tip is

no longer coated with in-

tact red cells.

4) Pipette 360 µLofcrystal

violet stain into the mix-

ture and mix fluids by

pipetting up and down

several times. The final

volume is now 500 µL.

b. For Platelets

1) Place a representative sam-

ple of the platelet in a clean

test tube.

2) Pipette 100 µLofthepla

telet sample into a clean

test tube.

3) Pipette 400 µLofcrystal

violet stain into the 100 µL

of platelets and mix fluids

by pipetting up and down

several times. The final

volume is now 500 µL.

2. Fit the hemocytometer with a cover-

slip and, using a pipette, load the

mixtureuntilthecountingareais

completely covered but not over-

flowing.

3. Cover the hemocytometer with a

moist lid to prevent evaporation (a

plastic petri dish into which a piece

of damp filter paper has been placed

works well) and let it rest undisturbed

for10to15minutes,toallowthe

white cells to settle in the counting

area of the chamber.

4. Removethemoistlid,placethe

hemocytometer on the microscope

and, using a 20×objective, count the

white cells present in the entire

50-µL volume of the counting cham-

ber.

5. Calculate and record results.

a. White cell concentration:

leukocytes/µL = (cells counted/50 µL) ×5

= cells counted/10

Methods Section 7: Quality Control 833

where 50 µListhevolume

counted and 5 is the dilution

factor resulting from the addi-

tion of lysing agent and stain.

b. Total white cell content of the

leukocyte-reduced component:

leukocytes/

component

leukocytes/ L

1000 L/mL

lume in mL

of the component

6. Record the component’s identity, the

date, and the identity of the person

performing the testing.

Notes

1. White cells deteriorate during refrig-

erated storage; counts on stored blood

or red cell components may give in-

accurate results.

2. Use of talc-free gloves is recom-

mended because talc particles that

contaminate the counting chamber

can be misread as white cells.

3. Experience identifying crystal-violet-

stained white cells can be obtained

by examining samples from compo-

nents that have not been leukocyte

reduced.

4. The accuracy of the counting method

can be validated from a reference

sample with a high white cell con-

tent that has been quantified by an-

other means. This reference sample

can be used for serial dilutions in

blood or a component that has been

rendered extremely leukocyte re-

duced by two passages through a

leukocyte reduction filter. Counts

obtained on the serially diluted sam-

ples can be compared with the ex-

pected concentration derived by cal-

culation.

5. This counting technique is not known

to be accurate at concentrations lower

than 1 white cell/µL.

References

1. Lutz P, Dzik WH. Large-volume hemocyto-

meter chamber for accurate counting of

white cells (WBCs) in WBC-reduced platelets;

validation and application for quality control

of WBC-reduced platelets prepared by

apheresis and filtration. Transfusion 1993;33:

409-12.

2. Dzik WH, Szuflad P. Method for counting

white cells in white cell-reduced red cell con-

centrates (letter). Transfusion 1993;33:272.

834 AABB Technical Manual

Appendices

835

Appendices

Appendix 1. Normal Values in Adults

Determination SI Units Conventional Units

Alanine aminotransferase 4-36 U/L at 37 C 4-36 U/L at 37 C

Bilirubin, total 2-21 µmol/L 0.1-1.2 mg/dL

Haptoglobin 0.6-2.7 g/L 60-270 mg/dL

Hematocrit

Males 0.40-0.54 40-54%

Females 0.38-0.47 38-47%

Hemoglobin

Males 135-180 g/L 13.5-18.0 g/dL

Females 120-160 g/L 12.0-16.0 g/dL

Hemoglobin A20.015-0.035 total Hb 1.5-3.5% total Hb

Hemoglobin F 0-0.01 total Hb <1% total Hb

Hemoglobin (plasma) 5-50 mg/L 0.5-5.0 mg/dL

Immunoglobulins

IgG 8.0-18.0 g/L 800-1801 mg/dL

IgA 1.1-5.6 g/L 113-563 mg/dL

IgM 0.5-2.2 g/L 54-222 mg/dL

IgD 5.0-30 mg/L 0.5-3.0 mg/dL

IgE 0.1-0.4 mg/L 0.01-0.04 mg/dL

Methemoglobin <0.01 total Hb <1% total Hb

Platelet count 150-450 ×109/L 150-450 ×103/µL

Red cells

Males 4.6-6.2 ×1012/L 4.6-6.2 ×106/µL

Females 4.2-5.4 ×1012/L 4.2-5.4 ×106/µL

Reticulocyte count 25-75 ×109/L 25-75 ×103/µL

Viscosity, relative 1.4-1.8 ×water 1.4-1.8 ×water

White cells 4.5-11.0 ×109/L 4.5-11.0 ×103/µL

836 AABB Technical Manual

Appendix 2. Selected Normal Values in Children

SI Units Conventional Units

Bilirubin (total)

Cord Preterm <30 mmol/L <1.8 mg/dL

Term <30 mmol/L <1.8 mg/dL

0-1 day Preterm <137 mmol/L <8 mg/dL

Term <103 mmol/L <6 mg/dL

1-2 days Preterm <205 mmol/L <12 mg/dL

Term <137 mmol/L <8 mg/dL

3-7 days Preterm <274 mmol/L <16 mg/dL

Term <205 mmol/L <12 mg/dL

7-30 days Preterm <205 mmol/L <12 mg/dL

Term <120 mmol/L <7 mg/dL

Thereafter Preterm <34 mmol/L <2 mg/dL

Term <17 mmol/L <1 mg/dL

Hemoglobin WBC Platelets

26-30 weeks’ gestation 11.0-15.8 g/dL 1.7-7.1 ×10 /L 180,000-327,000/µL

Term 13.5-19.5 g/dL 9-30 ×10 /L 192,000/µL (mean)

1-3 days 14.5-22.5 g/dL 9.4-34 ×10 /L 252,000/µL (mean)

2 weeks 13.4-19.8 g/dL 5-20 ×10 /L

1 month 10.7-17.1 g/dL 4-19.5 ×10 /L

2 months 9.4-13.0 g/dL

6 months 11.1-14.1 g/dL 6-17.5 ×10 /L

6 months-2 years 10.5-13.5 g/dL 6-17 ×10 /L 150,000-350,000/µL

2-6 years 11.5-13.5 g/dL 5-15.5 ×10 /L 150,000-350,000/µL

6-12 years 11.5-15.5 g/dL 4.5-13.5 ×10 /L 150,000-350,000/µL

12-18 years

Male 13.0-16.9 g/dL 4.5-13.5 ×10 /L 150,000-350,000/µL

Female 12.0-16.0 g/dL 4.5-13.5 ×10 /L 150,000-350,000/µL

Appendices 837

Appendix 2. Selected Normal Values in Children (cont’d)

IgG IgM IgA

Newborn 831-1231 mg/dL 6-16 mg/dL <3 mg/dL

1-3 months 312-549 mg/dL 19-41 mg/dL 8-34 mg/dL

4-6 months 241-613 mg/dL 26-60 mg/dL 10-46 mg/dL

7-12 months 442-880 mg/dL 31-77 mg/dL 19-55 mg/dL

13-24 months 553-971 mg/dL 35-81 mg/dL 26-74 mg/dL

25-36 months 709-1075 mg/dL 42-80 mg/dL 34-108 mg/dL

3-5 years 701-1157 mg/dL 38-74 mg/dL 66-120 mg/dL

6-8 years 667-1179 mg/dL 40-80 mg/dL 79-169 mg/dL

9-11 years 889-1359 mg/dL 46-112 mg/dL 71-191 mg/dL

12-16 years 822-1070 mg/dL 39-79 mg/dL 85-211 mg/dL

Activated Partial Thromboplastin Time

Preterm 70 seconds

Full-term 45-65 seconds

Prothrombin Time

Preterm 12-21 seconds

Full-term 13-20 seconds

Reprinted with permission from The Harriet Lane Handbook. 15th ed. St. Louis, MO: Mosby, 2000.

Appendix 3. Typical Normal Values in Tests of Hemostasis and Coagulation

(Adults)

Test Normal Value

Activated partial thromboplastin time 25-35 seconds

Bleeding time 2-8 minutes

Coagulation factors 500-1500 U/L

Fibrin degradation products <10 mg/L

Fibrinogen 2.0-4.0 g/L

Plasma D-dimers <200 mg/L

Protein C 70-1400 U/L

Protein S (total) 70-1400 U/L

Prothrombin time 10-13 seconds

Thrombin time 17-25 seconds

Reprinted with permission from Henry JB. Clinical diagnosis and management by laboratory methods. 20th ed. Phila-

delphia: WB Saunders, 2001.

838 AABB Technical Manual

Appendix 4. Coagulation Factor Values in Platelet Concentrates

Factor/

Protein

Normal

Range Day 0 Day 1 Day 2 Day 3 Day 4 Day 5

II % 78-122 104 91-96 96 85-94 90 90

V % 47-153 78-98 69-78 50 36-47 28 24-35

VII % 51-168 108 93-117 88 80-103 75 72

VIII % 48-152 68-126 85-99 76 68-76 75 39-70

IX % 62-138 72-105 100-106 95 91-98 93 63-97

X % 58-142 66-101 93-94 92 85-88 84 60-83

XI % 52-148 91-111 106-108 103 96-98 101 86-110

XII % 46-126 117 107-112 116 106-123 123 131

C % 57-128 106 102 101 98 99 100

S % 83-167 95 75 61 40 32 31

Antithrombin % 88-126 103 99 101 102 103 97

Plasminogen % 60-140 140 133 126 122 124 117

Fibrinogen

mg/dL

198-434 217-308 278-313 310 265-323 302 221-299

Ristocetin

cofactor %

50-150 106 124 125 133 116 127

Note: Coagulation factor % = 100 x coagulation factor units/mL.

Reproduced with permission from Brecher ME, ed. Collected questions and answers. 6th ed. Bethesda, MD:

AABB, 2000.

Appendices 839

Appendix 5. Approximate Normal Values for Red Cell, Plasma, and Blood

Volumes

Infant1Adult2

Premature

Term Birth at

72 hours Male Female

Red Cell Volume mL/kg 50 40 26 24

Plasma Volume mL/kg 58 47 40 36

Blood Volume mL/kg 108 87 66 60

The adult values should be modified to correct

for:

1. Below age 18: increase values by 10%.

2. Weight loss:

a. Marked loss within 6 months—calcula-

tions made at original weight.

b. Gradual loss over a longer time—calcu-

lations made at present weight and

raised 10% to 15%.

3. Obese and short: values are reduced by

10%.

4. Elderly: values are reduced by 10%.

5. Pregnancy3:

Estimation of Body Surface Area4:

BSA(m ) Ht(cm) Wt(kg)

3600 or Ht(in) Wt(lb)

3131

2=××

Blood Volume (BV)5:

BV = 2740 mL/m2—males

BV = 2370 mL/m2—females

Hematocrit6:

Venous hematocrit = Hv(blood obtained by

vein or finger puncture)

Whole-body hematocrit = HB

HB=(Hv)×(0.91)

References

1. Miller D. Normal values and examination of the

blood: Perinatal period, infancy, childhood and ado-

lescence. In: Miller DR, Baehner RL, McMillan CW,

Miller LP, eds. Blood diseases of infancy and child-

hood. St. Louis: C.V. Mosby, 1984:21,22.

2. Albert SN. Blood volume. Springfield, IL: Charles C.

Thomas, 1963:26.

3. Peck TM, Arias F. Hematologic changes associated

with pregnancy. Clin Obstet Gynecol 1979;22:788.

4. Mosteller RD. Simplfied calculation of body-surface

area. N Engl J Med 1987;317:1098.

5. Shoemaker WC. Fluids and electrolytes in the

acutely ill adult. In: Shoemaker WC, Ayres S,

Grenvik A, et al, eds. Textbook of critical care. 2nd

ed. Philadelphia: WB Saunders Co., 1989:1130.

6. Mollison PL, Englefriet CP, Contreras M. Blood

transfusion in clinical medicine. 9th ed. Oxford:

Blackwell Scientific Publications, 1993.

840 AABB Technical Manual

Appendix 6. Blood Group Antigens Assigned to Systems

In 1980, the International Society of Blood Transfusion (ISBT) formed a Working Party on Terminology

for Red Cell Surface Antigens. The task of this group was to devise a uniform nomenclature that would

be both eye- and machine-readable. The numeric system proposed by this group was not intended to re-

place traditional terminology but, instead, to enable communication using computer systems where

numbers are necessary. ISBT terminology uses uppercase letters and Arabic numbers for system and

antigen codes. Each system, collection, or series of antigens is given a number (eg, ABO system = 001),

and each antigen within the system is given a number (eg, A = 001, B = 002). Sinistral zeros may be

omitted. Thus, in ISBT terminology, the A antigen would be written using computer code as 001001 or

1.1, or using the system symbol, as ABO1.

Periodically, the Working Party meets to update assignment of antigens to systems, collections, and se-

ries. The table below lists the blood group systems and the antigens assigned to those systems. Other

red cell antigens are assigned to collections and to series of high- and low-incidence antigens. Although

all terms in the table are acceptable, the

Technical Manual

and

TRANSFUSION

choose to use traditional

terminology in most cases. Further information on blood group terminology, which antigens are assigned

to the collections, and the series of high- and low-incidence antigens can be found in the references.

System (ISBT

Symbol/Number) Antigen (ISBT Number)

ABO (ABO/001) A(ABO1)

B (ABO2)

A,B (ABO3)

A1(ABO4)

MNSs or MNS or M(MNS1) M

e(MNS13) Dantu (MNS25) ERIK (MNS37)

MN (MNS/002) N (MNS2) Mta(MNS14) Hop (MNS26) Osa(MNS38)

S(MNS3) St

a(MNS15) Nob (MNS27) ENEP (MNS39)

s(MNS4) Ri

a(MNS16) Ena(MNS28) ENEH (MNS40)

U(MNS5) Cl

a(MNS17) ENKT (MNS29) HAG (MNS41)

He (MNS6) Nya(MNS18) ‘N’ (MNS30) ENAV (MNS42)

Mia(MNS7) Hut (MNS19) Or (MNS31) MARS (MNS43)

Mc(MNS8) Hil (MNS20) Dane (MNS32)

Vw (MNS9) Mv(MNS21) TSEN (MNS33)

Mur (MNS10) Far (MNS22) MINY (MNS34)

Mg(MNS11) sD(MNS23) MUT (MNS35)

Vr (MNS12) Mit (MNS24) SAT (MNS36)

P (P/003) P1(P1)

Appendices 841

(cont’d)

Rh (RH/004) D (RH1) Hro(RH17) hrB(RH31) Nou (RH44)

C (RH2) Hr (RH18) Rh32 (RH32) Riv (RH45)

E (RH3) hrS(RH19) Rh33 (RH33) Sec (RH46)

C (RH4) VS (RH20) HrB(RH34) Dav (RH47)

e (RH5) CG(RH21) Rh35 (RH35) JAL (RH48)

f (RH6) CE (RH22) Bea(RH36) STEM (RH49)

Ce (RH7) DW(RH23) Evans (RH37) FPTT (RH50)

CW(RH8) c-like (RH26) Rh39 (RH39) MAR (RH51)

CX(RH9) cE (RH27) Tar (RH40) BARC (RH52)

V (RH10) hrH(RH28) Rh41 (RH41) JAHK (RH53)

EW(RH11) Rh29 (RH29) Rh42 (RH42) DAK (RH54)

G (RH12) Goa(RH30) Crawford (RH43) LOCR (RH55)

CENR (RH56)

Lutheran (LU/005) Lua(LU1) Lu6 (LU6) Lu12 (LU12) Aua(LU18)

Lub(LU2) Lu7 (LU7) Lu13 (LU13) Aub(LU19)

Lu3 (LU3) Lu8 (LU8) Lu14 (LU14) Lu20 (LU20)

Lu4 (LU4) Lu9 (LU9) Lu16 (LU16) Lu21 (LU21)

Lu5 (LU5) Lu11 (LU11) Lu17 (LU17)

Kell (KEL/006) K (KEL1) Ula(KEL10) K18 (KEL18) VLAN (KEL25)

k (KEL2) K11 (KEL11) K19 (KEL19) TOU (KEL26)

Kpa(KEL3) K12 (KEL12) Km (KEL20) RAZ (KEL27)

Kpb(KEL4) K13 (KEL13) Kpc(KEL21) VONG (KEL28)

Ku (KEL5) K14 (KEL14) K22 (KEL22)

Jsa(KEL6) K16 (KEL16) K23 (KEL23)

Jsb(KEL7) Wka(KEL17) K24 (KEL24)

Lewis (LE/007) Lea(LE1) LebH (LE4)

Leb(LE2) ALeb(LE5)

Leab (LE3) BLeb(LE6)

Duffy (FY/008) Fya(FY1) Fy4 (FY4)

Fyb(FY2) Fy5 (FY5)

Fy3 (FY3) Fy6 (FY6)

Kidd (JK/009) Jka(JK1)

Jkb(JK2)

Jk3 (JK3)

Appendix 6. Blood Group Antigens Assigned to Systems (cont’d)

System (ISBT

Symbol/Number) Antigen (ISBT Number)

842 AABB Technical Manual

Diego (DI/010) Dia(DI1) WARR (DI7) Vga(DI13) Fra(DI20)

Dib(DI2) ELO (DI8) Swa(DI14) SW1 (DI21)

Wra(DI3) Wu (DI9) BOW (DI15)

Wrb(DI4) Bpa(DI10) NFLD (DI16)

Wda(DI5) Moa(DI11) Jna(DI17)

Rba(DI6) Hga(DI12) KREP (DI18)

Tra(DI19)*

Yt or Cartwright Yta(YT1)

(YT/011) Ytb(YT2)

Xg (XG/012) Xga(XG1) CD99 (XG2)

Scianna (SC/013) Sc1 (SC1) Rd (SC4)

Sc2 (SC2) STAR (SC5)

Sc3 (SC3)

Dombrock (DO/014) Doa(DO1)

Dob(DO2)

Gya(DO3)

Hy (DO4)

Joa(DO5)

Colton (CO/015) Coa(CO1)

Cob(CO2)

Co3 (CO3)

LW or Landsteiner- LWa(LW5)

Wiener (LW/016) LWab (LW6)

LWb(LW7)

Chido/Rodgers Ch1 (CH/RG1) Rg1 (CH/RG11)

(CH/RG /017) Ch2 (CH/RG2) Rg2 (CH/RG12)

Ch3 (CH/RG3)

Ch4 (CH/RG4)

Ch5 (CH/RG5)

Ch6 (CH/RG6)

WH (CH/RG7)

H (H/018) H (H1)

Kx (XK/019) Kx (XK1)

Appendix 6. Blood Group Antigens Assigned to Systems (cont’d)

System (ISBT

Symbol/Number) Antigen (ISBT Number)

Appendices 843

Gerbich (GE/020) Ge2 (GE2) Wb (GE5) Dha(GE8)

Ge3 (GE3) Lsa(GE6) GEIS (GE9)

Ge4 (GE4) Ana(GE7)

Cromer (CR/021) Cra(CROM1) Dra(CROM5) WESb(CROM9) ZENA (CROM13)

Tca(CROM2) Esa(CROM6) UMC (CROM10)

Tcb(CROM3) IFC (CROM7) GUTI (CROM11)

Tcc(CROM4) WESa(CROM8) SERF (CROM12)

Knops (KN/022) Kna(KN1) McCa(KN3) Yka(KN5) Sl2 (KN7)

Knb(KN2) Sla(KN4) McCb(KN6) Sl3 (KN8)*

Indian (IN/023) Ina(IN1)

Inb(IN2)

Ok (OK/024) Oka(OK1)

Raph (RAPH/025) MER2 (RAPH1)

JMH or John Milton

Hagen

(JMH/026)

JMH (JMH1)

I (I/027) I(I1)

Globoside

(GLOB/028)

P (GLOB1)

GIL (GIL/029) GIL (GIL1)

*Provisional.

Daniels GL, Anstee DJ, Cartron JP, et al. International Society of Blood Transfusion working party on terminol-

ogy for red cell surface antigens. Vox Sang 2001;80:193-6.

Daniels GL, Fletcher A, Garratty G, et al. Blood group terminology 2004. From the ISBT committee on terminol-

ogy for red cell surface antigens. Vox Sang 2004;87:304-16.

Garratty G, Dzik W, Issitt PD, et al. Terminology for blood group antigens and genes—historical origins and

guidelines in the new millennium. Transfusion 2000;40:477-89.

Issitt PD, Anstee DJ. Applied blood group serology. 4th ed. Durham, NC: Montgomery Scientific, 1998.

Appendix 6. Blood Group Antigens Assigned to Systems (cont’d)

System (ISBT

Symbol/Number) Antigen (ISBT Number)

844 AABB Technical Manual

Appendix 7. Examples of Gene, Antigen, and Phenotype Terms

System Genes Antigens Phenotypes

ABO

1A2BAA

1A2B

DCEce

D C E c e D+C+E–c+e+

MNSs

MNSs M+N+S–s+

P1P1+ P1–

Lewis

Le le

LeaLebLe(a+)Le(a–b+)

Kell

KkKp

aJsaK–k+Kp(a+)Js(a–)

Kell

K1 K2 K3 K:–1,2,–3

Scianna

Sc1 Sc2 Sc:–1,–2,–3

Kidd

JkaJkbJk3 Jk(a+)Jk(a+b+)Jk:3

Modified from:

Denomme G, Lomas-Francis C, Storry J, Reid ME. Approaches to blood group molecular genotyping and its ap-

plications. In: Stowell C, Dzik W, eds. Emerging diagnostic and therapeutic technologies in transfusion medi-

cine. Bethesda, MD: AABB Press, 2003:95-129.

Garratty G, Dzik W, Issitt PD, et al. Terminology for blood group antigens and genes—historical origins and

guidelines in the new millennium. Transfusion 2000;40:477-89.

Zelinski T. Chromosomal localization of human blood group genes. In: Silberstein LE, ed. Molecular and func-

tional aspects of blood group antigens. Bethesda, MD: AABB, 1995:41-73.

Appendix 8. Examples of Correct and Incorrect Terminology*

Term Description Correct Terminology Incorrect Terminology

Phenotype Fy(a+) Fya+,Fy

(a+), Fya(+),Fy

a+, Fya(+), Duffya+,

Duffya-positive

Phenotype Fy(a+b–) Fya+b–,Fy

(a+b–),Fy

a(+)b(–), Fya(+)b(–)

Antibody Anti-FyaAnti Fya, Anti-Duffy

Antigen K Kell (name of system)

Antibody Anti-k Anti-Cellano

Phenotype K:1, K:–1 K1+, K:1+, K(1), K:(1), K1–, K:1–, K1-negative

Phenotypes A Rh+, B Rh– A+ (means positive for A antigen)

B– (means negative for B antigen)

Phenotype M+N– M(+), MM (implies unproved genotype)

Phenotype Rh:–1,–2,–3,4,5 Rh:–1,–2,–3,+4,+5

Rh:1–,2–,3–,4+,5+

Note: The examples shown may not represent the only correct terminologies. In the Rh system, for example,

use of CDE terminology is also acceptable and is more commonly used. The example demonstrates the correct

usage if numeric terminology is used.

*Issitt L. Blood group nomenclature. In: Blood groups: Refresher and updates. Bethesda, MD: AABB, 1995.

Appendices 845

Appendix 9. Distribution of ABO/Rh Phenotypes by Race or Ethnicity*

Phenotype Distribution (%)†

Race or

Ethnicity Number O Rh+ O Rh– A Rh+ A Rh– B Rh+ B Rh– AB Rh+ AB Rh–

White non-Hispanic 2,215,623 37.2 8.0 33.0 6.8 9.1 1.8 3.4 0.7

Hispanic‡259,233 52.6 3.9 28.7 2.4 9.2 0.7 2.3 0.2

Black non-Hispanic 236,050 46.6 3.6 24.0 1.9 18.4 1.3 4.0 0.3

Asian§126,780 39.0 0.7 27.3 0.5 25.0 0.4 7.0 0.1

North American Indian 19,664 50.0 4.7 31.3 3.8 7.0 0.9 2.2 0.3

All donors 3,086,215 39.8 6.9 31.5 5.6 10.6 1.6 3.5 0.6

*Used with permission from Garratty G, Glynn SA, McEntire R, et al for the Retrovirus Epidemiology Donor Study. ABO and Rh(D) phenotype frequencies of different racial/ethnic

groups in the United States. Transfusion 2004;44:703-6.

†Percentages may not add up to 100.0% because of rounding.

‡Hispanic includes Mexican (68.8%), Puerto Rican (5.0%), Cuban (1.6%), and other Hispanic donors (24.6%).

§Asian includes Chinese (29.8%), Filipino (24.1%), Indian (13.8%), Japanese (12.7%), Korean (12.5%), and Vietnamese (7.1%) donors.

846 AABB Technical Manual

Appendix 10. Suggested Quality Control Performance Intervals

Equipment and Reagents Frequency

I. Refrigerators/Freezers/Platelet Incubators

A. Refrigerators

1. Recorder Daily

2. Manual temperature Daily

3. Alarm system board (if applicable) Daily

4. Temperature charts (review daily) Weekly

5. Alarm activation Quarterly

B. Freezers

1. Recorder Daily

2. Manual temperature Daily

3. Alarm system board (if applicable) Daily

4. Temperature charts (review daily) Weekly

5. Alarm activation Quarterly

C. Platelet incubators

1. Recorder Daily

2. Manual temperature Daily

3. Temperature charts (review daily) Weekly

4. Alarm activation Quarterly

D. Ambient platelet storage area Every 4 hours

II. Laboratory Equipment

A. Centrifuges/cell washers

1. Speed Quarterly

2. Timer Quarterly

3. Function Yearly

4. Tube fill level (serologic) Day of use

5. Saline fill volume (serologic) Weekly

6. Volume of antihuman globulin dispensed

(if applicable)

Monthly

7. Temperature check (refrigerated centrifuge) Day of use

8. Temperature verification (refrigerated centrifuge) Monthly

B. Heating blocks/Waterbaths/View boxes

1. Temperature Day of use

2. Quadrant/area checks Periodically

C. Component thawing devices Day of use

D. pH meters Day of use

E. Blood irradiators

1. Calibration Yearly

2. Turntable (visual each time of use) Yearly

3. Timer Monthly/quarterly

4. Source decay Dependent on source type

5. Leak test Twice yearly

6. Dose delivery check (with indicator) Each irradiator use

7. Dose delivery verification

a. Cesium-137 Yearly

b. Cobalt-60 Twice yearly

c. Other source As specified by manufacturer

Appendices 847

Appendix 10. Suggested Quality Control Performance Intervals (cont’d)

Equipment and Reagents Frequency

F. Thermometers (vs NIST-certified or traceable)

1. Liquid-in-glass Yearly

2. Electronic Monthly

G. Timers/clocks Yearly

H. Pipette recalibration Yearly

I. Sterile connecting device

1. Weld check Each use

2. Function Yearly

J. Blood warmers

1. Effluent temperature Quarterly

2. Heater temperature Quarterly

3. Alarm activation Quarterly

III. Blood Collection Equipment

A. Whole blood equipment

1. Agitators Day of use

2. Balances/scales Day of use

3. Gram weight (vs NIST-certified) Yearly

B. Microhematocrit centrifuge

1. Centrifuge timer check Quarterly

2. Calibration Yearly

C. Cell counters/hemoglobinometers Day of use

D. Blood pressure cuffs Periodically

E. Apheresis equipment

Checklist requirements As specified by the

manufacturer

IV. Reagents

A. Red cells Day of use

B. Antisera Day of use

C. Antiglobulin serum Day of use

D. Transfusion-transmissible disease marker testing Each test run

V. Miscellaneous

A. Copper sulfate specific gravity Day of use

B. Shipping containers for blood transport

(usually at temperature extremes)

Twice yearly

Note: The frequencies listed above are suggested intervals, not requirements. For any new piece of equipment,

installation, operational, and process qualification must be performed. After the equipment has been suitably

qualified for use, ongoing quality control (QC) testing should be performed. Depending upon the operational

and process qualification methodology, the ongoing QC may initially be performed at a greater frequency than

one ultimately wishes to use. Once a track record of appropriate in-range QC results has been established (ei-

ther during equipment qualification or the ongoing QC), the frequency of testing can be reduced, but, at a mini-

mum, the frequency must comply with the manufacturer’s suggested intervals. If no such guidance is provided

by the manufacturer, the intervals given in this table would be appropriate to use.

848 AABB Technical Manual

Appendix 11. Directory of Organizations

AABB

8101 Glenbrook Road

Bethesda, MD 20814-2749

(301) 907-6977

FAX: (301) 907-6895

www.aabb.org

American Association of Tissue Banks (AATB)

1320 Old Chain Bridge Road, Suite 450

McLean, VA 22101

(703) 827-9582

FAX: (703) 356-2198

www.aatb.org

American Medical Association (AMA)

515 N. State Street

Chicago, IL 60610

(800) 621-8335

www.ama-assn.org

American Red Cross National Headquarters (ARC)

2025 E Street, NW

Washington, DC 20006

(202) 303-4498

Disaster Assistance: (866) 438-4636

www.redcross.org

American Society for Apheresis (ASFA)

570 West 7th Avenue, Suite 402

Vancouver, BC, Canada V5Z 1B3

(604) 484-2851

FAX: (604) 874-4378

www.apheresis.org

American Society for Clinical Pathology (ASCP)

2100 West Harrison Street

Chicago, IL 60612-3798

(312) 738-1336

Outside Illinois: (800) 621-4142

FAX: (312) 738-1619

www.ascp.org

American Society for Histocompatibility

and Immunogenetics (ASHI)

15000 Commerce Parkway, Suite C

Mount Laurel, NJ 08054

(856) 638-0428

FAX: (856) 439-0525

www.ashi-hla.org

American Society of Anesthesiologists (ASA)

520 N. Northwest Highway

Park Ridge, IL 60068-2573

(847) 825-5586

FAX: (847) 825-1692

www.asahq.org

American Society of Hematology (ASH)

1900 M Street, NW, Suite 200

Washington, DC 20036

(202) 776-0544

FAX: (202) 776-0545

www.hematology.org

America’s Blood Centers (ABC)

725 15th Street, NW

Suite 700, The Folger Building

Washington, DC 20005

(202) 393-5725

FAX: (202) 393-1282

www.americasblood.org

Armed Services Blood Program Office (ASBPO)

5109 Leesburg Pike, Suite 698

Falls Church, VA 22041-3258

(703) 681-8024

FAX: (703) 681-7541

www.tricare.osd.mil/asbpo

Association of Donor Recruitment Professionals

(ADRP)

P.O. Box 540524

Grand Prairie, TX 75054-0524

www.adrp.org

College of American Pathologists (CAP)

325 Waukegan Road

Northfield, IL 60093-2750

(800) 323-4040

FAX: (847) 832-8000

www.cap.org

Foundation for the Accreditation of Cellular Therapy

(FACT)

University of Nebraska Medical Center

986065 Nebraska Medical Center

Omaha, NE 68198-6065

(402) 559-1950

FAX: (402) 559-1951

www.factwebsite.org

ICCBBA, Inc.

204 St. Charles Way, Unit 179E

York, PA 17402

(717) 845-4790

FAX: (717) 845-9727

www.iccbba.com

850 AABB Technical Manual

Appendix 12. Resources for Safety Information

Centers for Disease Control and Prevention (CDC)

Office of Health and Safety, Biosafety Branch

Mail Stop F-05

1600 Clifton Road

Atlanta, GA 30333

(404) 639-2453

FAX: (404) 639-2294

www.cdc.gov

Clinical and Laboratory Standards Institute (CLSI)

940 West Valley Road, Suite 1400

Wayne, PA 19087-1898

(610) 688-0100

FAX: (610) 688-0700

www.clsi.org

Department of Transportation (DOT)

Office of Hazardous Materials Standards

Research and Special Programs Administration

DHM-10

400 7th Street, SW

Washington, DC 20590-0001

(202) 366-8553

FAX: (202) 366-3012

www.dot.gov

Environmental Protection Agency (EPA)

Chemical Emergency Preparedness and Prevention

Office (5104A)

1200 Pennsylvania Avenue, NW

Washington, DC 20460

(800) 424-9346

In Washington, DC metropolitan area: (703) 412-9810

www.epa.gov

Environmental Protection Agency (EPA)

Office of Solid Waste (5305W)

1200 Pennsylvania Avenue, NW

Washington, DC 20460

(800) 424-9346

www.epa.gov/osw

Food and Drug Administration (FDA)

Center for Biologics Evaluation and Research

Division of Blood Applications, HFM-370

1401 Rockville Pike

Rockville, MD 20852-1448

(301) 827-3524

FAX: (301) 827-3535

www.fda.gov/cber

International Air Transport Association (IATA)

1776 K Street, NW, Suite 400

Washington, DC 20006

(202) 293-9292

FAX: (202) 293-8448

www.iata.org

International Civil Aviation Organization (ICAO)

999 University Street

Montreal, Quebec

Canada H3C 5H7

(514) 954-8220

FAX: (514) 954-6376

www.icao.int

National Fire Protection Association (NFPA)

1 Batterymarch Park

Quincy, MA 02169-7471

(617) 770-3000

FAX: (617) 770-0700

www.nfpa.org

National Institute for Occupational Safety and

Health (NIOSH)

Education and Information Division

4676 Columbia Parkway

Cincinnati, OH 45226-1998

(800) 356-4674

Outside the US: (513) 533-8328

Clinicians’ Post-Exposure Prophylaxis Hotline:

(888) 448-4911

FAX: (513) 533-8588

www.cdc.gov/niosh

National Institutes of Health (NIH)

Division of Safety

Building 13, Room 3K04

Bethesda, MD 20892

(301) 496-2346

FAX: (301) 402-0313

www.nih.gov

Nuclear Regulatory Commission (NRC)

Office of Public Affairs

Washington, DC 20555

(800) 368-5642

In Washington, DC metropolitan area: (301) 415-8200

FAX: (301) 415-2234

www.nrc.gov

Appendices 849

Appendix 11. Directory of Organizations (cont'd)

International Society for Cellular Therapy (ISCT)

570 West 7th Avenue, Suite 402

Vancouver, BC, Canada V5Z 1B3

(604) 874-4366

FAX: (604) 874-4378

www.celltherapy.org

International Society of Blood Transfusion (ISBT)

Central Office

C/O Jan van Goyenkade 11

1075 HP Amsterdam

The Netherlands

+ 31 (0) 20 679 3411

FAX: + 31 (0) 20 673 7306

www.isbt-web.org

Joint Commission on Accreditation of Healthcare

Organizations (JCAHO)

One Renaissance Boulevard

Oakbrook Terrace, IL 60181

(630) 792-5000

FAX: (630) 792-5005

www.jcaho.org

National Hemophilia Foundation (NHF)

116 West 32nd Street, 11th Floor

New York, NY 10001

(212) 328-3700

FAX: (212) 328-3777

www.hemophilia.org

National Marrow Donor Program (NMDP)

3001 Broadway Street NE, Suite 500

Minneapolis, MN 55413-1753

(800) 627-7692

Outside the US: (612) 627-5800

www.marrow.org

Plasma Protein Therapeutics Association (PPTA)

147 Old Solomons Island Road, Suite 100

Annapolis, MD 21401

(410) 263-8296

FAX: (410) 263-2298

www.plasmainfo.org

United Network for Organ Sharing (UNOS)

700 North 4th Street

P.O. Box 2484

Richmond, VA 23218

(804) 782-4800

FAX: (804) 782-4817

www.unos.org

Note: Contact information changes rapidly. Therefore, the data listed above may not be current for the entire life of this publi-

cation.

Appendices 851

Note: Contact information changes rapidly. Therefore, the data listed above may not be current for the entire life of this publi-

cation.

Occupational Safety and Health Administration (OSHA)

Office of Communications

Room N-3647

200 Constitution Avenue, NW

Washington, DC 20210

(202) 693-1999

Workplace safety and health-related questions:

(800) 321-6742

www.osha.gov

US Postal Service (USPS)

Headquarters, Room 9301

475 L’Enfant Plaza, SW

Washington, DC 20260

(800) 275-8777

www.usps.com

Appendix 12. Resources for Safety Information (cont'd)

Index

Page numbers in italics refer to tabular or illustrative material.

A, B, H substances, 290-291, 298, 301, 445,

736-739

ABO compatibility

of bone grafts, 623-624

of Cryoprecipitated AHF, 411, 500-501

of Granulocytes Pheresis, 411

of HPC transplants, 598-599, 600, 601-602

of kidney transplants, 401

of liver transplants, 402, 628

of plasma products, 411, 496, 498

of platelet components, 361-362, 411,

489-490, 569

of Red Blood Cells, 411, 418, 486

of solid organ transplants, 402, 627, 629-630

of Whole Blood, 411, 486

ABO hemolytic disease of the fetus and new-

born, 536, 537, 538, 545

ABO system (ISBT 001), 289-303

acquired B phenotype, 298, 301-302

antibodies, 294-296

age variations, 295, 298, 299

anti-A and anti-B, 294-296

anti-A1, 295-296, 302

anti-A,B, 295, 296

in HDFN, 536, 538, 545

passively acquired, 563

reactivity, 295

antigens, 293

in newborns, 293

nomenclature, 226, 840, 844

on platelets, 361-362, 397

weakly expressed, 297-298, 300-301

B(A) phenotype, 298, 301

discovery of, 289

genetics and biochemistry, 229-230, 235,

290-293, 844

phenotypes, 844, 845

soluble substances, 290-291, 298, 301, 445,

736-739

subgroups, 293-294

in ABO discrepancies, 302

confirmation of by adsorption/elution,

735-736

testing for, 296

ABO testing

of autologous blood, 122

of blood components, 164, 165

in children, 296, 415, 545, 563, 574

with cold autoagglutinins, 299, 302-303, 469

comparison with previous records, 413, 526

confirmationofweakAorBsubgroup,

735-736

853

Index

of cord blood, 545, 595

discrepancies, 296-303, 469

absence of expected antibodies, 301

absence of expected antigens, 300-301

causes, 297

mixed-field agglutination, 298, 299

resolving, 300-303

specimen-related problems, 297-299

technical errors, 299-300

unexpected red cell reactions, 301-302

unexpected serum reactions, 302-303

hemolysis in, 295

microplate tests, 733-735

for organ transplantation, 621, 629

of Platelets Pheresis, 142

in prenatal studies, 538

reagents, 296

of recipients, 122, 410, 411

routine, 290, 296

slide tests, 731-732

in transfusion reaction evaluation, 654

tube tests, 732-733

ABTI antigen, 355

Accidents, 45-46

ACE (angiotensin-converting enzyme) inhibi-

tion, 636, 645

Acid-elution stain (Kleihauer-Betke), 550-551,

794-796

Acid glycine/EDTA, 446, 469, 747-748

Acidosis, in neonates, 561

Acquired antigens, 298, 301-302

Activated partial thromboplastin time (aPTT)

in massive transfusion, 511

monitoring hemostasis with, 494, 496

normal values, 837

in vitamin K deficiency, 498

Active failures, 26

Acute lung injury (ALI), 647

Acute normovolemic hemodilution (ANH), 117,

126-130

clinical studies, 128, 129, 130

physiologic considerations, 127-128

practical considerations, 128, 130

selection of patients, 131

Acute transfusion reactions. See Transfusion re-

actions

Adaptive (acquired) immunity, 243-244

ADCC (antibody-dependent cellular

cytotoxicity), 255, 441

Additives

anticoagulants-preservatives for red cells,

176, 186, 564-565

in serologic testing, 276-277, 437-438, 443,

753-754

Adhesion molecules, 246, 269

Adsol (AS-1), 176, 186, 565

Adsorption

allogeneic, 471-472, 781-782

autologous cold, 438, 466, 472, 775-776

autologous warm, 470-471, 779-781

differential, 781-782

and elution, 300-301, 448, 735-736

methods, 768-769, 775-776, 779-784

polyethylene glycol, 783-784

purposes, 446-447

with rabbit red cells, 439, 466

Adsorption separation, 140

Adult T-cell lymphoma/leukemia (ATL), 682

Adverse reactions

to apheresis, 141, 150-153

to DMSO, 607

in donors, 19, 107-109

to G-CSF, 594-595

management, 19, 20, 21

to transfusions

action for, 531

autologous, 123

infectious, 667-703

noninfectious, 633-661

records, 660-661

reporting, 19, 20, 21 , 661, 702-703

AET (2-aminoethylisothiouronium bromide),

342, 446

Affinity constant (Ko), 273

Age

of blood samples, 410, 431, 433

of donor, 98

effect on ABO antigens/antibodies, 293, 295,

298, 299

Agglutination

defined, 271

factors affecting, 272-275

antigen-antibody proportions, 274-

275

chemical bonding, 273

equilibrium (affinity) constant, 273

incubation time, 274, 444

ionic strength, 274

854 AABB Technical Manual

pH, 274, 444

temperature, 273-274, 336, 443

grading reactions, 412, 728-729

inhibition, 275-276, 444-445

mixed-field, 298, 299, 348, 357

stages, 272-275

tests for granulocyte antibodies, 380

Agreements, 10, 177

AHF. See Cryoprecipitated Antihemophilic Fac-

tor

AHG test. See Antiglobulin test

AIDS (acquired immunodeficiency syndrome),

675-676, 677. See also HIV

AIHA. See Autoimmune hemolytic anemia

Air embolism, 636, 651-652

AITP (autoimmune thrombocytopenic

purpura), 158, 373-374, 492, 553-554

Alanine aminotransferase (ALT), 164, 673, 835

Alarm systems, 185, 198-199, 823-826, 846

Albumin

antibodies to ingredients in, 437-438

as colloid replacement, 507-508

physiology, 507

recombinant, 219

as replacement fluid in apheresis, 150, 151

in serologic testing, 276, 427, 753

use in exchange transfusion, 567

Aldehyde dehydrogenase (ALDH), 603

ALI (acute lung injury), 647

Aliquoting components, 193-194, 564, 571

Allele-specific oligonucleotide (ASO) hybridiza-

tion. See Sequence-specific oligonucleotide

probes

Alleles, 225, 227. See also specific blood groups

blank, 389

dosage effect, 225, 227

frequencies, 227-229

rare, 233, 234

Allelic, defined, 241

Allergic reactions

in apheresis, 152

to latex, 46-47

to transfusions, 522, 634, 644-647

Alloantibodies, red cell. See also Antibody detec-

tion; Antibody identification; specific blood

groups

in ABO discrepancies, 299, 303

with autoantibodies, 438-439, 470-472

in bone graft patients, 623

clinical significance, 411, 418, 423-424, 439, 441

dosage effect, 225, 227, 327, 345, 425, 431

to high-incidence antigens, 356-357,

435-436, 441-442, 547

in liver transplant patients, 629

to low-incidence antigens, 340, 358, 436-437,

546

multiple, 236, 434-435, 441-442, 449

in selection of units, 418, 439-443

serologic behavior, 336

in sickle cell disease, 576

temperature of reaction, 273-274, 336, 443

in thalassemia patients, 576

titration, 449, 539-540, 761-764, 796-798

Allogeneic adsorption, 471-472, 781-782

Allogeneic HPC transplantation

ABO incompatibilities, 598-599, 600, 601-602

cell processing, 596-597, 598, 601-604

collection of products, 591-596

defined, 582

donor evaluation, 589-591

evaluation and QC of products, 607-608

freezing and storage of products, 604-606

graft-vs-host disease in, 586-587

of HPC-A (apheresis), 587-588, 592, 594-595

of HPC-C (cord blood), 588-589, 595-596

of HPC-M (marrow), 583, 585-587, 591-592

indications for, 582

infectious disease testing, 590-591

matched, unrelated transplantation,

585-586, 589

mobilization of HPCs, 587, 594-595

nonmyeloablative, 582-583

positive DAT after, 455

regulations, 608

related transplantation, 587-588, 589

standards, 609

thawing and infusion of products, 607

transfusion support in, 591-592, 599, 600,

601

transportation and shipping of products,

606-607

Allografts. See Organ donation and transplanta-

tion; Tissue transplantation

Alloimmune hemolytic anemia. See Hemolytic

disease of the fetus and newborn; Hemolytic

transfusion reactions

Alloimmunization. See also Alloantibodies, red cell

direct/indirect allorecognition, 265

Index 855

HLA, 265, 266, 362-366, 397-398, 637

in sickle cell disease, 575, 576

as transfusion complication, 265, 637,

656-657

Allotypic, defined, 269

α-methyldopa, 455, 475-476

ALT (alanine aminotransferase), 164, 673, 835

AMD 3100, 594

American Rare Donor Program (ARDP), 441-442,

769-770

American trypanosomiasis (Chagas’ disease),

697-698, 700

2-aminoethylisothiouronium bromide (AET),

342, 446

Amniocentesis, 539, 540, 541, 549

Amniotic fluid analysis, 540-541, 542

Ana(Gerbich) antigen, 352, 843

Anaphylactic transfusion reactions, 644-647

evaluation, 655

frequency, 645

management, 635

pathophysiology, 635, 644-645

prevention, 646-647

symptoms, 639, 644-645

treatment, 645-646

Anaphylactoid transfusion reactions, 644, 645

Anaphylatoxins, 262, 639

Anemia. See also Hemolytic disease of the fetus

and newborn

alloimmune hemolytic, 459

autoimmune hemolytic, 458-477, 509-510

cold agglutinin syndrome, 459, 460, 464-

466

DAT-negative AIHA, 459, 468

drug-induced hemolytic, 459, 460, 472-477,

481-482

IgM warm AIHA, 464

mixed-type AIHA, 459, 460, 466-467

in neonates, 558, 559, 563-564

oxygen compensation in, 484

paroxysmal cold hemoglobinuria, 459, 460,

467-468

screening donors for, 102, 799

sickle-cell disease, 119, 155-156, 508-509,

574-576

in thalassemia, 508

treatment, 487-488

warm autoimmune hemolytic anemia, 459,

460, 461-464

Angiotensin-converting enzyme (ACE) inhibi-

tion, 636, 645

ANH. See Acute normovolemic hemodilution

Anhydrous, defined, 724

Ankylosing spondylitis, 403

Anti-A and anti-B, 294-296

Anti-A1, 293, 295-296, 302

Anti-A,B, 295, 296

Anti-C3b, -C3d, 279, 280, 456

Anti-CMV, 170

Anti-D

active vs passive antibody, 548

antibody titrations, 539-540

in D+ individuals, 327

discovery of, 315-316

dosage effect, 327

in HDFN, 330, 535, 536-537, 539-540

in liver transplantation, 628-629

partial D, 322-323

reagents, 328-330

and weak D, 323

Anti-Delta, 670

Anti-HAV, 671

Anti-HBc (antibody to hepatitis B core antigen)

blood component testing, 164, 166, 675

look-back for, 675

marker of infection, 669, 670

reentry of donors with, 675

as surrogate marker, 672

testing transplant donors for, 621

Anti-HBe (antibody to hepatitis B e antigen),

669, 670

Anti-HBs (antibody to hepatitis B surface anti-

gen), 669, 670

Anti-HCV (antibody to hepatitis C virus)

blood component testing, 164, 166, 170

marker of infection, 670-671, 672-673

reentry of donors with, 673, 674, 675

supplemental testing, 170

testing organ donors for, 620, 621, 623

Anti-HEV, 671

Anti-HIV-1, 2

reentry of donors with positive tests, 674,

681

supplemental testing, 169-170

testing blood donors for, 164, 166, 169-170,

676, 679-681

testing organ donors for, 620, 621,

623

856 AABB Technical Manual

Anti-HTLV-I, II

blood component testing, 120, 164, 166, 170,

683

donors for organ transplants, 620

supplemental testing, 170

Anti-IgA, 645, 646, 655

Anti-IgG, 279, 280, 427, 438, 456

Anti-K1, 536, 538, 540

Anti-Ku, 343

Anti-Pr, 777, 778

Antibodies. See also Alloantibodies; Antigen-

antibody reactions; Autoantibodies; other

Antibody entries; specific blood groups

complement-binding, 280

concomitant, 327-328, 441

defined, 269

distinguishing IgG and IgM, 764-765

drug-induced, 374-377, 472-477, 481-482,

786-791

equilibrium constant (Ko), 273

high-titer, low avidity, 449, 762, 765-

766

HLA

in antibody identifications, 433

and Bg antigens, 358

detection, 365-366, 397

in platelet refractoriness, 362-365,

397-398, 491

in transfusion reactions, 398-400, 637,

647, 656

monoclonal

in ABO testing, 296

anti-D, 328, 329-330

assays utilizing, 284, 373, 380

in autologous tumor purging, 597-598

for reagent use, 266-267

in neonates, 560

platelet

autoantibodies, 373-374

clinical importance, 370-373

detecting, 370-373, 374, 375, 377, 552

drug-induced, 374-377

HLA, 266, 362-366, 397-398

platelet-specific, 366, 367-368, 370-373,

552

production, 249

reagent, 266-267

to reagent components, 298, 437-438

temperature of reaction, 273-274, 443

Antibody-dependent cellular cytotoxicity

(ADCC), 255, 441

Antibody detection

antiglobulin test, 277-281, 282, 283

with autoantibodies, 438-439, 463-464,

470-472

in autologous blood, 122

autologous control, 412, 427, 438-439

automated testing, 285

in blood components, 165

in children, 415, 563, 574

column agglutination for, 278, 284-285

in cord blood, 595

ELISA for, 286

enhancement, 276-277, 427, 443-444,

753-754

enzyme-linked immunosorbent assay, 286

frequency of testing, 442-443, 463-464

of granulocyte autoantibodies, 380

of HLA antibodies, 365-366, 397

immunofluorescence, 285-286

indirect antiglobulin test, 277, 278, 752-754

interpreting reactions, 415-416, 417

MAIEA assay, 284, 286

in organ transplantation, 629

of platelet antibodies, 370-373, 374, 375, 377

in Platelets Pheresis, 142

in positive DAT evaluation, 456

practical considerations, 412

in prenatal evaluations, 539

in pretransfusion testing, 411-413, 417

prewarming technique, 308, 438, 754-755

reagents, 278-280, 425, 427, 468

solid-phase red cell adherence tests, 283-284

specimen requirements, 424

in transfusion reaction evaluation, 654

Antibody identification, 427-450

ABO type of red cells tested, 434

adsorption, 446-447, 448, 470-472

anomalous reactions, 437-438

antibodies to high-incidence antigens,

356-357, 435-436, 441-442, 547

antibodies to low-incidence antigens, 340,

358, 436-437, 546

with antibodies to reagent components,

437-438

with autoantibodies, 438-439, 463-464,

470-472

autologous control, 427, 429-430, 438-439

Index 857

elution, 447-448, 456-458

enhancement techniques, 276-277, 427,

443-444, 753-754

flowchart for, 432

frequency of testing, 442-443, 463-464

inactivation of antigens, 445-446, 766-767

inhibition tests, 444-445

interpreting results, 428-429

medical history in, 424

multiple antibodies, 236, 434-435, 441-442,

449

no discernible specificity, 433-434

phenotyping autologous red cells, 429-430,

439

with positive DAT, 427, 436, 438-439, 456

in prenatal evaluations, 539

prewarming technique, 308, 438, 754-755

probability values, 429, 430

reagents, 278-280, 425, 426, 427, 468

and selection of blood, 418, 439-443, 463

specimen requirements, 424

sulfhydryl reagents in, 446, 448-449, 744-745,

766-767

titration studies, 449, 539-540, 761-764,

796-798

in transfusion reaction evaluation, 654

using immunohematology reference labs,

439

variations in antigen expression, 431, 433,

468

Antibody screens. See Antibody detection

Antibody specificity prediction (ASP) method,

365

Anticoagulant-preservative solutions, 178-179

CPD, CP2D, CPDA-1, 176, 178, 186, 564,

565

red cell changes during storage, 185, 186,

187, 431, 433

shelflifeofcomponents,178, 188, 189-190

Antifibrinolytic agents, 513-514

Antigen-antibody reactions. See also Antibody

detection; Antibody identification

agglutination, 271, 272-276

hemolysis, 271-272

precipitation, 272

prozone, 272, 275

Antigen-presenting cells (APCs), 269

Antigens. See also specific blood groups

acquired, 298, 301-302

blood group nomenclature, 238-239,

318-319, 840-844

CD, 246, 247-248

collections, 335, 355-356

defined, 269

depressed, 341-342, 468

distribution in population, 335, 337

granulocyte/neutrophil, 377-380

high-incidence

antibodies to, 356-357, 435-436, 441-442,

547

of Cromer system, 353, 354

defined, 335

of Gerbich system, 352

GIL, 355

of Knops system, 354

of Lutheran system, 347

not assigned to a system or collection,

356-357

with positive DAT, 436

selecting blood negative for, 441-442

of Vel collection, 355-356

HLA, 362, 385, 388-389, 390-391

inactivation, 342, 445-446, 766-767

low-incidence, 357-358, 436-437, 546

antibodies to, 340, 358, 436-437, 546

of Cromer system, 353, 354

defined, 335

of Gerbich system, 352

of Lutheran system, 347

of MNS system, 227, 228, 337-338, 340

not assigned to a system or collection,

357

of Scianna system, 350

platelet, 361-363, 366, 367-368, 397, 552

public, 392

typing donor units for, 418, 440-441, 657,

745-748

variations in expression of, 431, 433, 468

Antiglobulin (AHG) test. See also Direct

antiglobulin test

in antibody detection/identification,

277-281, 282, 283

complement in, 280-281, 453

in crossmatching, 413, 414

direct, 278, 454-458, 760-761

indirect, 278, 752-754

principles, 277-278

reagents, 278-280, 427, 454, 468

858 AABB Technical Manual

sources of error, 282, 283

use of IgG-coated cells, 281, 412

Antiprotease concentrates, 505-506

Antipyretics, 522

Antithrombin, 500, 505-506

AnWj antigen, 356-357

APCs (antigen-presenting cells), 269

Apheresis

complications, 141, 150-153

for component collection, 140-144

donation intervals, 141, 143

equipment, 139-140, 152, 847

HPCs collected by, 581, 587-588, 592-595

monitoring cell counts, 832

records, 142

separation techniques, 139-140

therapeutic, 144-158

indications for, 146-148, 153-158

photopheresis, 158

plasma volumes exchanged, 145, 149

removal of normal plasma constituents,

149-150

removal of pathologic substances, 145,

148-149

replacement fluids, 150, 151

SPA immunoadsorption, 158

vascular access, 145, 150

Aprotinin, 502, 513-514

aPTT. See Activated partial thromboplastin time

Arachis hypogaea (anti-T), 743

ARDP (American Rare Donor Program), 441-442,

769-770

Arrhythmias, 650

Arterial transplants, 624

Articular cartridge transplants, 624

AS (additive solutions), 176, 186, 564-565

ASP (antibody specificity prediction) method,

365

Assessments, 22-24

blood utilization, 23, 36-38, 514

competency, 8

external, 23-24

internal, 22

proficiency testing, 24

quality indicators, 22-23, 31

transfusion oversight, 514

Ataantigen, 356

ATL (adult T-cell lymphoma/leukemia), 682

Audits, transfusion, 23

Auto controls. See Autologous controls

Autoadsorption. See Adsorption

Autoantibodies

cold

ABO discrepancies with, 299, 302-303,

469

adsorption, 438, 466, 472, 775-776

with alloantibodies, 438-439, 472

anti-I/i, 306, 307-308, 438, 465-466

anti-IH, 308, 434, 777, 778

in cold agglutinin syndrome, 281, 307,

465-466

and complement, 281

determining specificity, 776-778

in mixed AIHA, 466, 467

in paroxysmal cold hemoglobinuria,

467

in Rh testing, 329, 469-470

titration, 458, 465, 778-779

use of sulfhydryl reagents with, 469-470,

744-745

in warm autoimmune hemolytic anemia,

461

in DAT-negative AIHA, 468

granulocyte, 380

platelet-specific, 373-374

posttransfusion, 657

warm

adsorption, 470-472, 779-781

with alloantibodies, 439, 470-472

drug-induced, 475-476

frequency of testing, 463-464

mimicking alloantibodies, 472

transfusion-stimulated, 462

transfusion with, 462-464

in warm autoimmune hemolytic anemia,

461-462

Autoclaving biohazardous waste, 57

Autoimmune hemolytic anemia (AIHA)

classification, 458, 459

cold agglutinin syndrome, 459, 460, 464-466

complement coating cells, 281, 460, 465

DAT-negative AIHA, 459, 468

IgM warm AIHA, 464

mixed-type AIHA, 459, 460, 466-467

paroxysmal cold hemoglobinuria, 459, 460,

467-468

serologic findings in, 460

transfusion in, 462-464, 467, 468, 509-510

Index 859

warm autoimmune hemolytic anemia, 459,

460, 461-464

Autoimmune neutropenia, 379-380

Autoimmune platelet disorders, 373-374

Autoimmune thrombocytopenic purpura

(AITP), 158, 373-374, 492, 553-554

Autologous adsorption

cold, 438, 466, 472, 775-776

warm, 470-471, 779-781

Autologous blood

acute normovolemic hemodilution, 117,

126-130

clinical studies, 128, 129, 130

physiologic considerations, 127-128

practical considerations, 128, 130

selection of patients, 131

bacterial contamination of, 693

categories, 117

for HIV-positive donors, 117, 681

intraoperative blood collection, 117, 130-133

clinical studies, 131-132

controversies in, 132-133

direct reinfusion, 133

equipment for, 132-133

practical considerations, 132

processing, 132-133

requirements and recommendations, 133

inventory management, 94-95

for patients needing rare blood types, 442

postoperative blood collection, 117, 133-135

preoperative blood donation, 117, 118-126

advantages/disadvantages, 118

adverse reactions, 123

aggressive phlebotomy, 125

collection, 121, 126

compliance requirements, 119-120

continuous quality improvement,

123-124

contraindications, 119

cost-effectiveness, 125, 127

donor deferrals, 120

donor screening, 121-122, 124-125

erythropoietin use, 125

establishing program, 120-124

labeling, 120, 122-123

medical interview, 122

in pediatric patients, 118-119

physician responsibility, 120-121

records, 111, 123

shipping, 120

storage, 123

supplemental iron use, 121

testing, 119-120, 122

timing and red cell regeneration during,

122

transfusionofunits,123

transfusion trigger, 125

volume collected, 122

voluntary standards, 119

weakDindonor,324

separation from transfused cells, 748-750

Autologous controls

in antibody identification, 427, 429-430,

438-439

positive, 417, 438-439

in pretransfusion testing, 412, 417, 454

Autologous HPC transplantation

autologous tumor purging, 597-598

cell processing, 596-604

collection of products, 591-596

defined, 582

donor evaluation, 589, 590-591

evaluation and QC of products, 607-608

freezing and storage of products, 604,

605-606

of HPC-A (apheresis), 587, 592-595

of HPC-C (cord blood), 589, 595-596

of HPC-M (marrow), 583, 591-592

infectious disease testing, 590-591

mobilization of HPCs, 592-594

regulations, 608

standards, 609

thawing and infusion of products, 607

transportation and shipping, 606-607

Autologous tumor purging, 597-598

Automated testing platforms, 285

Autosomal dominant and recessive traits, 233,

234

5b antigen, 378

B-cell receptor (BCR), 249

B lymphocytes, 249-250, 252-253

receptors/markers on, 249-253

stimulation of, 254-255

β2-microglobulin, 245

B(A) phenotype, 298, 301

Babesiosis, 695-696, 700

860 AABB Technical Manual

Bacterial contamination, 690-695

clinical considerations, 692-693

fatalities associated with, 690-692

of HPCs, 602-603

organisms involved, 691, 692

platelet-associated, 690-692, 694-695

preventive measures for, 693-694

of reagents, 331, 332

of Red Blood Cells, 691

testing components for, 195, 655, 693, 694

in transfusion-associated sepsis, 635, 643,

655, 691-692

transfusion risk of, 700

Bacteriologic studies

of components, 195, 655, 693, 694

of hematopoietic products, 602-603

in transfusion reaction evaluation, 655

Bags, blood, 104-105

centrifugation of, 179-180

for frozen storage, 182

returning to lab, 531-532

Barcode labels, 171

Basophils, 255

BCR (B-cell receptor), 249

Bennett-Goodspeed (Bg) antigens, 358, 391

Bilirubin

in HDFN, 536, 540-541, 542, 547

hyperbilirubinemia, exchange transfusion

for, 566-567

total, normal values, 835, 836

Bioassays, 65

Biohazardous waste

defined, 55-56

disposal, 56-57

treating, 57

Biologic product deviations, 19, 21

Biological products, 717

Biological safety cabinets (BSCs), 50, 51, 52-53

Biosafety, 49-57

biosafety levels and precautions, 49-50,

54-55, 77

Bloodborne Pathogen Standard, 49

decontamination, 51, 54

emergency response plan, 55-57

engineering controls, 50-51

hazard identification and communication,

labeling, 719

personal protective equipment, 54

safe work practices, 54-55

shipping dangerous goods, 716-722

Standard Precautions, 49

storage, 54

training, 50

waste management, 55-57

Biphasic hemolysin of paroxysmal cold

hemoglobinuria, 467

Blank alleles, 364

Bleeding

indication for transfusion, 486

microvascular, 511, 651

with platelet defects, 490

transfusion-related, 639

Bleeding time, 488, 489, 837

Blocking phenomenon, 330

Blood administration

assessment, 37-38

blood warmers for, 522, 529, 560, 650, 847

compatibility testing, 524

component preparation in, 522, 523

delays in starting, 525

delivering blood to transfusion area, 524-

525

electromechanical pumps for, 522, 523,

529-530, 565

emergency release, 522-523

errors, 525-526, 527, 641, 642, 653, 654

filters for, 528-529, 565-566

identification procedures, 525-526

infusion sets for, 527-528, 565-566

IV solutions, 530

in neonates, 565-566

patient care during, 530-531

patient consent, 521-522

patient considerations in, 522

patient education, 521-522

post-administration events, 531-532

prescription for, 522-523

pressure devices for, 530

quality assurance, 532

reaction evaluation, 531, 652-656

special instructions for, 522-523

starting the transfusion, 526-527

venous access, 523-524, 565-566

Blood bags, 104-105

centrifugation of, 179-180

for frozen storage, 182

returning to lab, 531-532

Index 861

Blood collection

adverse donor reactions, 19, 107-109

anticoagulants and preservatives, 178-179,

186

blood containers, 104-105

of blood samples, 105-106, 408-409, 801-804

care of donor, 106

of components by apheresis, 140-144

equipment quality control, 847

fatalities related to, 19

identification in, 98, 105

intraoperative, 117, 130-133

phlebotomy, 105-106, 178, 693, 801-804

postoperative, 117, 133-135

preoperative, 126

preoperative autologous, 118-126

preparing venipuncture site, 105, 800-801

records, 97-98, 165

safety, 41, 51

volume collected, 101, 122, 178-179

of whole blood, 178-179

Blood component selection

ABO/Rh compatibility in, 411, 418, 486,

489-490, 496, 498

after non-group-specific transfusions,

419-420

with alloantibodies, 418, 439-443

for exchange transfusions, 546-547, 567

for intrauterine transfusions, 544

for massive transfusions, 419, 510-511

platelets, 363-365, 489-490, 569

rare types, 441-442

red cell components, 411, 486

in urgent situations, 419, 510-511

in warm autoimmune hemolytic anemia,

463

Blood components. See also Blood collection;

Blood component selection; specific compo-

nents

aliquoting, 193-194, 564, 571

appearance, 194, 525, 655

bacterial contamination, 195, 643, 655,

690-695

for children, 564-572, 574-577

CMV-negative, 95, 172

disposition, 196

distribution, handling and dispensing, 36-37

for exchange transfusion, 567

expiration and shelf life, 178, 188, 189-190

freezing, 180-183, 807-812

for hemostasis, 496, 497, 498-507, 570-572

identification, 105, 416, 524-526, 527

inspection, 93-94, 194-195, 416, 418, 525

irradiated, 183, 192-193, 493

issuing, 196-197, 416, 418, 524-525

labeling, 15, 170-172, 416, 440

leukocyte reduction, 180, 190, 492-493,

573-574

ordering, 36, 91-94, 522-523, 526

outdating, factors affecting, 90-91

pathogen inactivation, 702

phenotyping, 440-441

pooling, 183-184, 193, 815

preparation, 173, 179-184, 522, 523, 804-818

quality control, 197-199, 202

quarantine, 173-174, 194

records, 165, 172-173, 416, 418

reissuing, 197, 525

rejuvenation, 194, 806-807

returning, 525

storage, 93-94, 184-185, 186, 187

testing

ABO and D, 164, 165

alanine aminotransferase, 164

antibody screening, 165

cytomegalovirus, 170

equipment requirements, 164-165

general requirements, 163-164

hepatitis, 669-673

HIV, 164, 166, 169-170, 679-681

HTLV, 120, 164, 166, 170, 683

records, 165

syphilis, 165-166, 695

viral markers, 166-170

thawing, 191-192, 809, 815

transportation and shipping, 66, 179,

195-196, 717, 722

volume reducing, 193, 490, 569, 817-818

washed, 193

Blood containers, 104-105, 179-180, 182, 564

Blood donation. See Blood collection

Blood exposure, 45, 54-55

Blood Group Antigen Mutation Database, 236

Blood group sugars, 445

Blood group systems. See also specific blood

groups

chromosomal assignments, 225, 226,

235-236

862 AABB Technical Manual

co-dominant traits in, 234-235

defined, 335

distribution of antigens, 335, 337

genetics

basic principles, 223-225

and heredity, 225-232

patterns of inheritance, 232-236

population genetics, 236-237

nomenclature, 238-239, 318-319, 840-

844

serologic behavior of antibodies, 336

Blood inspections

before release, 194-195, 416, 418, 525

of inventory, 93-94

Blood loss

dilutional coagulopathy in, 499-500, 651

due to platelet dysfunction, 490

in preterm neonates, 559

transfusion in, 486-487

Blood ordering practices

assessment, 36

improving, 91-94

physicians’ orders, 522-523, 526

for routine vs emergency orders, 93, 414

Blood pressure, 633

of donors, 102

hypertension, 633

hypotension

in donors, 107

in recipients, 585, 586, 591, 595

in therapeutic apheresis, 152

treatment, 592

Blood pressure cuffs, 847

Blood samples, 409-410

age of, 410, 431, 433

appearance, 410

frozen, 184-185

hemolyzed, 410

identification and collection, 105-106,

408-409, 801-804

incompletely clotted, 409, 722-723

labeling, 409, 719

lipemic, 410

packaging, 717-718, 720-721

requirements for testing, 424, 454

retention and storage, 410

transportation and shipment, 66, 717-721

Blood spills, 55, 56

Blood transfusions. See Transfusions

Blood utilization

assessments, 23, 36-38

blood ordering practices, 36, 91-94, 414

inventory levels, 89-90, 93

maximum surgical blood order schedules,

92-93, 414

outdating factors, 90-91

routine vs emergency orders, 93

of special components, 94-95

units processed, transfused and outdated in

2001, 91

Blood volume, 558-559, 839

Blood warmers, 522, 529, 560, 650, 847

Bloodborne Pathogen Standard, 49

Bombay (Oh) phenotype, 304

Bonds, chemical, 273

Bone banking, 623, 625

infectious disease transmission in, 619

preservation and dating periods for, 624

Bone marrow. See Marrow transplantation

Bovine aprotinin, 502, 513-514

Bovine spongiform encephalopathy (BSE), 690

Burkitt’s lymphoma, 687

C/c antigens and antibodies (Rh system), 316,

841

cis product antigens, 324

concomitant antibodies, 327-328, 441

dosage in, 317

expression of, 320

gene complexes, 319

in HDFN, 536

incidence of, 317, 321

phenotypes, 320

testing for, 330-331, 441

C1-esterase inhibitor, 500

Calcium, 151-152, 561, 636, 649-650

Calculations

for CRYO dose, 501

for Factor VIII dose, 503-504

for multiple alloantibodies, 236, 441

Calibration

of cell washers, 830-832

defined, 30

of platelet separation centrifuges, 179,

826-828

of serologic centrifuges, 828-830

of thermometers, 198, 821-823

Index 863

CAP (College of American Pathologists), 514

Capture-P, 372

Cardiovascular system. See Heart and heart valves

Carriers, defined, 233

Cartwright (Yt) system (ISBT 011), 226, 336, 348,

349, 842

CAS. See Cold agglutinin syndrome

Catheters

for apheresis, 145

for HPC collection, 593, 594

for neonates, 565, 568

for transfusions, 523-524

Cause-and-effect diagrams, 26

CBER (FDA Center for Biologics Evaluation and

Research), 19, 702-703

CCE (counterflow centrifugal elutriation),

596-597

CCI (corrected platelet count increment), 362,

363, 489

CD (cluster of differentiation) markers, 246,

247-248, 250

CD34

analysis/enumeration, 603

collection targets, 593-594

defined, 592

selection of, 596-597

Cefotetan antibodies, 477

Cell adhesion molecules, 246, 269

Cell counters, 847

Cell counts

in apheresis components, 832

in HPC products, 607-608

Cell selection techniques, 596-598

Cell therapy. See Hematopoietic progenitor cell

transplantation

Cell washers, 830-832, 846

Center for Biologics Evaluation and Research

(CBER), 19, 702-703

Central venous catheters, 524, 593

Centrifugation

apheresis separation by, 139-140

in component preparation, 179-180

counterflow elutriation, 596-597

in separating autologous cells, 748-749

standardization of variables, 716

Centrifuges

calibrating, 179, 826-830

continuous-flow, 596-597

QC performance intervals, 846, 847

Centromere, 224, 241

Cephalosporins, 476

detection of antibodies to, 786-788

in positive DAT, 455, 474, 476, 477

Cephalothin (Keflin), 476

CFR (Code of Federal Regulations), 1, 32

CGD (chronic granulomatous disease), 343

Ch (Chido/Rodgers) antigens, 351-352, 842

Chagas’ disease, 697-698, 700

Change control, defined, 30

Charts, run and control, 23

Check cells, 281, 412

Chemical bonds, 273

Chemical Hygiene Plan (CHP), 57-58

Chemical safety, 57-63

chemical categories, 82-83

chemical data sheet, 78-79

chemical hygiene officer, 58

chemical spills, 61-62, 84-88

chemicals found in blood banks, 80-81

engineering controls, 61

general principles, 57-58

hazard communication, 59

hazardous vapors, 62, 63

health hazards categories, 58, 59

labeling and signs, 59-60

material data safety sheets, 59, 60-61, 78-79

personal protective equipment, 61

safe work practices, 61, 82-83

spills, 61-62, 84-88

training, 58-59

waste disposal, 63

Chemiluminescence assay, 380, 441

Chemokines, 594

Chido/Rodgers system (ISBT 017), 351-352

antibodies, 352

antigens, 351-352, 842

genes, 226

plasma inhibition studies, 445, 765-766

Children. See also Hemolytic disease of the fetus

and newborn

ABO antigens/antibodies in, 293, 295, 298,

420

ABO discrepancies in, 298

anemia in, 558, 559, 563-564

autologous collection from, 118-119

blood volume of, 558-559, 839

compatibility testing in, 415, 562-563, 574

cytomegalovirus in, 562

864 AABB Technical Manual

DIC in, 567, 572

erythropoiesis in, 557-558, 559

extracorporeal membrane oxygenation in,

572-573, 574

graft-vs-host disease in, 560-561

hematopoietic transplantation in, 587, 593

hypothermia in, 560

immunologic status, 560-561

leukocyte reduction for, 573-574, 576

Lewis antigens in, 306

metabolic problems in, 561-562

normal laboratory values in, 836-837

paroxysmal cold hemoglobinuria in, 468

plasma volume of, 839

polycythemia in, 572

red cell volume of, 839

size of, 558-559

transfusions in

administration, 565-566

aliquoting for small volumes, 193-194,

564, 571

Cryoprecipitated AHF, 572

education and consent, 521

to enhance hemostasis, 570-572

exchange, 546-547, 560, 566-568

FFP, 571, 576-577

Granulocytes, 569-570

with hemoglobinopathies, 574-576

indications for, 563-564

older infants and children, 574-577

Platelets, 490, 552-553, 568-569, 576-577

Red Blood Cells, 564-568

with sickle cell disease, 574-576

with thalassemia, 575-576

volumes for, 558, 564, 568

variations in antigen expression, 431

Chills with transfusions, 633, 634, 643

Chimerism

blood group, 237, 299

hematopoietic, 298, 601

and posttransfusion GVHD, 399

transfusion, 298, 399

Chloroquine diphosphate, 446, 469, 746-747

Chorionic villus sampling, 539

CHP (chemical hygiene plan), 57-58

Christmas disease, 505

Chromatid, 241

Chromatin, 223, 241

Chromosomes. See also Genes

defined, 223-224, 241

DNA structure in, 203-204

locations of blood group genes, 225, 226,

235-236

Chronic granulomatous disease (CGD), 343

Chronic inflammatory demyelinating

polyneuropathy (CIDP), 156

Circulatory overload, 636, 648-649

Circulatory shock, 633

Cis product antigens, 319-320, 324, 342

Citrate toxicity, 151, 636, 649-650

CJD (Creutzfeldt-Jakob disease), 689-690

Class I, II, III antigens (HLA), 244-246, 386-394

biochemistry and structure, 390-391

biologic function, 393-394

defined, 269, 385

genetics, 386-387

nomenclature, 392-393

Clinical specimens

defined, 717

labeling, 718, 719

shipping, 717-718, 720-722

Clocks, 847

Clone, defined, 269

Cluster of differentiation (CD) markers, 246,

247-248

CMV. See Cytomegalovirus

Co (Colton) antigens and antibodies, 336, 349,

351

Co-dominant traits, 233, 234-235, 241

Coagulation

in hemolytic transfusion reactions, 640, 642

in liver transplants, 629

in massive transfusion, 511, 651

monitoring hemostasis, 494, 496

normal test values, 837, 838

physiologic principles, 493-494

role of platelets, 488-489

Coagulation factors

changes in red cell storage, 187-188

concentrates, 496, 497

in Cryoprecipitated AHF, 500-502

Factor VIIa concentrates, 497

Factor VIII concentrates, 497, 503-504

Factor IX concentrates, 497, 505

minimum levels needed for hemostasis, 495

in neonates, 571

normal values, 837

in plasma, 496, 498-500

Index 865

in platelet concentrates, 838

replacement, 496, 497, 498-508

support during massive transfusion, 511

virus inactivation in concentrates, 701

for vitamin K deficiency, 497, 498-499

for warfarin reversal, 497, 498-499

Coagulopathy

dilutional, 499-500, 567, 651

in hemolytic transfusion reactions, 640,

642

in liver disease, 499

in liver transplantation, 629

in massive transfusions, 511, 651

in neonates, 571

treatment with heparin, 642

in vitamin K deficiency, 498-499

in warfarin reversal, 498-499

Code of Federal Regulations (CFR), 1, 32

Codons, 206-207

Cold-acid elutions, 457, 772

Cold agglutinin syndrome (CAS), 464-466

adsorption procedures, 466

classification, 459

complement in, 281

pretransfusion testing in, 466

serologic findings in, 460, 465

specificity of autoantibodies, 307, 465-466

Cold agglutinin titer, 458, 465, 778-779

Cold autoadsorption

in cold agglutinin syndrome, 308, 466

for detection of alloantibodies, 438, 472

method, 775-776

in resolving ABO discrepancies, 303

Cold autoantibodies

ABO discrepancies with, 299, 302-303, 469

adsorption, 438, 466, 472, 775-776

with alloantibodies, 438-439, 472

anti-I/i, 306, 307-308, 438, 465-466

anti-IH, 308, 434, 777, 778

in cold agglutinin syndrome, 281, 307,

465-466

and complement, 281

determining specificity, 776-778

in mixed AIHA, 466, 467

in paroxysmal cold hemoglobinuria, 467

in Rh testing, 329, 469-470

titration, 458, 465, 778-779

use of sulfhydryl reagents with, 469-470,

744-745

in warm autoimmune hemolytic anemia,

461

Cold hemagglutinin disease (CHD). See Cold ag-

glutinin syndrome

Cold reactive alloantibodies, 273-274, 299, 303

Cold stress. See Hypothermia

Collectionofblood.See Blood collection

Collections, antigen, 335, 355-356

College of American Pathologists (CAP), 514

Colloid solutions, 486, 507-508, 572

Colony-forming cell assays, 603-604

Colony-stimulating factors, 264

Colton system (ISBT 015), 226, 336, 349, 351, 842

Column agglutination technology, 278, 284-285

Compatibility testing. See Pretransfusion testing

Competency assessments, 8

Complement, 259-262

in acute transfusion reactions, 639-640

alternative pathway, 260, 261

in antiglobulin testing, 280-281, 453

in autoimmune hemolytic anemia, 281, 460,

465

C1-esterase inhibitor, 500

classical pathway, 259-261

complement receptors, 262, 354

membrane attack complex, 261-262, 263

physiologic effects of activation, 262

and positive DAT, 453

regulation of activation, 262, 263

Complications. See Adverse reactions

Computer crossmatch, 413, 414-415

Computer systems

alternative systems, 18-19

backups, 19

for record storage, 17-19

security, 18

validation, 13-14, 415

Confidential unit exclusion, 101

Confidentiality of records, 18

Consent

for apheresis, 140, 142

for blood donation, 103

for transfusion, 521-522

for transplantation, 620-621

Containers

for biological specimens, 717

for blood collection, 104-105, 179-180

damaged or leaking, 720

freezer storage bags, 182

866 AABB Technical Manual

quality control, 847

for shipments, 195, 607, 717-718

Contamination. See Bacterial contamination

Continuous-flow centrifuges, 596-597

Continuous Quality Improvement, 123-124

Contracts, 10

Control charts, 23, 30

Controls

autologous, 412, 427, 438-439, 454

for high-protein reagents, 328-329

IgG-coated cells, 281, 412

for indirect antiglobulin tests, 754

for low-protein reagents, 330

in viral marker testing, 167-169

Convulsions in donors, 108

Copper sulfate

quality control, 819-821, 847

in screening donors, 102, 799-800

Cord blood

antigen expression on, 431, 433

HPC-C transplantation, 588-589

autologous, 589

collection, 588, 595-596

colony-forming cell assays, 603-604

cord blood banks, 589

diseases treated with, 583, 584

engraftment in, 588-589

evaluation and quality control, 607-

608

freezing and storage, 604, 605-606

infectious disease testing, 591

microbial cultures, 602-603

processing, 602

regulations, 608

related donors, 589

standards, 609

stem cell enumeration, 603

thawing and infusion, 607

transportation and shipping, 606-607

serologic testing, 544-546, 595

Cordocentesis, 539, 541-542, 552

Corneal transplants, 619, 621, 624

Corrected platelet count increment (CCI), 362,

363, 489

Corrective action, 24-25

Corticosteroids, 144

Cost blood group collection, 352, 355

Counterflow centrifugal elutriation (CCE),

596-597

CP2D (citrate-phosphate-dextrose-dextrose),

176, 178

CPD (citrate-phosphate-dextrose), 176, 178, 186

CPDA-1 (citrate-phosphate-dextrose-adenine),

176, 178, 564-565

CR (complement receptors), 262, 354

Cra(Cromer) antigen/antibody, 353, 354, 843

CREG (cross-reactive groups), 392

Creutzfeldt-Jakob disease (CJD), 689-690

Cromer system (ISBT 021), 226, 353, 354, 843

Cross-reactive groups (CREGs), 392

Crossmatch-to-transfusion (C:T) ratios, 92, 93

Crossmatching

antiglobulin test in, 413, 414

for children, 415, 546, 563

computer, 413, 414-415

HLA, 397, 398, 401, 402, 492

immediate-spin, 413, 414, 751-752

“in-vivo,” 509

interpretation of results, 415-416, 417

platelets, 364, 365, 398

in pretransfusion testing, 412, 413-415, 417

in transfusion reaction evaluation, 654, 655

Crossover, gene, 208, 209, 230-231, 232, 241, 389

CRYO. See Cryoprecipitated Antihemophilic Fac-

tor

Cryoglobulinemia, 156

Cryoprecipitate Reduced Plasma, 177, 190, 496

Cryoprecipitated Antihemophilic Factor (CRYO)

ABO compatibility, 411, 500-501

calculating dose, 501

for coagulation factor replacement, 496

description, 177

for disseminated intravascular coagulation, 502

expiration dates, 189

for fibrinogen abnormalities, 501-502

for hemophilia A, 502-503

indications for use, 499, 500-503

inspection, 194

inventory management, 94

for neonates, 558, 572

pooled, 183-184, 191, 193, 815

preparation, 814-815

quality control, 199, 202

room temperature storage, 185

thawing, 191, 815

topical use, 502

transportation and shipping, 196

for von Willebrand syndromes, 501

Index 867

Cryopreservation

agents, 181, 604

computer-controlled, 605

effects of, 181

of hematopoietic progenitor cells, 604-606

passive controlled-rate, 605-606

of platelets, 183

of red cells, 181-183, 807-812

storage bags, 182

Crystalloids, 150, 151, 486, 572

Cs (Cost) antigens and antibodies, 352, 355

C:T (crossmatch-to-transfusion) ratios, 92, 93

Cultures, bacterial, 195, 602-603, 655, 693, 694

Customer relations, 9-10

CXCR4, 594

Cytokines

defined, 269

in immune response, 251, 254, 262-263, 264

in transfusion reactions, 640, 643

Cytomegalovirus (CMV), 686-687

associated with hepatitis, 667, 668

blood component testing for, 170

clinical manifestations, 686

and leukocyte reduction, 687

in neonates, 562

prevention, 687

transfusion-transmitted, 686-687

in transplantation, 590, 591, 621, 629, 630

Cytomegalovirus (CMV)-negative products, 95

granulocytes, 492, 570

for intrauterine transfusion, 544, 553

labeling, 172

Dantigen.See also Rh system; Rh testing

clinical significance, 316

discovery of, 315-316

expression of, 319-320

in HDFN, 330, 536-537

ISBT classification, 841

partial D, 322-323, 538

testing, 165, 328-330, 414

weak D, 322-324

in autologous donations, 324

in donors, 165, 323

“microscopic,” 550

partial D, 322-323

as position effect, 302

in pregnancy, 538-539

quantitative, 322

in recipients, 323-324, 411

Dangerous goods, 716-722

DAT. See Direct antiglobulin test

DAT-negative autoimmune hemolytic anemia,

459, 468

DDAVP (desmopressin), 513

as fibrinolytic inhibitor, 514

in hemophilia A, 503, 513

indications for, 513

in liver disease, 499

in von Willebrand syndrome, 501, 513

Decay-accelerating factor (DAF), 353

Decontamination of biohazardous waste, 51, 54,

Deferrals, donor

of autologous donors, 120

for babesiosis, 696

for Creutzfeldt-Jakob disease, 689, 690

for drugs taken by donor, 100-101, 113

for hepatitis, 674, 703

for HIV, 674, 680, 681, 703

for HTLV, 683, 684, 685, 703

implicated in posttransfusion infections, 703

for Lyme disease, 696-697

for malaria, 697

records of, 98, 173

Deglycerolized Red Blood Cells

checking adequacy of deglycerolization,

812-813

expiration date, 189

preparation, 191-192, 809

refreezing, 183

of sickle cell trait, 192

storage, 192

Delayed hemolytic transfusion reactions

(DHTR), 346, 575, 637, 656-657

Delayed serologic transfusion reaction (DSTR),

656

Delayed transfusion reactions. See also Transfu-

sion reactions

alloimmunization, 637, 656-657

graft-vs-host disease, 399, 560, 637, 657-659

hemolytic, 346, 575, 637, 656-657

immunomodulation, 638, 660

iron overload, 638, 660

Kidd antibodies in, 346, 656

posttransfusion purpura, 366, 370, 638, 659-660

Density gradient separation, 601

868 AABB Technical Manual

Derivatives. See also specific derivatives

for coagulation factor replacement, 496

virus inactivation, 496, 699, 701

Desensitization therapy, 505

Designoutput,defined,4,30

Designated donations, 98, 103-104

Desmopressin. See DDAVP

Deviations, 19, 20, 21

Dextrose solution, 530

Dha(Gerbich) antigen, 352, 843

DHTR (delayed hemolytic transfusion reac-

tions), 346, 575, 637, 656-657

Di (Diego) antigens and antibodies, 336, 348, 349

Diagnostic specimens

defined, 717

labeling, 718, 719

shipping, 717-718, 719, 720-722

DIC. See Disseminated intravascular coagula-

tion

Dichloromethane/methylene chloride elutions,

457, 775

Diego system (ISBT 010), 348

antigens/antibodies, 336, 348, 842

chromosomal location of genes, 226

and En(a–), 338

in HDFN, 348

phenotype frequencies, 349

in transfusion reactions, 348

Differential centrifugation, 179-180

Differential warm adsorption, 781-782

Digitonin acid elutions, 457

Dilution

of % solutions, 726-727

of serum, 725-726

Dilutional coagulopathy, 499-500, 567, 651

Dimethylsulfoxide (DMSO), 604, 607

2,3-Diphosphoglycerate. See 2,3-DPG

Direct antiglobulin test (DAT)

in classifying AIHA, 458

complement in, 280-281

method, 760-761

positive test

in antibody identification, 427, 436,

438-439

causes, 453-454

in cold agglutinin syndrome, 460, 465

drug-induced, 455, 472-477, 481-482

elution with, 456-458, 745-746

evaluation, 454-458, 480

in HDFN, 545-546

in IgM warm AIHA, 464

medical history in, 454-456

in mixed-type AIHA, 460, 466

in paroxysmal cold hemoglobinuria, 460,

467

red cell testing with, 745-747

serologic studies, 456

in warm autoimmune hemolytic anemia,

459, 460, 461

in pretransfusion testing, 412, 454

principles, 278

reagents, 278-280 , 427, 454, 468

specimens, 454

in transfusion reaction evaluation, 653-654

in transplantation, 455, 629

vs autologous control, 427

Directed donations, 94-95, 537-538

Disaster planning, 67-68

Disinfectants, 51, 54

Disposal

of biohazardous waste, 56-57

of blood components, 196

of chemical waste, 63

of radioactive waste, 66

Disposition of blood components, 196

Disseminated intravascular coagulation (DIC)

in neonates, 567, 572

in transfusion reactions, 640, 642

treatment for, 502, 567

Dithiothreitol. See DTT

Diversion, 693

DMSO (dimethylsulfoxide), 604, 607

DNA (deoxyribonucleic acid)

libraries, 217

molecular techniques

cloning, 217, 222

isolation of nucleic acids, 209-211

microarrays, 218

polymerase chain reaction technique,

211, 212, 213-214, 222, 394-396

profiling (typing or fingerprinting), 216, 222

recombinant proteins, 218-219, 222

restriction endonucleases, 214, 215

restriction fragment length polymor-

phism analysis, 214, 215, 222

sequencing, 217-218, 222, 396

structure, 203-204

transcription, 204, 205

Index 869

Do (Dombrock) antigens and antibodies, 336,

349, 350-351, 842

Documents, 14-17. See also Records

management, 14-15, 17

quality system, 14, 16

types, 15

Dolichos biflorus lectin, 293, 296, 302, 743-744

Dombrock system (ISBT 014)

antibodies, 336, 350-351

antigens, 350, 842

genes, 226

phenotype frequencies, 349

Dominant traits, 232-233, 234, 241

Donath-Landsteiner test, 458, 467, 784-785

Donation intervals for apheresis, 141, 143

Donation process. See Blood collection

Donor area, 55, 97

Donor deferrals

of autologous donors, 120

for babesiosis, 696

for Creutzfeldt-Jakob disease, 689, 690

for drugs taken by donor, 100-101, 113

for hepatitis, 674, 703

for HIV, 674, 680, 681, 703

for HTLV, 683, 684, 685, 703

implicated in posttransfusion infections, 703

for Lyme disease, 696-697

for malaria, 697

for positive infectious disease tests, 680, 683,

684, 685

records, 98, 173

Donor history questionnaire, 110-112, 114-118

Donors

adverse reactions in, 19, 107-109

autologous, 103, 121-122, 124-125, 131, 135,

693

care of, after phlebotomy, 106

confidential unit exclusion, 101

consent, 103, 620-621

designated donations, 98, 103-104

directed donations, 104, 537-538

drugs taken by, 100-101, 113, 115

eligibility, 99-100

family members as, 442

hematocrit, 102

for hematopoietic transplantation, 589-591

hemoglobin levels, 102, 799-800

for HLA-matched platelets, 398

hypotension in, 107

implicated in posttransfusion infection, 703

information provided to, 98-99, 114

medical history, 100-101, 110-112, 693

notification of abnormal tests, 99, 103, 703

for organ and tissue transplantation, 618,

620-623

physical examination of, 100, 101-103, 620

for plateletpheresis, 141

records, 165, 173

red cell density, 102

reentry with positive screening tests, 673,

674, 675, 681-682

registration, 97-98

report of illness after donation, 103

testing

ABO, 165, 290, 296, 413

alanine aminotransferase, 164

antibody detection, 165

for blood group antigens, 418, 440-441,

745-748

cytomegalovirus, 170

equipment requirements, 164-165

general requirements, 163-164

hepatitis, 120, 166-170, 669-673

HIV, 120, 164, 166-170, 679-681

HTLV, 120, 683

repeat, 413

Rh, 165, 328-332, 413

syphilis, 120, 165-166, 695

viral markers, 166-170

weak D, 165, 323, 328

weight, 101, 143

Doppler flow studies, 542

Dosage effect

in antibody identification, 425, 431

in Duffy system, 345

genetics, 225, 227

in Rh system, 327

Dosimeters, 64-65

Dot blot, 213, 395

2,3-DPG

in neonatal transfusions, 561-562

in red cell storage lesion, 185, 187

in tissue oxygenation, 484, 511-512

Dra(Cromer) antigen, 353, 354, 843

Drug-induced immune hemolytic anemia,

472-477

autoantibodies, 474, 475-476

classifications, 473-476

870 AABB Technical Manual

drug adsorption mechanism, 473, 474, 475

drug-dependent antibodies, 473, 474-475

drugs associated with, 476, 481-482

laboratory investigation, 460, 476-477,

786-791

metabolites, 477

nonimmunologic protein adsorption, 476

theories of, 472-473

Drugs

administered for leukapheresis, 143-144

alteration of pharmacodynamics in

apheresis, 150-151

for autologous tumor purging, 597

as cause of thrombocytopenia, 374-377

for febrile transfusion reactions, 644

in positive DAT, 455, 472-477, 481-482

to prevent allergic transfusion reactions, 646

taken by donors, 100-101, 113, 115

Dry ice, 196, 719, 720-721

Dry shippers, 607

DSTR (delayed serologic transfusion reaction),

656

DTT (dithiothreitol)

applications for, 448-449

differentiating IgG from IgM, 764-765

to disperse autoagglutination, 302, 469, 470,

744-745

inactivating blood group antigens, 342, 445,

446, 766-767

Duffy system (ISBT 008), 343-345

antibodies, 336, 345, 844

antigens, 343-344, 841

biochemistry, 344-345

chromosomal locations of genes, 226

effect of enzymes on, 276, 336, 345

in HDFN, 345

and malaria, 337, 344

phenotypes and frequencies, 344, 844

in transfusion reactions, 345

Dura mater transplants, 619, 624

Dysfibrinogenemias, 501-502

E/e antigens and antibodies

cis product antigens, 324

concomitant antibodies, 327-328, 441

dosage in, 317

expression of, 320

gene complexes, 319

in HDFN, 536

incidence of, 317, 321

phenotypes, 320

testing for, 330-331, 441

E/e antigens and antibodies (Rh system), 316,

841

EACA (epsilon aminocaproic acid), 513-514

EBV (Epstein-Barr virus), 667, 668, 687-689

ECMO (extracorporeal membrane oxygenation),

572-573, 574

Education

donor, 98-99, 114, 117-118

patient, 521-522

EIA. See Enzyme-linked immunosorbent assay

ELBW (extremely low birthweight) neonates,

557, 568. See also Neonates

Elderly people, 295, 299

Electrical safety, 48-49

Electromechanical infusion devices, 522,

529-530, 565

ELISA. See Enzyme-linked immunosorbent as-

say

Elutions

with adsorption, 300-301, 448, 735-736

in antibody identification, 447-448

in cold agglutinin syndrome, 460, 465

in IgM warm AIHA, 464

methods, 457, 771-775

cold-acid, 457, 772

dichloromethane/methylene chloride,

457, 775

digitonin-acid, 457

gentle-heat, 745-746

glycine-HCl/EDTA, 772-773

heat, 457, 773-774

Lui freeze thaw, 457, 774

in mixed-type AIHA, 460, 467

in paroxysmal cold hemoglobinuria, 460, 467

in positive DAT evaluation, 456-458

technical factors in, 447-448

uses, 448

in warm antibody AIHA, 460, 461

Embolism, air, 636, 651-652

Emergency procedures

blood orders, 93

identification of patients, 408

issue of blood, 419, 510-511, 522-523

in pretransfusion testing, 419

Emergency release, 510-511, 522-523

Index 871

Emergency response plans, 44

for biohazardous waste, 55-57

for chemicals, 61-62, 63

for electrical emergency, 49

for fires, 48

for radiation safety, 66

Emergency showers, 61

Emergency supplies for donor area, 108

Employees. See Personnel

En(a–) red cells, 338

Engineering controls

for biosafety, 50-51, 52-53, 54

for chemical safety, 61

for electrical safety, 48

for fire prevention, 47

general guidelines, 43-44, 73-76

for radiation safety, 65-66

Engraftment data, 608

Enhancement of antibodies

albumin additives, 276, 427, 753

alteration of pH, 274, 444

in antibody detection/identification,

276-277, 427, 443-444, 753-754

combined adsorption/elution, 300-301, 448,

735-736

effect of incubation times, 274, 444

enzymes, 276, 443, 445 , 446, 756-760

increased serum-to-cell ratio, 444

LISS and LISS additives, 274, 275, 277, 427,

443, 753, 754

polyethylene glycol, 276-277, 427, 443, 753

temperature reduction, 443

Environmental chambers, 185, 197-198, 846

Enzyme-linked immunosorbent assay

(EIA/ELISA)

in anti-HTLV testing, 683

in antibody detection, 286

in antigen-antibody detection, 286

for Chagas’ disease testing, 698

for heparin-dependent antibodies, 377

in hepatitis testing, 672-673

in HIV testing, 679

in viral marker testing, 166

Enzymes, proteolytic

in adsorption, 470, 471, 781-782

in antibody detection/identification, 276,

443, 445, 446

effect on blood group antigens, 276, 336,

339-340, 345

evaluating treated red cells, 758-759

ficin preparation, 756

one-stage technique, 759

papain preparation, 756-757

in resolving ABO discrepancies, 300, 301

standardization of procedures, 757-758

two-stage technique, 759-760

Eosinophils, 255

Epistasis, 236

Epitope, defined, 269

EPO. See Erythropoietin

Epsilon aminocaproic acid (EACA), 513-514

Epstein-Barr virus (EBV), 667, 668, 687-688

Equilibrium constant (Ko), 273

Equipment. See also specific equipment

for apheresis, 139-140, 152

calibration, 821-823, 826-830

for component testing, 164-165

critical, 10

management, 10-11

personal protective, 43-44, 73-75

quality control

automatic cell washers, 830-832

centrifuge calibration, 826-830

continuous temperature monitoring sys-

tems, 197-198

freezer alarms, 198-199, 824-826, 846

performance intervals, 846-847

refrigerator alarms, 198-199, 823-824, 846

thermometers, 198, 821-823, 847

for transfusions, 523-524, 527-530

validation, 11, 13

Er blood group collection, 355

Ergonomics, 41-42

Errors

in ABO typing, 299-300

in antiglobulin testing, 282, 283

identification, 525-526, 527, 641, 642, 653,

654

Erythroblastosis fetalis. See Hemolytic disease of

the fetus and newborn

Erythropoiesis, 557-558, 559

Erythropoietin (EPO)

in anemia treatment, 219, 488

in autologous donations, 125, 219

in newborns and infants, 558, 559

recombinant, 125, 219, 488, 512, 559

as transfusion alternative, 512

Esa(Cromer) antigen, 353, 354, 843

872 AABB Technical Manual

Ethnic groups, differences in

in ABO system, 845

in Cromer system, 353

in distribution of antigens, 335, 337

in Duffy system, 337, 344, 345

in HLA system, 585

in Kell system, 341, 343

in Kidd system, 337

in Lutheran system, 347

in Rh system, 316, 317, 320, 321, 325

Etiologic agents, 719

Exchange transfusions

with antibody against high-incidence anti-

gen, 547

blood selection for, 546-547, 567

blood warming in, 560

in DIC, 567

in HDFN, 546-547

for hyperbilirubinemia, 566-567

methods used, 568

“partial,” 563

for removal of toxins, 567

in sickle cell disease, 574

vascular access, 568

volume and hematocrit, 568

Executive management, 6

Exons, 206

Expiration dates

checking before issue, 525, 526

of components, 188, 189-190

of tissues and organs, 624

Exposure control plan, 49, 50

External assessments, 23-24

External controls in viral marker testing, 167-169

Extracorporeal membrane oxygenation (ECMO),

572-573, 574

Extracorporeal photochemotherapy, 158

Eye washes, 50, 75

Face shields, 74

Facilities

design and workflow, 40

ergonomics, 41-42

housekeeping, 40-41

mobile sites, 41

organizational management, 6-7

restricted areas, 41

safety, 27, 39-42

FACT standards, 609

Factor V, 188

Factor VIIa, 497, 505

Factor VIII

calculating dose, 503-504

concentrates, 497, 503-504

deficiency, 503

for hemophilia A, 502-504

inhibitors to, 504-505

in stored blood, 188

virus inactivation, 710

for von Willebrand syndromes, 501

Factor IX, 497, 498, 504, 505

Failures, active and latent, 26-27

Fainting in donors, 107

False-positive/false-negative results

in antiglobulin testing, 282, 283

in Rh testing, 329, 330, 331-332

Fascialatatransplants,624

Fatalities

during apheresis, 153

due to bacterial contamination, 690-692

due to transfusions, 641, 645, 661, 691,

702-703

employees, 46

reporting, 19, 46, 661, 702-703

FDA. See Food and Drug Administration

Febrile nonhemolytic transfusion reactions

(FNHTR), 643-644

granulocyte antibodies in, 379

HLA antibodies in, 398-399

management, 634

manifestations, 643-644

pathophysiology, 634, 643-644

preventing, 576, 644

treatment, 644

Fetal hemoglobin (hemoglobin F), 550, 558, 835

Fetomaternal hemorrhage (FMH)

as immunizing event, 536

Kleihauer-Betke acid-elution test, 550-551,

794-796

microscopic weak D test, 550

postpartum evaluation, 548, 549-551

rosette test, 550, 793-794

Fetus. See also Hemolytic disease of the fetus

and newborn

erythropoiesis in, 557-558, 559

hematocrit, 541

Rh typing, 539

Index 873

Fever, transfusion-associated, 398-399, 633, 634,

643

FFP. See Fresh Frozen Plasma

Fibrin degradation products, 837

Fibrin sealant, 502

Fibrinogen

abnormalities, 501-502

CRYO for replacement of, 500, 501

in monitoring coagulation, 494, 511

normal values, 837

Fibrinolytic inhibitors, 513-514

Ficin, preparation of, 756. See also Enzymes,

proteolytic

Filgrastim. See Granulocyte colony-stimulating

factor

Filters

leukocyte-reduction, 180, 190, 528-529

microaggregate, 528, 565-566

standard in-line, 527-528

Fire safety, 47-48

Fish-bone diagrams, 26

Fisher/Race Rh terminology, 319

Flow cytometry

in CD34 analysis/enumeration, 603

in granuloctye autoantibody testing, 380

in HLA crossmatch, 397, 401

in platelet antibody detection, 372, 375, 376

to predict clinical significance of antibodies,

441

Flow PRA, 366

Fluids, replacement, 150, 151

Fluosol, 513

FMH. See Fetomaternal hemorrhage

FNHTR. See Febrile nonhemolytic transfusion

reactions

Food and Drug Administration (FDA)

cGMP training, 8

quality assurance regulations, 1, 2

regulations for autologous blood, 119-120

regulations for tissue transplantation, 626

reporting adverse events to, 19, 20, 21, 661,

702-703

Forensic testing, 402

Forms, 15, 17. See also Documents; Records

FoundationfortheAccreditationofCellTherapy

(FACT), 609

Freeze-thaw elutions, 457, 774

Freezers, 185

alarm systems for, 185, 198-199, 824-826

quality control, 197-199, 846

temperature monitoring systems for,

197-198

thermometers for, 198

Freezing

blood samples, 184-185

cryoprotective agents, 181, 604

freeze-thaw damage, 181

Fresh Frozen Plasma, 180-181, 814

hematopoietic progenitor cells, 604-606

Platelets, 183

Red Blood Cells, 181-183, 807-812

refreezing deglycerolized cells, 183

skin grafts, 624, 625

storage bags for, 182

Fresh Frozen Plasma (FFP)

ABO compatibility, 411, 496, 498

coagulation factors in, 496

collection by apheresis, 141, 142

description, 177

expiration dates, 189-190

indications for, 496, 498-500

inspection, 194

inventory management, 94

misuse, 500

for neonates, 558, 571

in older infants and children, 576

preparation, 180-181, 813-814

small-volume, 571

thawing, 191

transfusion thresholds, 494, 496

transportation and shipping, 196

virus inactivation, 701-702

Frozen components. See specific components

Fume hoods, 61

Fy (Duffy) antigens and antibodies, 336,

343-344, 345, 841

G antigen (Rh system), 324-325, 841

G-CSF. See Granulocyte colony-stimulating fac-

tor

GBV-C virus, 668

Ge (Gerbich) antigens and antibodies, 352-353,

843

GEIS antigen, 352, 843

Gel test, 285

Gene chips, 218

Gene therapy, 217, 219-220

874 AABB Technical Manual

Genes

blood group terminology, 844

chromosomal assignment, 225, 226, 235-236

defined, 241

DNA structure, 203-204

frequencies, 227-229, 320-321

gene conversion, 209, 210

major histocompatibility complex, 244-246,

386-389

modifier, 235-236

nucleotide insertions and deletions, 208

nucleotide substitutions in, 207-208, 228

obligatory, 237

single crossover, 208, 209, 230-231, 232, 241, 389

suppressor, 235-236

variability, 208-209, 210

Genetics of blood groups

of ABO system, 229-230, 235, 290-293

alleles, 225, 227-229

basic principles, 223-225

definitions of terms, 241

of H system, 290-293

and heredity, 225-232

of HLA system, 386-389

Lewis system, 305

of MNS system, 338

nomenclature, 238-239, 318-319, 840-844

patterns of inheritance, 232-236

polymorphism in, 207-208

population genetics, 236-237

of Rh system, 316-318

Genotypes. See also specific blood groups

defined, 233

frequencies, 227-228

nomenclature for, 238-239

Gentle heat elutions, 745-746

Gerbich system (ISBT 020), 226, 352-353, 843

GIL system (ISBT 029), 226, 355, 843

Glanzmann’s thrombasthenia Type I, 366, 369

Gleevec (imatinib mesylate), 220

Globoside (GLOB) blood group, 226, 308, 843

Gloves, 46, 55, 73-74, 106

Glycerolization of red cells, 181-182, 808-809,

810-812

Glycine-HCl/EDTA

to disperse autoagglutination, 469

to dissociate IgG from red cells, 469, 747-748

elutions, 772-773

to inactivate red cell antigens, 446

Glycine max (anti-T, -Tn), 743

Glycophorins, 338, 339

GM-CSF (granulocyte-macrophage col-

ony-stimulating factor), 264, 512, 592, 594

Goggles, safety, 74

Gov antigens, 370

Grading test results, 412, 728-729

Graft failure, 598

Graft-vs-host disease (GVHD)

acute/chronic, 586

and graft-vs-leukemia effect, 586-587

in hematopoietic transplantation, 585-588

HLA system in, 399, 400

in neonates, 560-561

transfusion-associated, 399, 400, 560-561,

637, 657-659

Graft-vs-leukemia (GVL) effect, 586-587

Gram-equivalent weight, 723

Gram-molecular weight, 723

Gram’s stain, 693

Granulocyte colony-stimulating factor (G-CSF)

and false-positive test results, 590

for HPC mobilization, 587, 592-595

in leukapheresis, 144

in neonatal sepsis, 570

production and function, 264

side effects of, 594-595

uses for, 218, 222, 512

Granulocyte-macrophage colony-stimulating

factor (GM-CSF), 264, 512, 592, 594

Granulocytes

alloantigens, 377-380, 647

antibodies to

in neonatal alloimmune neutropenia, 379

testing for, 380

in TRALI, 379-380, 647, 656

in immune system, 255-256

Granulocytes Pheresis

ABO compatibility, 411

collection, 143-144

description, 177-178

expiration dates, 189

indications/contraindications for use, 492,

570

irradiation, 144, 492

laboratory testing, 144

for neonates, 558, 569-570

quality control, 202

storage, 144

Index 875

transfusion, 143, 492, 569-570

transportation and shipping, 196

validation, 35

Group O serum (anti-A,B), 295

Growth factors, recombinant

for HPC mobilization, 587, 592-595

in leukapheresis, 144

as transfusion alternative, 512, 559

uses for, 219, 222

GTI PAKAUTO, 374

Guillain-Barré syndrome, 156

GUTI (Cromer) antigen/antibody, 353, 354, 843

GVHD. See Graft-vs-host disease

GVL (graft-vs-leukemia) effect, 586-587

Gya(Dombrock) antigen, 350, 842

H system (ISBT 018), 303-304

anti-H, 304, 434

anti-IH, 308, 434, 777, 778

biochemistry and genetics, 290-293

chromosomal locations of genes, 226

H antigen, 290, 303-304, 361, 842

Ohphenotype (Bombay), 304

para-Bombay phenotypes, 304

saliva testing for substances, 301, 736-739

HAM (HTLV-associated myelopathy), 682

Hand-washing, 50, 75

Haplotypes

ancestral, 389

defined, 231

of HLA system, 387-388, 389, 400, 402

of Rh system, 318-319

Haptoglobin, 835

Hardy-Weinberg law, 227-229

HAV (hepatitis A virus), 667-668, 671, 700

Hazard Communication Standard, 59-60

Hazard identification and communication, 43

for biosafety, 50

for chemical safety, 59-61

for electrical safety, 48

for fire prevention, 47

Hazardous areas of facilities, 41

Hazardous materials

biohazards, 49-57

chemicals, 57-63

radioactive, 63-66

safety plan for, 42-47

transportation and shipping, 66, 716-722

waste management, 55-57, 67

HBeAg (hepatitis B e antigen), 669, 670

HBsAg. See Hepatitis B surface antigen

HBV. See Hepatitis B virus

HCV. See Hepatitis C virus

HDFN. See Hemolytic disease of the fetus and

newborn

HDV (hepatitis D virus), 668, 670

Heart and heart valves

arrhythmias in recipients, 650

cardiac arrest in donors, 108

cardiac output, 485-486

disease of, 119, 487-488

infectious disease transmission, 619

preservation, 624

transplantation, 402, 625, 629

Heat elutions, 457, 745-746, 773-774

Heating blocks, 846

Hemapheresis practitioner (HP), 139

Hematocrit

in autologous blood donors, 125, 126

in blood donors, 102

of blood in exchange transfusions, 568

fetal, 541

in neonates, 572

normal values, 835

of red cell components, 805

transfusion trigger, 487

Hematoma, in donors, 108

Hematopoietic growth factors

for HPC mobilization, 587, 592-595

for leukapheresis, 144

as transfusion alternative, 512, 559

uses for, 218-219, 222

Hematopoietic progenitor cell (HPC) transplan-

tation, 581-609

ABO incompatibility, 598-599, 600, 601-602

ABO typing discrepancies in, 299

allogeneic, 582, 583, 585-588, 589-590,

594-595

autologous, 582, 583, 587, 589, 592-594, 597

autologous tumor purging, 597-598

cell processing, 596-604

chimerism in, 298, 601

collection of products, 144, 591-596

colony-forming cell assays, 603-604

diseases treated with, 583, 584

donor eligibility, 589-591

876 AABB Technical Manual

evaluation and QC of products, 607-608

freezing and storage of cells, 604-606, 624

and graft-vs-host disease, 585, 586-587

HLA testing, 400-401, 585-587

of HPC-A (apheresis), 581, 587-588, 592-595

of HPC-C (cord blood), 588-589, 595-596,

602

of HPC-M (marrow), 583, 585-587, 591-592

indications for, 582

infectious disease testing, 590-591

matched, unrelated donors, 585-586, 589

microbial cultures, 602-603

mobilization of HPCs, 587, 592-595

nonmyeloablative, 582-583

positive DAT after, 455

processing, 596-598, 601-604

red cell depletion, 601

regulations, 608

related donors, 587-588, 589

selection of CD34+ cells, 596-597

sources of cells for, 583, 584-589

standards, 609

stem cell enumeration, 603

suitability criteria, 606

syngeneic, 582, 587

T-cell depletion, 586, 598

thawing and infusion of products, 607

transfusion support, 591-592, 599, 600, 601

transportation and shipping, 606-607

types, 581-582

xenogeneic, 582, 583

Hemoglobin

in autologous donors, 125

in blood donors, 102

bovine, 513

fetal (F), 550, 558, 835

in neonates, 558, 563, 572, 836

normal values, 835, 836

and oxygen, 185, 187, 483-485

recombinant, 219, 513

S, 544, 567, 574, 575

in transfusion reaction evaluation, 654-655

as transfusion trigger, 483, 486-488

Hemoglobin solutions, 512-513

Hemoglobinometers, 847

Hemoglobinopathies, 508-509, 574-576. See also

specific hemoglobinopathies

Hemolysis

in ABO testing, 295

in antibody detection, 412

defined, 271-272

immune-mediated

ABO/Rh typing problems with, 469-470

alloimmune hemolytic anemia, 459

antibodies associated with, 336

antibody detection with, 470-472

classification of anemias, 458, 459

cold agglutinin syndrome, 459, 460,

464-466

DAT-negative AIHA, 459, 468

defined, 458-459

drug-induced, 459, 460, 472-477, 481-482

in hemolytic transfusion reactions,

639-642

IgM warm AIHA, 464

intravascular/extravascular, 265-266, 267,

458

mixed-type AIHA, 459, 460, 466-467

paroxysmal cold hemoglobinuria, 459,

460, 467-468

warm antibody autoimmune hemolytic

anemia, 459, 460, 461-464

non-immune mediated, 152, 636, 642-643

passenger lymphocyte, 628, 629-630, 657

in patient samples, 410

and positive DAT evaluation, 455

in transfusion reaction evaluation, 653, 655

Hemolytic anemias. See also Hemolytic disease

of the fetus and newborn

alloimmune, 459

classification of, 458, 459

cold agglutinin syndrome, 459, 460, 464-466

DAT-negative AIHA, 459, 468

drug-induced, 459, 460, 472-477, 481-482

IgM warm AIHA, 426, 427, 428, 429-431

mixed-type AIHA, 459, 460, 466-467

paroxysmal cold hemoglobinuria, 459, 460,

467-468

serologic findings in, 460

warm autoimmune hemolytic anemia, 459,

460, 461-464

Hemolytic disease of the fetus and newborn

(HDFN), 535-551

ABO, 536, 537, 538, 545

ABO discrepancies in, 302

antibodies associated with, 336, 536-537,

545-546

elutions in, 456

Index 877

exchange transfusion in, 546-547

intrauterine transfusion in, 541, 543-544

maternal immunization in, 536-538, 542-543

measuring severity of

amniotic fluid analysis, 540-541, 542

Doppler flow studies, 542

maternal antibody titer, 449, 539-540,

761-764, 796-798

percutaneous umbilical blood sampling,

541-542

pathophysiology, 535-536

postpartum evaluation, 544-546

prenatal evaluation, 538-540

Rh testing in, 330, 538-539, 545

use of Rh Immune Globulin, 547-551

Hemolytic transfusion reactions (HTR)

antibodies associated with, 336

coagulation activation in, 640, 642

complement activation in, 639-640

cytokines in, 640

delayed, 346, 575, 637, 656-657

due to ABO incompatibility, 639, 640, 641

elutions in, 456

frequency, 641

HLA antibodies in, 399-400

management, 634

non-immune, 642-643

pathophysiology, 639-641

prevention, 642

renal failure in, 640-641

shock in, 640

signs and symptoms of, 633, 639-641

treatment, 641-642

Hemolytic-uremic syndrome (HUS), 154-155

Hemophilia A, 502-505

calculating Factor VIII dose, 503-504

factor replacement for, 504

inheritance of, 233-234

inhibitors of Factor VIII, 504-505

Hemophilia B, 505

Hemorrhage, intraventricular, 569

Hemorrhagic disease of the newborn, 571

Hemostasis

in acute normovolemic hemodilution, 128

coagulation factors needed for, 495

components for, 496, 497, 498-508, 570-572

during massive transfusion, 511, 651

monitoring, 494, 496

in neonates, 570-571

normal test values, 837

physiologic principles, 488-489 , 493-494

Heparin, 375-377, 505, 642, 723

Heparin co-factor, 505-506

Heparin-induced thrombocytopenia (HIT),

375-377, 492

Hepatitis, 667-675

associated with CMV or EBV, 668

chronic carriers, 668-669

clinical manifestations, 668-669

donors implicated in, 703

markers, 669, 670-671, 672-673, 674

non A, non B (NANB), 673, 699

prophylaxis, 45, 668, 672, 703

quarantine and recipient tracing, 675

reentry of donors with, 673, 674, 675

reporting cases, 702

risk of, 673, 675, 700

surrogate markers, 673

vaccination for, 668

viruses, 667-668

Hepatitis A virus (HAV), 667-668, 671, 700

Hepatitis B core antigen, antibody to (anti-HBc)

blood component testing, 164, 166, 675

look-back for, 675

marker of infection, 669, 670

reentry of donors with, 675

as surrogate marker, 672

testing transplant donors for, 621

Hepatitis B e antigen (HBeAg), 669, 670

Hepatitis B immune globulin (HBIG), 45, 668,

703

Hepatitis B surface antigen (HBsAg)

antibody to (anti-HBs), 669, 670

blood component testing, 164, 166, 169

chronic carriers, 668-669

look-back for, 675

marker of infection, 669, 670, 672

reentry of donors with positive tests, 674, 675

testing organ and tissue donors for, 620, 621,

623

Hepatitis B virus (HBV)

chronic carriers, 668-669

clinical manifestations, 667, 668-669

employee exposure to, 45

HBV DNA, 669, 672

look-back for, 675

markers of infection, 120, 669, 670, 672, 674

NAT testing, 669, 672

878 AABB Technical Manual

prophylaxis for, 45, 668, 672, 703

reentry of donors with, 674, 675

testing transplantation donors for, 590

transfusion risk of, 672, 679, 700

Hepatitis C virus, antibody to (anti-HCV)

blood component testing, 164, 166, 170

marker of infection, 670-671, 672-673

reentry of donors with, 673, 674, 675

supplemental tests, 170

testing organ donors for, 620, 621, 623

Hepatitis C virus (HCV)

autologous blood testing, 120

blood component testing, 164, 166-167, 170

chronic carriers, 669

clinical manifestations, 667, 668, 669

employee exposure to, 45

false-positive test results, 590

infections due to IGIV, 699

look-back for, 675

markers of infection, 669, 670-671, 672-673,

674

NAT testing, 164, 166, 170, 213, 590, 673, 675

reentry of donors with, 673, 674, 675

testing transplantation donors for, 590, 620,

621, 623

transfusion risk of, 673, 675, 699, 700, 701

Hepatitis D virus (HDV), 668, 670

Hepatitis E virus (HEV), 667-668, 671

Hepatitis G virus (HGV), 668

Hereditary angioneurotic edema, 500

Hereditary hemochromatosis (HH), 387

Heredity, genetics of

alleles, 225, 227-229

Hardy-Weinberg Law, 227-229

independent assortment, 229-230, 231

linkage, 230-231, 232

linkage disequilibrium, 231-232, 337, 389

segregation, 229, 230

Herpesviruses, 686-688. See also

Cytomegalovirus

HES (hydroxyethyl starch), 143-144

Heterozygous, defined, 225

HEV (hepatitis E virus), 667-668, 671

HGV (hepatitis G virus), 668

HH (hereditary hemochromatosis), 387

HHV-6/HHV-8 (human herpesviruses), 688

High-incidence antigens

antibodies to, 356-357, 435-436, 441-442,

547

of Cromer system, 353, 354

defined, 335

of Gerbich system, 352

GIL, 355

of Knops system, 354

of Lutheran system, 347

not assigned to a system or collection,

356-357

selecting blood negative for, 441-442

of Vel collection, 355-356

High-titer, low-avidity antibodies, 449, 762,

765-766

HIT (heparin-induced thrombocytopenia),

375-377, 492

HIV (human immunodeficiency virus), 675-682

in AIDS, 675-676

in autologous donors, 117, 681

clinical manifestations, 99, 676-677

confirmatory tests for, 169-170, 679-681

donors implicated in, 703

employee exposure to, 45

false-positive test results, 590

HIV-1, 675, 676, 678

HIV-2, and HIV-1, group O, 675, 677-679

information provided to donors about, 99

nucleic acid testing, 164, 166-167, 213, 590,

678-681

recipient tracing (look-back), 681-682

reentry of donors with positive screens, 674,

681

reporting cases, 702

risk factors for, 677

testing components for, 164, 166-167,

169-170, 213, 679-681

testing transplantation donors for, 590, 620,

621, 623

transfusion considerations, 678-679

transfusion risks of, 679, 700, 701

Hives, 634, 644-647

HLA-B27, 403

HLA Matchmaker, 365

HLA system, 385-404

alloimmunization to platelets, 265, 266,

362-366, 397-398, 637

antibody detection, 365-366, 397

antigens, 362, 385, 388-389, 390-391

and Bg antigens, 358

biochemistry, 390-391

biologic function, 393-394

Index 879

and chimerism, 399

crossmatching, 397, 398, 401, 402, 492

disease associations, 402-404

in forensic testing, 402

genetics, 386-389

in graft-vs-host disease, 399, 400

major histocompatibility complex, 244-246,

386-389

matching

granulocytes, 492

in HPC transplantation, 388, 400-401,

585-586

in organ transplantation, 387-388,

401-402

of platelets, 363-365, 398

nomenclature, 391-393

in parentage testing, 402

in platelet refractoriness, 362-365, 397-398,

491

structure of molecules, 244-246, 390-391

tissue distribution, 390-391

and transfusion, 397-400

in transfusion reactions, 398-400, 637, 643,

656

in transfusion-related acute lung injury, 399,

647

and transplantation, 388, 400-402

typing, 394-397, 595, 621

HMIWI (hospital/medical/infectious waste in-

cinerators), 57

HNA antigens, 378

Homozygous, defined, 225

Homozygous type II familial hypercholesterol-

emia, 140, 157

Housekeeping, 40-41

HPA antigens, 366, 367-368, 369-370, 552

HPCs. See Hematopoietic progenitor cell trans-

plantation

HTLA (high-titer, low-avidity antibodies), 449,

762, 765-766

HTLV-associated myelopathy (HAM), 682

HTLV (human T-cell lymphotropic virus),

682-683

autologous blood testing, 120

blood component testing, 164, 166, 170, 674,

683

clinical manifestations, 682

donors implicated in, 703

quarantine and look-back, 683

testing transplantation donors for, 590, 620

transfusion risk, 700

transmission, 682

Type I, II, 682

HTR. See Hemolytic transfusion reactions

Human albumin (5% and 25%), 507

Human Genome Project, 217

Human granulocytic erlichiosis, 696

Human herpesviruses, 688

Human immunodeficiency virus. See HIV

Human monocytic erlichiosis, 696

Human resources, 7-9

Human T-cell lymphotropic virus. See HTLV

Human thrombin, 502

Hy (Dombrock) antigen, 350, 842

Hydatid cyst fluid, 310, 445

Hydroxyethyl starch (HES), 143-144

Hydroxyurea, 575

Hyper-IgM immune deficiency, 255

Hyperbilirubinemia, 536, 566-567

Hypercholesterolemia, familial, 140, 157

Hyperhemolytic syndrome, 575

Hyperkalemia, 650

Hyperleukocytosis, 154

Hypertension, 633

Hypertransfusion programs, 575-576

Hyperventilation, in donors, 107

Hyperviscosity, serum, 153-154

Hypoalbuminemia, 507

Hypocalcemia, 561, 636, 649-650

Hypofibrinogenemia, 500, 501

Hypokalemia, 650

Hypotension

associated with ACE inhibition, 636, 645

in donors, 107

in recipients, 633, 640, 641, 645

in therapeutic apheresis, 152

treatment, 646

Hypothermia, 511, 560, 637, 650

Hypovolemia, 141, 152

I/i antigens and antibodies, 306-308

in ABO discrepancies, 302

chromosomal locations of genes, 226

in cold agglutinin syndrome, 307, 465-466

complex reactivity, 308

ISBT nomenclature, 843

880 AABB Technical Manual

in pregnancy, 539

serologic behavior, 307-308, 777, 778

IAT. See Indirect antiglobulin test

ID-NAT (individual donor NAT), 669, 678, 680, 685

Identification

of blood samples, 409

of components, 105, 416, 524-526, 527

of donors, 98, 105

of equipment, 11

errors in, 525-526, 527, 641, 642, 653, 654

of persons issuing blood, 416, 525

of phlebotomists, 409

of recipients, 407, 408-409, 418, 524-526, 527

traceability of components, 173

Idiopathic thrombocytopenic purpura (ITP),

158, 373-374, 492, 553-554

Idiotypes, 256, 269

IFC (Cromer) antigen, 353, 354, 843

IgA, 259

antibodies to, 645, 646, 655

normal values, 835, 837

polymers, 258, 259

properties, 253

secretory, 258, 259

IgD, 253, 259, 835

IgE, 259

in allergic transfusion reactions, 644-645

normal values, 835

properties, 253

IgG, 258

anti-A and -B, 295

in autoimmune hemolytic anemias,

458-459, 460

complement activation by, 260

dissociation of, by chloroquine, 469, 746-747

distinguishing from IgM, 764-765

in neonates, 560

normal values, 835, 837

in positive DAT, 280, 453, 461

properties, 253

subclasses, 258, 458-459

temperature of reaction, 274

IgG-coated cells (check cells), 281, 412

IGIV. See Immunoglobulin, Intravenous

IgM, 258

anti-A and -B, 295

in antiglobulin testing, 281

cold-reactive autoagglutinins, 302-303, 465,

466, 467, 469, 776-778

complement activation by, 260

dispersing autoagglutination, 302, 469-470,

744-745

distinguishing from IgG, 764-765

normal values, 835, 837

polymers, 257, 258

properties, 253

temperature of reactions, 274

IgM warm AIHA, 464

IgSF (immunoglobulin superfamily), 244, 245,

246

IL (interleukins), 264, 512, 640

Imatinib mesylate (Gleevec), 220

Immediate-spin crossmatch, 413, 414, 751-752

Immune complexes

in antigen-antibody reactions, 280, 281

demonstration of, 788-789

mechanism of drug-induced antibodies,

473, 474-475

Immune-mediated hemolysis. See also

Hemolytic disease of the fetus and newborn

ABO/Rh typing problems with, 469-470

alloimmune hemolytic anemia, 459

classification of anemias, 458, 459

cold agglutinin syndrome, 459, 460, 464-466

DAT-negative AIHA, 459, 468

defined, 458-459

drug-induced, 459, 460, 472-477, 481-482

in hemolytic transfusion reactions, 639-642

intravascular/extravascular, 265-266, 267,

458

mixed-type AIHA, 459, 460, 466-467

paroxysmal cold hemoglobinuria, 459, 460,

467-468

warm autoimmune hemolytic anemia, 459,

460, 461-464

Immune serum globulin (ISG), 668

Immune system

cell adhesion molecules in, 246

cells of, 249-250, 251, 252-256

cluster of differentiation molecules in, 246,

247-248

complement in, 259-262, 263

cytokines in, 262-263, 264

defined, 269

and drug-dependent antibodies, 472-473

in HLA alloimmunization to platelets, 265,

266

immune response, 243-246, 247-248

Index 881

immunoglobulin superfamily in, 244, 245

immunoglobulins in, 253, 256-259

innate and adaptive immunity, 243-244

lymphocytes in, 249-250, 251, 252-255

major histocompatibility complex in,

244-246

of neonates, 560-561

organs of, 249

phagocytic cells in, 255-256

in production of reagent antibodies, 266-267

receptors/markers, 250

in red cell alloimmunization, 265

in red cell destruction, 265-266, 267

signal transduction in, 246

soluble components, 256-263

Immune thrombocytopenic purpura (ITP), 158,

373-374, 492, 553-554

Immune tolerance, 237

Immunity. See Immune system

Immunization, maternal

antibody titers, 449, 539-540, 761-764,

796-798

mechanisms, 536-538

prenatal evaluation, 538-542

suppression, 542-543

Immunocompromised patients, 298-299

Immunofluorescence tests

in antigen-antibody detection, 285-286

in confirming HIV tests, 169

for granulocyte antibodies, 380

for platelet antibody detection, 372

Immunogen, defined, 269

Immunoglobulin, Intravenous (IGIV), 505

ABO discrepancies with, 299

for idiopathic thrombocytopenic purpura, 554

indications for, 505, 506

for neonatal immune thrombocytopenia,

552-553

positive DAT with, 456

for posttransfusion purpura, 659-660

to suppress maternal alloimmunization, 543

virus inactivation in, 699

Immunoglobulin products

hepatitis B immune globulin (HBIG), 45,

668, 703

immune serum globulin (ISG), 668

immunoglobulin, intravenous (IGIV), 505,

506, 543

virus inactivation in, 699, 701

Immunoglobulin superfamily (IgSF), 244, 245,

246

Immunoglobulins, 256-259. See also specific

immunoglobulins

classes, 258-259

Fab and Fc fragments, 256-257

functions, 256

interchain bonds, 256

J chains, 258

normal values, 835, 837

polymers, 257-258

properties, 253

secretory component, 258

structure, 256, 257

Immunohematology reference laboratories, 439

Immunomagnetic cell separation, 596, 598

Immunomodulation, 638, 660

In (Indian) antigens and antibodies, 349, 354,

843

“In-vivo” crossmatch, 441, 509

Incinerators, hospital/medical/infectious waste

(HMIWI), 57

Incompletely clotted specimens, 409, 722-723

Incubation

temperatures, 443, 715

times, 274, 444

Independent assortment, 229-230, 231

Indian system (ISBT 023), 226, 349, 354, 843

Indirect antiglobulin test (IAT)

false-positive/false-negative results, 282, 283

methods, 752-754

principles, 278

reagents, 278-280, 425, 427, 468

role of complement, 280-281

use of additives, 276-277, 443, 753-754

use of IgG-coated cells, 281, 412

Infants. See Children; Neonates

Infectious disease testing

of autologous blood, 119-120

of blood components, 166-170

external controls in, 167-169

of granulocytes, 144

for hepatitis, 669, 670-671, 672-673, 674

for HIV, 164, 166-167, 169-170, 213, 679-681

of HPC donors, 590-591

for HTLV, 120, 164, 166, 170, 683

invalidation of results, 167-169

neutralization, 169

notification of abnormal tests, 703

882 AABB Technical Manual

in organ and tissue transplantation, 621-623

of Platelets Pheresis, 142

records of, 173

supplemental tests, 169-170

surrogate markers, 673

for syphilis, 120, 165-166, 695

Infectious diseases. See also Transfusion-trans-

mitted diseases

as apheresis complication, 152

in blood donors, 99

safety precautions for, 49-57

transfusion-transmitted, 667-699, 700,

701-703

transmitted by allografts, 617, 619-620

Infectious mononucleosis, 687

Infectious substances

defined, 716-717

labeling, 718, 719

shipping, 717-718, 720-722

waste, 55-57

Inflammation, 262

Informed consent

for apheresis, 140, 142

of blood donors, 103

for transfusion, 521-522

for transplantation, 620-621

Infusions. See also Transfusions

of hematopoietic components, 607

infusion pumps, 522, 523, 529-530, 565

infusion sets, 527-529, 565-566

rates for, 531, 566

Inheritance patterns

autosomal dominant, 233, 234

autosomal recessive, 233, 234

blood group co-dominant, 234-235

chromosomal assignment, 226, 235-236

dominant and recessive, 232-233

of major histocompatibility complex,

387-389

sex-linked dominant or co-dominant, 233,

234

sex-linked recessive, 233-234

Inhibition tests

agglutination, 275-276

in antibody identification, 444-445

for Chido/Rodgers, 445, 765-766

for secretor status, 737-739

Inhibitors

of complement activation, 262, 263

to Factor VIII, 504-505

fibrinolytic, 513-514

Injuries, 45-46, 51

Innate immunity, 243-244

Inspections

of components before release, 194-195, 416,

418, 525

external assessments, 23-24

of incoming supplies, 10

of inventory, 93-94

Integrins, 246, 369

Interferon γ(IFNγ), 264

Interleukins, 264, 512, 640

Internal assessments, 22

Internal controls, 167

Internal event report, 19, 20

International Organization for Standardization

(ISO), 1-2, 3

Intervening sequences, 206

Intraoperative blood collection, 117, 130-133

clinical studies, 131-132

controversies in, 132-133

direct reinfusion, 133

equipment for, 132-133

practical considerations, 132

processing, 132-133

requirements and recommendations, 133

Intrauterine transfusions, 541, 543-544

Intraventricular hemorrhage, 569

Introns, 206

Inventory

counts and inspection, 93-94

determining levels, 89-90

minimum and ideal levels, 89

and outdating, 90-91

routine vs emergency orders, 93

of special products, 94-95

Ionic strength, 274

Iron, supplemental, 121

Iron overload, 638, 660

Irradiated blood components, 192-193

expiration dates, 183, 189, 192

Granulocytes, 144, 492

HPC products, 592

indications for, 183, 193, 493, 658-659

for intrauterine transfusion, 544

inventory management, 95

labeling, 172

platelet products, 144, 189, 192-193, 492, 553

Index 883

potassium leak in, 561

to prevent TA-GVHD, 183, 192, 493, 658-659

quality control, 202

Red Blood Cells, 189, 192, 193, 493

to reduce platelet alloimmunization, 365, 398

ultraviolet B, 365, 398

washing, 561

Irradiators, blood, 65-66, 192, 846

ISBT (International Society of Blood Transfu-

sion)

128 barcode labeling, 171

nomenclature

for platelet antigens, 366, 367-368

for red cell antigens, 226, 238-239,

840-843

ISG (immune serum globulin), 668

Ishikawa diagrams, 26

ISO 9001 standards, 1-2, 3

Isotype switching, 252

Issuing blood

delivering blood to patient area, 524-525

identification of recipient and component,

418, 524-526

policies and procedures of, 416, 418, 524-525

reissue, 197, 525

from transfusion service, 196-197

in urgent situations, 419, 510-511, 522-523

ITP (idiopathic thrombocytopenic purpura),

158, 373-374, 492, 553-554

IV solutions, 530

J chains, 258

Jaundice, 566, 639

JCAHO (Joint Commission on Accreditation of

Healthcare Organizations), 25, 514

Jk (Kidd) antigens and antibodies, 336, 345-346,

841

JMH antigen/antibodies, 355, 843

Joa(Dombrock) antigen, 350, 842

Job descriptions, 7

John Milton Hagen (JMH) system (ISBT 026),

226, 355, 843

Joint Commission on Accreditation of

Healthcare Organizations (JCAHO), 25, 514

Jraantigen, 356

Js (Kell) antigens and antibodies, 336, 341, 343,

841

Juran’s Quality Trilogy, 2, 4

K/k (Kell) antigens and antibodies, 336, 340-342,

343, 841

Kaposi’s sarcoma-associated herpesvirus, 688

Kell system (ISBT 006), 340-343

anti-Ku, 343

antibodies, 336, 343, 844

antigens, 340-342, 468, 841, 844

biochemistry, 342

genes, 226, 844

in HDFN, 343, 536, 540

inheritance, 229-230

Kx antigen, 226, 342, 842

McLeod phenotype, 342-343

phenotypes and frequencies, 341, 844

in transfusion reactions, 343

Kernicterus, 536, 566

Kidd system (ISBT 009), 345-346

allele frequencies in, 227-229

antibodies, 336, 346

antigens, 345-346, 841, 844

chromosomal locations of genes, 226

genes, 844

in HDFN, 346

inheritance patterns of, 235

phenotypes and frequencies, 345, 346, 844

in transfusion reactions, 346, 656

Kidney

diseases treated with apheresis, 156-157

failure, 640-641

transplantation, 401-402, 619, 624, 627

Kinetic PCR, 213

Kleihauer-Betke acid-elution test, 550-551,

794-796

Kn (Knops) antigens, 354, 843

Knops system (ISBT 022), 226, 352, 354, 843

Kp (Kell) antigens and antibodies, 336, 341, 343,

841

Kx antigen (ISBT 019), 226, 342, 842

Labeling

aliquots, 564

for antigen typing, 440

autologous blood, 120, 122-123

biohazardous materials, 50, 719

blood components, 15, 170-172, 416, 440

blood samples, 409

884 AABB Technical Manual

dry ice, 719

hazardous chemicals, 59-60

ISBT 128 system, 171

reagents, 715

shipments, 718, 719

Laboratory coats, 73

Lactated Ringer’s solution, 530

Lan antigen, 356

Landsteiner-Wiener blood group (ISBT 016),

226, 327, 351, 842

Latent failures, 26-27

Latex allergies, 46-47

Law of mass action, 273

LCR (ligase chain reaction), 213, 214

LDL apheresis, 140, 157

Le (Lewis) antigens/antibodies, 304-306, 336,

844

Leach phenotype, 352

Lectins

Dolichos biflorus (anti-A1), 293, 296, 302,

743-744

preparation and use, 743-744

Ulex europaeus, 304, 743-744

Leishmania sp., 699

Leukapheresis, 143-144, 154, 587, 593

Leukocyte-reduction filters, 180, 190, 528-529

Leukocytes-reduced components

for children, 573-574, 576

expiration dates, 189

indications for, 493

labeling, 172

leukocyte content in, 492-493

Platelets, 180, 184, 189, 199, 202, 365, 493,

817

Platelets Pheresis, 35, 202, 493

poststorage, 190

prestorage, 180, 184, 199, 805-806, 817

to prevent bacterial contamination, 693

to prevent CMV infection, 687

quality control, 202, 832-834

Red Blood Cells, 180, 189, 199, 202, 493,

805-806

to reduce platelet alloimmunization, 265,

365

Whole Blood, 180

Lewis blood group (ISBT 007), 304-306

antibodies, 305-306, 336, 434, 539

antigens, 304-305, 841

in children, 306

genes, 226, 305

Lewis substance, 445

phenotypes, 305

saliva testing for, 445, 736-739

transfusion practices, 306

Ligands, 269

Ligase chain reaction (LCR), 213, 214

Liley graphs, 540, 541

Linkage, 230-231, 232

Linkage disequilibrium, 231-232, 337, 389

Lipemic samples, 410

Liquid-in-glass thermometers, 821-822

Liquid nitrogen

shipping, 718, 719

storage, 185, 189, 606

Liquid Plasma, 177, 189

LISS (low-ionic-strength saline)

antibodies to ingredients in, 437-438

and antigen-antibody proportions, 275, 277

to enhance antigen-antibody reactions, 277,

427, 443, 753, 754

and incubation time, 274

Liver

disease, 499

transplantation, 402, 619, 624, 627-629

LKE (Luke) antigen, 308, 310, 311

Locus, defined, 241

Long distance PCR, 213, 214

Look-back

for hepatitis, 675

for HIV, 681-682

for HTLV, 683

Low-incidence antigens

antibodies to, 340, 358, 436-437, 546

of Cromer system, 353, 354

defined, 335

of Diego system, 348

of Gerbich system, 352

of Lutheran system, 347

of MNS system, 227, 228, 337-338, 340

not assigned to a system or collection, 357

of Scianna system, 350

Low-ionic-strength saline. See LISS

Low-volume units, 101, 122, 179

Lsa(Gerbich) antigen, 352, 843

Lu (Lutheran) antigens and antibodies, 336,

347-348, 841

Lui freeze-thaw elution, 457, 774

Luke (LKE) antigen, 308, 310, 311

Index 885

Lung transplants, 402

Lutheran system (ISBT 005), 347-348

antibodies, 336, 347-348

antigens, 347, 841

biochemistry, 347

chromosomal location of genes, 226

in HDFN, 348

phenotypes and frequencies, 347

LW system (ISBT 016), 226, 327, 351, 842

Lyme disease, 696-697

Lymphocytes

B cells, 249-250, 251, 252-253

NK (natural killer) cells, 255

passenger, 628, 629-630, 657

T cells, 253-254

Lymphocytotoxicity assays, 365-366, 395, 396-397

Lyonization, 224, 241

Macrophage chemoattractant protein (MCP),

640

Macrophage colony-stimulating factor (M-CSF),

264

Macrophages, 250, 255, 256

Magnetic cell separation, 596

MAIEA (monoclonal antibody-specific immobi-

lization of erythrocyte antigens) assay, 284,

286

MAIGA (monoclonal antibody-specific immobi-

lization of granulocyte antigens), 380

MAIPA (monoclonal antibody-specific immobi-

lization of platelet antigen), 373

Major histocompatibility complex (MHC). See

also HLA system

Class I, II, III molecules, 244-246, 390-391

defined, 269

organization of regions, 386-387

patterns of inheritance, 387-389

restriction, 393

Malaria, 697, 700

Management

assessment, 38

of critical supplies and services, 9-10

of documents and records, 14-19

of equipment, 10-11

of facilities, 6-7

of organization, 6-7

of personnel, 7-9

of safety program, 45

Manual, safety, 45

Marrow transplantation

ABO incompatibilities, 598-599, 600, 601-

602

ABO typing discrepancies, 299

allogeneic, 582, 583, 585-587

autologous, 583

bacterial contamination, 602-603

collection of marrow, 591-592

diseases treated with, 583, 584

donor databases, 585

donor evaluation, 589-591

engraftment in, 588-589

evaluation and quality control, 607-608

freezing and storage of product, 604-606

and graft-vs-host disease, 585-587

HLA testing, 400-401, 585-586

infectious disease testing, 590-591

matched, unrelated donor, 585-586

nonmyeloablative, 582-583

positive DAT after, 455

processing, 596-598, 601-604

red cell depletion, 601

regulations, 608

standards, 609

thawing and infusion, 607

transportation and shipping product,

606-607

MART antigen, 378, 379

Masks, 75

Massive transfusions

2,3-DPG levels in, 511-512

changing blood types, 511, 628-629

citrate toxicity in, 649-650

coagulopathy in, 511, 651

emergency issue, 510-511

hyper- and hypokalemia in, 650

hypothermia in, 511, 650

selection of blood, 419, 510-511

tissue oxygenation in, 511

Material data safety sheets (MSDS), 59, 60-61,

78-79

Materials, critical, 9

Maternal immunization

antibody titers, 449, 539-540, 761-764,

796-798

mechanisms, 536-538

prenatal evaluation, 538-542

suppression, 542-543

886 AABB Technical Manual

Maximum surgical blood order schedules

(MSBOS), 92-93, 414

McCoy (McC) antigens, 352, 354

McLeod phenotype, 342-343

MCP (macrophage chemoattractant protein),

640

2-ME (2-mercaptoethanol)

applications for, 448-449

to disperse autoagglutination, 469, 470,

744-745

inactivation of Kell antigens, 342

Mechanical barrier systems, 527

Mechanical hemolysis, 152, 642

Medical history

in allogeneic donor selection, 100-101,

110-112

in antibody identification, 424

in autologous donations, 122

in cord blood collection, 595

in evaluation of positive DAT, 454-456

in prenatal evaluations, 538

in tissue and organ transplantation, 618,

620

Medical waste, 55, 56, 57

Medications. See Drugs

Meiosis, 224-225, 241

Membrane attack complex (MAC), 261-262, 263

MER2 (Raph) antigen, 354-355, 843

2-mercaptoethanol. See 2-ME

Meryman method of red cell cryopreservation,

807-810

Messenger RNA (mRNA)

isolation of, 210-211

processing, 204-206

translation, 206-207

Metabolic abnormalities, in neonates, 561-562

Methemoglobin, 835

Methylene chloride (dichloromethane) elutions,

457, 775

MHC. See Major histocompatibility complex

MHO4 clone, 298, 301

Microaggregate filters, 528, 565-566

Microangiopathic hemolysis, 502

Microarrays, 218

Microbead array assay, 395-396

Microfilariasis, 699

Microhematocrit centrifuges, 847

Microlymphocytotoxicity tests, 365-366, 395,

396-397

Microplate tests

for ABO group, 733-735

in antigen-antibody detection, 283-284

for Rh typing, 328, 741

Microscopic weak D test, 550

Microvascular bleeding (MVB), 511, 651

Miltenberger system, 338

Missense responses (mutations), 208, 227

Mitosis, 224, 225, 241

Mixed-field agglutination, 298, 299, 348, 357

Mixed lymphocyte culture (reaction)

(MLC/MLR), 397

Mixed type AIHA, 466-467

classification, 459

serologic findings in, 460, 466-467

specificity of autoantibodies, 467

transfusion in, 467

MLC (mixed lymphocyte culture), 397

MLR (mixed lymphocyte reaction), 397

MNS system (ISBT 002), 337-340

antibodies, 303, 336, 340, 444

antigens

linkage disequilibrium in, 231, 232, 337

linkage in, 232

low-incidence, 227, 228, 337-338, 340

M, N, S, s, U, 337

nomenclature, 840, 844

phenotypes and frequencies, 337, 844

biochemistry, 338-339

effect of enzymes on, 276, 339-340

genes, 226, 227, 228, 338, 844

in HDFN, 340

hybrid molecules, 338

in transfusion reactions, 340

MoAb. See Monoclonal antibodies

Mobile blood collection, 41, 179

Mobilization of hematopoietic progenitor cells,

587, 592-595

Modifier genes, 235-236

Molar solutions, 723

Mole, defined, 723

Molecular biology, 203-220

DNA and mRNA, 203-207

genetic variability, 208-209, 210

polymorphism, genetic mechanisms in,

207-208

techniques

DNA cloning, 217, 222

DNA microarrays, 218

Index 887

DNA profiling, 216-217, 222

DNA sequencing, 217-218, 222, 396

gene therapy, 219-220

isolation of nucleic acids, 209-211

phage display/repertoire cloning, 222

polymerase chain reaction, 211, 212,

213-214, 215, 222, 394-396

protein and RNA targeted inactivation, 220

recombinant proteins, 218-219, 222

restriction endonucleases, 214, 216

restriction fragment length polymor-

phism analysis, 214, 216, 222

Monoclonal antibodies

in ABO testing, 296

anti-D, 328, 329-330

assays utilizing, 284, 373, 380

in autologous tumor purging, 597-598

for reagent use, 266-267

Monoclonal antibody-specific immobilization

of granulocyte antigens (MAIGA), 380

Monoclonal antibody-specific immobilization

of platelet antigens assay (MAIPA), 373

Monoclonal antibody-specific immobilization

of erythrocyte antigens (MAIEA) assay, 284,

286

Monocyte monolayer assay, 441

Monocytes, 250, 255, 256

Mononuclear phagocytic system, 255

Monospecific AHG reagents, 280

MP-NAT (minipooled NAT)

for HCV, 669

for HIV, 678, 679

for parvovirus, 689

for West Nile virus, 685

MPHA (mixed passive hemagglutination assay),

371-372

mRNA. See Messenger RNA

MSBOS (maximum surgical blood order sched-

ules), 92-93, 414

MSDS (material data safety sheets), 59, 60-61,

78-79

Multiplex PCR, 213

Muscular spasms, in donors, 108

Mutations, genetic, 208, 227, 228

Myasthenia gravis, 156

N antigen/antibody, 336, 337, 340, 840

NA/NB neutrophil antigens, 378

NAIT. See Neonatal alloimmune

thrombocytopenia

NAN (neonatal alloimmune neutropenia), 379

Nasopharyngeal carcinoma, 687

NAT. See Nucleic acid amplification test

National Marrow Donor Program (NMDP), 585,

589

Natural killer (NK) cells, 255

Nausea, 107-108, 639

Needles, 51, 523, 565

Negative selection, 597-598

Neonatal alloimmune neutropenia (NAN), 379

Neonatal alloimmune thrombocytopenia

(NAIT), 551-553

management after delivery, 553

platelet-specific antigens in, 366, 552

prenatal considerations for, 552-553

scheduling therapy, 553

serologic testing in, 552

sources of platelets for, 553

Neonatal immune thrombocytopenia (NIT)

alloimmune (NAIT), 366, 551-553

secondary to maternal ITP, 553-554

Neonatal polycythemia, 572

Neonates. See also Hemolytic disease of the fetus

and newborn

ABO antigens/antibodies in, 293, 295, 298

ABO discrepancies in, 299

ABO/Rh typing in, 415, 545

anemia in, 558, 559, 563-564

antigen variations in, 431

blood volume, 558-559, 839

compatibility testing in, 415, 562-563

cytomegalovirus infection in, 562

DIC in, 567, 572

direct antiglobulin testing in, 545-546

erythropoiesis in, 557-558, 559

extracorporeal membrane oxygenation in,

572-573, 574

graft-vs-host disease in, 560-561

hypothermia in, 560

immune thrombocytopenia in, 551-554

immunologic status, 560-561

leukocyte reduction for, 573-574

Lewis antigens in, 306

low birthweight, 557

metabolic problems in, 561-562

neonatal alloimmune neutropenia, 379

normal laboratory values in, 836-837

888 AABB Technical Manual

plasma volume in, 839

polycythemia in, 572

red cell volume in, 839

size of, 558-559

transfusions in

administration, 565-566

Cryoprecipitated AHF, 558, 572

effect of additive/preservative solutions,

564-565

to enhance hemostasis, 570-572

exchange, 546-547, 560, 566-568

of Fresh Frozen Plasma, 558, 571

Granulocytes, 558, 569-570

indications for, 563-564

Platelets, 490, 552-553, 558, 568-569

Red Blood Cells, 558, 564-568

volumes for, 558, 564, 568, 571

Neutralization

in antibody identification, 445

of Sdaantigen, 357, 445, 767-768

in viral marker testing, 169

Neutropenia, 379-380

Neutrophils

alloantigens, 377-380, 647

antibodies to

in neonatal alloimmune neutropenia, 379

testing for, 380

in TRALI, 379-380, 647, 656

in immune system, 255

NIT. See Neonatal immune thrombocytopenia

NK (natural killer) cells, 255

Nomenclature

of blood group systems, 238-239, 318-319,

840-844

CD (clusters of differentiation), 246, 247-248

of HLA system, 391-393

ISBT, 226, 238-239, 840-843

of platelet antigens, 366, 367-368

of Rh system, 315, 317, 318-319

Non-A, non-B hepatitis (NANB), 673, 699

Non-immune hemolysis, 636, 642-643

Nonconforming products or services

management, 19, 20, 21

quarantine, 173-174, 194

Nonimmunologic protein adsorption, 476

Nonsense response (mutations), 208, 227

Normal solutions, 723

Nuclear Regulatory Commission (NRC), 41, 64,

65, 66

Nucleic acid amplification test (NAT)

in HBV testing, 669, 672

in HCV testing, 164, 166, 213, 590, 672, 673,

675, 701

in HIV testing, 164, 166-167, 213, 590,

678-681

in HTLV testing, 683

in parvovirus testing, 688-689

in viral maker testing, 166-167

in West Nile virus testing, 213

Nucleic acids

isolation of, 209-211

sequencing, 217-218, 222

Nucleotides

defined, 203

insertion and deletion, 208

sequences, 204, 205

substitution, 207-208, 228

Nutricel (AS-3), 176, 186, 565

Obligatory gene, 237

Occupational Safety and Health Administration

(OSHA), 41

Officers

chemical hygiene, 58

safety, 42-43

Ohphenotype (Bombay), 304

Ok system (ISBT 024), 226, 354, 843

Oligonucleotide probes, sequence-specific

(SSOP), 214, 373, 395-396

OND antigen, 378, 379

“Open” system, 189, 192

Opsonization, 262

Optisol (AS-5), 176, 186, 565

Oral thermometers, electronic, 822-823

Ordering policies, 36, 91-94, 414

Organ donation and transplantation

ABO compatibility in, 401, 402, 627, 629-630

consent for, 620-621

cytomegalovirus in, 629, 630

disease transmission in, 617, 619-620

donor eligibility, 620-621

of heart, 629

HLA testing, 401-402

of kidney, 401-402, 619, 624, 627

of liver, 402, 627-629

of pancreas, 401-402, 629

positive DAT after, 455

Index 889

preservation conditions and dating periods,

624

records, 626

recovery of tissue, 621

regulations, 626

risk reduction in, 618, 620

serologic testing for, 621-623, 629

skills and experience appropriate for, 618

transfusion support, 627, 629-630

types of donors for, 622

Organisms

in bacterial contamination, 691, 692

genetically modified, 717

Organizational management, 6-7

Organizations, 848-850. See also specific organi-

zations

regulating quality systems, 1-2

regulating safety, 39-40, 71-72

Organs of immune system, 249

Orientation program, 8

OSHA (Occupational Safety and Health Admin-

istration), 41

Outdating, 90-91

Outpatients, 408-409

Oxygen

compensation for anemia, 484

delivery of, in ANH, 127-128

dissociation in red cell storage, 185, 187

in massive transfusion, 511

measuring adequacy of supply, 484-485

supply and demand, 483-484

treating inadequate supply of, 485

Oxygen therapeutics, 512-513

P blood group (ISBT 003), 308-311

anti-P1, 303, 310-311

antibodies, 311, 468

antigens, 308-309, 310, 311, 840, 844

association with PCH, 468

biochemistry and genetics, 309-310, 844

chromosomal locations of genes, 226

hydatid cyst fluid/P1 substance, 310, 445

phenotypes, 309, 844

P1A1 and P1A2 (HPA-1) antigens, 366, 367, 369,

552

p24 antigen, 679

Packaging biological materials, 717-718, 720-721

PAD. See Preoperative blood donation

Pain at infusion site, 633

Pancreatic transplantation, 401-402, 620, 624,

629

Panel-reactive antibody (PRA), 362, 397, 401

Panels, red cell, 425, 426

Papain, preparation of, 756-757. See also En-

zymes, proteolytic

Para-Bombay phenotype, 304

Parasitic worms, 698-699

Parentage testing

genetics, 236-237

HLA testing in, 402

linkage disequilibrium in, 389

Paresthesias, 141, 151

Pareto analysis, 27

Paroxysmal cold hemoglobinuria (PCH),

467-468

classification, 459

serologic findings in, 460, 467

specificity of autoantibodies, 469

transfusion in, 467

Paroxysmal nocturnal hemoglobinuria, 378

Partial D, 322-323

Parvovirus B19, 688-689, 700

Passenger lymphocyte hemolysis, 628, 629-630,

657

Paternity testing. See Parentage testing

Pathogen inactivation, 702

Patients. See Recipients

PCH. See Paroxysmal cold hemoglobinuria

PCR. See Polymerase chain reaction

PCR-SSO/PCR-SSOP. See Sequence-specific

oligonucleotide probes

Pedi packs, 564

Pediatric patients. See Children; Neonates

Peer review, 23, 514

PEG. See Polyethylene glycol

Penicillin, 455, 474, 477, 786-788

Percentage solutions, 723, 726-727

Percutaneous umbilical blood sampling (PUBS),

541-542

Performance improvement standards, 25

Pericardium transplants, 619

Personal protective equipment (PPE), 43-44

for biosafety, 54

for chemical safety, 61

face shields, 74

gloves, 46, 55, 73-74, 106

laboratory coats, 73

890 AABB Technical Manual

masks, 75

safety goggles, 74

uniforms, 73

Personnel

accidents and injuries in, 45-46

competency assessment, 8

hepatitis prophylaxis for, 45, 703

job descriptions for, 7

latex allergies in, 46-47

medical first aid and follow-up, 45

orientation program, 8

protective equipment for, 43-45, 73-75

records, 19

safety monitoring programs, 45

selection of, 7

staffing levels, 8-9

training

biosafety, 50

chemical safety, 58-59

computer systems, 32

electrical safety, 48

FDA cGMP, 8

fire safety, 47

general safety, 43, 44

new employee, 8

radiation safety, 65

PF4 ELISA, 377

pH, 274, 431, 444

pH meters, 846

Phage display/repertoire cloning, 222, 267

Phagocytic cells, 255-256, 262

Phagocytosis, 262, 269

Pharmacologic alternatives to transfusion,

512-514

Pharmacologic purging, 597

Phenotypes.Seealsospecific blood groups

of autologous red cells, 429-430, 439

calculations for combined phenotypes, 236,

441

defined, 233

of donor units, 440-441

frequencies, 236

nomenclature for, 238-239, 844

Phlebotomy

adverse reactions to, 19, 107-109

aggressive, in autologous donations, 125

care of donors after, 106

collection of blood samples, 105-106,

408-409, 801-804

of donors, 105-106, 800-804

prevention of contamination in, 693

Phosphate buffer, 728

Photochemical inactivation, 702

Photopheresis, 158

Physical examination of donors, 100, 101-103, 620

Physicians’ orders, 522-523, 526

Physician’s responsibilities, in autologous pro-

gram, 120-121

Physiologic anemia of infancy, 558, 559, 563-564

Physiologic jaundice, 566

Phytanic acid disease, 157-158

Pipettes, recalibration, 847

PlAantigens, 366, 552

Plasma

ABO compatibility, 411, 496, 498

avoiding use, 701

coagulation factor replacement with, 496,

498-500

collection by apheresis, 141, 142-143

components

derivatives/substitutes, 496, 503-508, 699,

701

Fresh Frozen Plasma, 142, 177, 496,

498-500

inventory management, 94

Liquid Plasma, 177, 189

Plasma, 177

Plasma, Cryoprecipitate Reduced, 177,

190, 496, 498

Plasma Frozen within 24 Hours after

Phlebotomy, 181, 189

Pooled Plasma, Solvent/deter-

gent-treated, 496, 702

Recovered Plasma (for manufacture), 177

Source Plasma, 142

Thawed Plasma, 189, 191

constituents removed in apheresis, 149-150

description, 177

expiration dates, 189-190

for pretransfusion testing, 409, 424

as replacement fluid in apheresis, 150, 151

use in children, 571

virus inactivation, 701-702

volume, normal value, 839

Plasma D-dimers, 837

Plasma exchange. See Therapeutic apheresis

Plasma inhibition of anti-Ch and-Rg, 445,

765-766

Index 891

Plasma protein fraction (PPF), 507

Plasmapheresis, 142-143, 500

Plasmids, 217

Platelet chambers, 185, 197-198, 846

Platelet concentrates (Platelets)

aliquoting, 193-194

bacterial growth in, 690-692, 694-695

biochemical changes during storage, 188

coagulation factors in, 838

description, 176-177, 489

expiration dates, 188, 189

freezing, 183

infectious risks of, 700

inspection, 194-195

inventory management, 94, 95

irradiated, 189, 192-193, 553

leukocyte-reduced, 184, 189, 190, 199, 202,

365, 493, 817

pooled, 184, 189, 193

preparation, 176-177, 815-817, 827-828

quality control, 199, 202

storage, 185, 188, 694-695

transfusions, 488-492

ABO/Rh matching, 361-362, 489-490, 569

in children, 490, 552-553, 558, 568-569,

576-577

contraindications to, 491-492

HLA alloimmunization to, 265, 266,

362-366, 397-398, 637

indications for, 490-491, 569

physiologic principles, 488-489

prophylactic, 490-491

refractoriness to, 362-365, 397-398, 491

therapeutic, 490

transportation and shipping, 196

volume reduction, 193, 490, 569, 817-818

washed, 193, 646

Platelet counts

in assessing hemostasis, 488

corrected platelet count increment, 362, 363,

489

in idiopathic thrombocytopenic purpura, 554

in massive transfusion, 511, 651

in NAIT, 552, 553

in neonates, 568-569

normal values, 835, 836

predicted platelet count increment, 362, 363,

489

transfusion triggers, 490-491, 569, 576

Platelet disorders

DDAVP in, 513

in liver disease, 499

thrombocytopenia

in blood loss, 499

drug-induced, 374-377

heparin-induced, 375-377, 492

idiopathic thrombocytopenic purpura,

158, 373-374, 492, 553-554

neonatal alloimmune thrombocytopenia,

511-513, 551-553

posttransfusion purpura, 366, 370, 638,

659-660

secondary to maternal ITP, 553-554

thrombotic thrombocytopenic purpura,

154-155, 158, 492, 500

treatment, 158, 490-491

Platelet incubators, 185, 197-198, 846

Platelet-rich plasma (PRP), 176, 177

Plateletpheresis, 140-142, 154

Platelets. See also Platelet concentrates; Platelet

disorders; Platelets Pheresis

antigens/antibodies

ABH, 361-362, 397

autoantibodies, 373-374

clinical importance, 370-373

detecting, 370-373, 374, 375, 377, 552

drug-induced, 374-377

HLA, 265, 362-366, 397-398

platelet-specific, 366, 367-368, 369-373, 552

assessing function, 488-489

corrected count increment, 362, 363, 489

life span and kinetics, 489

membrane glycoproteins, 369-370

normal values, 835, 836

predicted platelet count increment, 362, 363,

489

refractoriness, 362-365

antibody specificity prediction method

for, 365

causes, 362-363, 364, 397-398, 491

defined, 362

finding compatible donors, 398

HLA-matched platelets for, 363-365, 398

preventing alloimmunization, 365-366

selecting platelets with, 363-365

Platelets Pheresis

ABO/Rh matching, 361-362, 411, 489-490, 590

bacterial growth in, 690-692, 694-695

892 AABB Technical Manual

collection, 140-141

crossmatching, 364, 365, 398

described, 489

donor selection and monitoring, 141

HLA matched, 95, 363-365, 398

infectious risks of, 700

inspection, 194-195

inventory management, 94, 95

laboratory testing, 142

leukocytes reduced, 142, 202, 493

product validation, 35

quality control, 202

records, 141

storage, 185, 694-695

transfusions, 488-492

ABO/Rh matching, 361-362, 411, 489-490

contraindications to, 491-492

HLA alloimmunization to, 265, 266,

362-366, 397-398, 637

physiologic principles, 488-489

prophylactic, 490-491

refractoriness to, 362-365, 397-398, 491

therapeutic, 490

transportation and shipping, 196

Pluripotent hematopoietic stem cell, 249, 251

Policies and procedures, 7, 11, 15

Polyagglutination, 298, 331, 743

Polycythemia, 572

Polyethylene glycol (PEG)

in antibody detection/identification,

276-277, 427, 443, 753-754

effect on incubation times, 274

use in adsorption, 783-784

Polymerase chain reaction (PCR)

applications, 213-214, 215, 222

in HLA typing, 394-396

kinetic, 213

ligase chain reaction, 213, 214

long distance, 213, 214

multiplex, 213

oligonucleotide probes, 214, 373, 395-396

in platelet typing, 373

reaction procedure, 211, 212, 213

reverse transcriptase, 214, 217

sequence-specific primers, 214, 373, 395, 396

in testing fetus for D antigen, 538

variations, 213

Polymorphisms, 207-209

Polymorphonuclear granulocytes, 255

Polyspecific AHG, 279-280, 427

Pooled components

Cryoprecipitated AHF, 183-184, 191, 193, 815

expiration, 183, 189

labeling, 172, 183

Platelets, 184, 189, 193

preparation, 183

Pooled plasma, solvent-detergent-treated, 496, 702

Population genetics, 236-237

Position effect, 319-320, 324

Postoperative blood collection, 117, 133-135

Posttransfusion platelet recovery (PPR), 362,

363, 489

Posttransfusion purpura (PTP), 366, 370, 638,

659-660

Potassium, 187, 561, 650

PPE. See Personal protective equipment

PPF (plasma protein fraction), 507

PPR (posttransfusion platelet recovery), 362,

363, 489

PRA (panel-reactive antibody), 362, 397, 401

Preadmission testing, 409

Precipitation, 272

Pregnancy. See also Hemolytic disease of the fe-

tus and newborn; Prenatal studies

autologous collection in, 119

as immunizing stimulus, 536-537, 552

Lewis antibodies in, 305

Premedication in transfusions, 522, 646-647

Prenatal studies

amniotic fluid analysis, 540-541, 542

antibody titration, 449, 539-540, 761-764,

796-798

Doppler flow studies, 542

maternal history, 538

in neonatal immune thrombocytopenia, 552

percutaneous umbilical blood sampling,

541-542

serologic studies, 538-539

typing the fetus, 539

Preoperative blood donation (PAD), 117,

118-126

advantages/disadvantages, 118

adverse reactions, 123

aggressive phlebotomy, 125

collection, 121

compliance requirements, 119-120

component collection, 126

continuous quality improvement, 123-124

Index 893

contraindications, 119

cost-effectiveness, 125, 127

donor deferrals, 120

donor screening, 121-122, 124-125

erythropoietin use, 125

establishing program, 120-124

labeling, 120, 122-123

medical interview, 122

in pediatric patients, 118-119

physician responsibility, 120-121

records, 123

shipping, 120

storage, 123

supplemental iron in, 121

testing, 119-120, 122

timing and red cell regeneration during, 122

transfusion, 123

transfusion trigger, 125

volume collected, 122

voluntary standards, 119

weakDindonor,324

Prescriptions for blood orders, 522-523, 526

Preservatives

anticoagulant-preservative solutions,

178-179

CPD, CP2D, CPDA-1, 178-179, 186, 564, 565

red cell changes during storage, 185, 186,

187, 431, 432

shelflifeofcomponents,178, 188,

189-190

reagent, antibodies to, 437

Pressure devices, 530

Pretransfusion testing, 407-420

after non-group-specific transfusions, 419-420

with autoantibodies, 469-472

of autologous blood, 122

blood labeling and release, 416, 418

blood samples, 409-410, 424

in children, 415, 562-563, 574

in cold agglutinin syndrome, 466

comparison with previous records, 413, 526

crossmatching, 413-415, 417

interpretation of results, 415-416, 417

in massive transfusions, 419, 510-511

patient identification, 407, 408-409

procedures included, 524

selection of units, 418-420

serologic testing, 410-413, 417

surgical blood orders, 91-93, 414

transfusion requests, 407-408, 522-523

type and screen, 91-93, 414

in urgent situations, 419, 510-511

Preventive action, 24-25

Prewarming technique, 308, 438, 754-755

Probability values in antibody identification,

429, 430

Problem identification and resolution, 25-27

Procainamide, 455

Procedures and policies, 7, 11, 15

Process capability, defined, 30

Process control, 30

Process flowcharting, 26, 27

Process improvement, 24-27

Process management, 11-14

computer system validation, 13-14

concepts, 4-5, 12

equipment validation, 13

process validation, 11-12

quality control, 14

validation plan, 12-13

Processes, 7, 11-12, 15

Production, principles of, 5

Products, nonconforming, 19, 20, 21

Proficiency testing (PT), 24

Promoter sequence, 204, 205

Protein C, 500, 507, 571, 837

Protein inactivation, 220

Protein S, 500, 507, 837

Protein synthesis, 204-207

Proteolytic enzymes. See Enzymes, proteolytic

Prothrombin time (PT)

in liver disease, 499

in massive transfusion, 511

in monitoring hemostasis, 494, 496

normal value of, 837

in vitamin K deficiency, 498

Prozone, 272, 275

PRP (platelet-rich plasma), 176, 177

Psoralen (S59), 702

PT. See Prothrombin time

PTP (posttransfusion purpura), 366, 370, 638,

659-660

Public antigens, 392

PUBS (percutaneous umbilical blood sampling),

541-542

Pulmonary edema, 648-649

Pulse, of donor, 102

Pumps, infusion, 522, 523, 529-530, 565

894 AABB Technical Manual

QSE(QualitySystemEssentials),3

Quad packs, 564

Qualification, defined, 30

Quality assurance (QA)

of blood administration, 527, 532

defined, 2, 30

Quality control (QC)

of blood components, 197-199, 202

apheresis, 832

Cryoprecipitated AHF, 199

leukocyte-reduced components, 199,

832-834

Platelets, 199, 694

Red Blood Cells, 199

Red Blood Cells, Deglycerolized, 812-813

of copper sulfate solution, 819-821, 847

defined, 2, 31

of equipment

automatic cell washers, 830-832

centrifuge calibration, 826-830

continuous temperature monitoring sys-

tems, 197-198

freezer alarms, 198-199, 824-826, 846

frequency of, 14, 846-847

refrigerator alarms, 198-199, 823-824, 846

thermometers, 198, 821-823, 847

of hematopoietic products, 607-608

in process control, 14

records, 14

unacceptable results for, 14

Quality improvement, 4

Quality indicators, 22-23, 31

Quality management, 2, 4-6, 31

Quality oversight, 6-7, 8

Quality System Essentials (QSEs), 3

Quality systems, 1-38

application of principles, 6-28

Code of Federal Regulations references, 32

common terms, 30-31

customer and supplier relations, 9-10

defined, 1-2

deviations and nonconforming products or

services, 19, 20, 21

documents and records, 14-15, 16, 17-19

equipment management, 10-11

human resources, 7-9

monitoring and assessment, 22-24, 36-38

organizational management, 6-7

process improvement, 24-27

process management, 11-14

quality assurance, 2

quality concepts, 2, 4-6

quality control, 2

quality management, 2

work environment, 27-28

Quarantine

of nonconforming components, 173-174

of repeatedly reactive units, 673, 674, 675,

683

of unusable products, 93-94

Race of donor, 98

Radiation safety, 63-66

blood irradiators, 65-66

effects of radiation, 63-64

emergency response plan, 66

exposure limits, 64

monitoring radiation, 64-65

radiation measurement units, 63

regulations, 64

safe work practices, 66

training, 65

waste management, 66

Raph system (ISBT 025), 226, 354, 843

Rapidly progressive glomerulonephritis (RPGN),

156-157

Rare Donor Program, 441-442, 769-770

Rd (Scianna) antigen, 349, 350, 842

Reagents

in ABO testing, 296

albumin additives, 276, 427, 753

antibodies to components of, 298, 437-438

antiglobulin, 278-280 , 427, 454, 468

chloroquine diphosphate, 446, 469, 746-

747

contamination of, 331, 332

for elutions, 457, 772-773, 775

enzymes, 276, 443, 445, 446, 756-760

glycine-HCl/EDTA, 446, 469, 747-748

labeling, 715

LISS and LISS additives, 274, 275, 277, 427,

443, 753, 754

monoclonal, 266-267, 296, 328, 329-330

for phenotyping, 440-441

polyethylene glycol (PEG), 276-277, 427, 443,

753-754

Index 895

preparation of, 715, 743-744, 756-757

quality control intervals, 847

red cells, 425, 426, 438

in Rh testing, 328-331

sulfhydryl, 448-449, 744-745, 764-765,

766-767

use of manufacturer’s directions, 11

ZZAP, 445, 446, 781-783

Receptors

on B cells, 249-253

complement, 262, 354

defined, 269

immunoglobulin superfamily, 244, 245

on macrophages and monocytes, 250,

255-256

for pathogens, P antigens as, 311

on phagocytic cells, 255-256

on T cells, 245, 254, 393, 394

Recessive traits, 233-234, 241

Recipients

ABO and Rh testing in, 122, 410, 411

care during transfusions, 530-531

education and consent, 521-522

identification, 407, 408-409, 416, 524-526,

527

phenotyping, 429-430, 439

records, 413, 416, 660-661

tracing (look-back), 675, 681-682, 683

weak D in, 323-324, 411

Recombinant human erythropoietin (rHuEPO),

219

in anemia treatment, 488

in autologous donations, 125

in neonates, 559

Recombinant immunoblot assay (RIBA), 170,

670-671, 672

Recombinant interleukin-11, 512

Recombinant proteins

for HPC mobilization, 587, 592-595

in leukapheresis, 144

as transfusion alternative, 512, 559

uses for, 218-219, 222

Records

archiving, 17-18

autologous donation/transfusion, 123

blood component, 165, 172-173, 416, 418

changing, 18

checking before blood release, 416, 526

confidentiality, 18

donor, 97-98, 103, 165, 173

electronic, 17-18

infectious disease testing, 173

linking personnel to, 19

management, 17-19

of patients with special needs, 661

plateletpheresis, 142

pretransfusion testing, 413

storage, 18

stored tissue allograft, 626

transfer, 173

transfusion, 416

transfusion complication, 660-661

Recovered Plasma, 177

Red blood cells

abnormalities in Rhnull, 326

alloimmunization to, 265, 637, 656-657

antigen nomenclature, 238-239, 318-319,

840-844

changes in storage, 185, 186, 187-188, 431,

433

immune-mediated destruction, 265-266

membrane components of, 290-291

normal values, 835

preparation of 3% suspension, 727

reagents, 425, 426, 438

removal from marrow, 601

separating autologous and transfused,

748-750

survival studies, 441, 509, 655

volume, normal value, 839

Red Blood Cells, Deglycerolized

adequacy of deglycerolization, 812-813

expiration date, 189

preparation, 191-192, 809

refreezing, 183

storage, 192

Red Blood Cells, Frozen

cryoprotective agents for, 181

expiration dates, 184, 189

freeze-thaw damage in, 181

preparation, 181-183, 807-812

storage, 184-185, 809

thawing and deglycerolizing, 191-192, 809

transportation and shipping, 196

Red Blood Cells, Pheresis, 35, 144

Red Blood Cells (RBCs)

ABO/Rh compatibility, 411, 418, 486

antigen-matched, 95

896 AABB Technical Manual

bacterial contamination, 691

collection by apheresis, 35, 143

description, 176

expiration dates, 189

freezing, 181-183, 807-812

infectious risks of, 700

irradiated, 189, 192, 193, 493

leukocytes-reduced, 189, 190, 199, 202, 493,

805

low volume, 101, 122, 179

open system, 189

preparation, 176, 804-807

quality control, 199, 202

rejuvenated, 189, 194, 806-807

storage, 185, 186, 187-188

substitutes, 512-513

transfusion, 483-488

in HPC transplantation, 591-592, 599,

600, 601

indications for, 485-488, 563-564

in neonates, 562-568, 588

physiologic principles, 483-485

selection of components, 411, 486

transportation and shipping, 195-196

washed, 189, 193

Red cell depletion, 601

Reentry protocols, 673, 674, 675, 681-682

Reference laboratories, 439

Refractoriness to platelets, 362-365

antibody specificity prediction method for,

365

causes, 362-363, 364, 397-398, 491

defined, 362

finding compatible donors, 398

HLA-matched platelets for, 363-365, 398

platelet crossmatching for, 365, 398

preventing alloimmunization, 365-366

selecting platelets with, 363-365

Refrigerators, 184

alarm systems for, 198-199, 823-824, 846

quality control, 197-199, 846

temperature monitoring systems for,

197-198

thermometers for, 198

Refsum’s disease, 157-158

Registration of donors, 97-98

Regulations

for autologous donations, 119-120

for hematopoietic transplantation, 608

quality-related CFR regulations, 32

for radiation safety, 64

for safety, 28, 39-40, 64, 71-72

for tissue transplantation, 626

for transport and shipping dangerous goods,

716-721

Reissuing blood products, 197, 525

Rejuvenated RBCs, 189, 194, 806-807

Relative risk (RR), 403-404

Release of blood

identification of recipient and component,

524-526

policies of, 416, 418

reissue, 197, 525

in urgent situations, 419, 510-511, 522-523

Remedial action, 25

Renal system

diseases treated with apheresis, 156-157

failure in transfusion reactions, 640-641

kidney transplantation, 401-402, 619, 624,

627

Reports

of deviations, nonconformances and com-

plications, 19, 20, 21

of fatalities, 19, 46, 661, 702-703

of injuries, 45-46

internal event, 19, 20

Requests

for autologous blood collection, 121

for transfusion, 407-408, 522-523

Requirement, defined, 31

Respiratory distress, 152, 639, 647-648

Restricted work areas, 41

Restriction endonucleases, 214, 215

Restriction fragment length polymorphism

analysis (RFLP), 214, 215, 222, 373

Reticulocyte counts, normal value, 835

Reverse line technique, 395

Reverse transcriptase PCR (RT-PCR), 214, 217

RFLP (restriction fragment length polymor-

phism analysis), 214, 215, 222, 373

Rg (Chido/Rodgers) antigens/antibodies,

351-352, 842

Rh Immune Globulin (RhIG), 547-551

in amniocentesis, 541, 549

antepartum administration of, 538, 541,

548

contraindications for, 549

dosage for, 547, 549, 550-551

Index 897

for fetomaternal hemorrhage, 549-551

in platelet transfusions, 490

in positive DAT, 456

postpartum administration of, 548-549

Rh system (ISBT 004), 315-331

antibodies, 327-329

anti-D in D+ individuals, 327

concomitant, 327-328, 441

directed at cis products, 324

dosage effect, 327

antigens

C, c, E, e, 316, 319, 320, 330-331

cis product, 324

D antigen, 315-316, 319-320

deleted phenotypes, 326

G antigen, 324-325

incidence, 317

nomenclature, 841, 844

partial D, 322-323

Rhmod, 326

Rhnull, 325-326

variants, 325

weak D, 322-324

association with LW, 351

in component selection, 418, 441, 486, 490,

511

ethnic differences in, 316, 317, 320, 321,

325

genes, 226, 320-321

genetics and biochemistry, 316-318

genotypes, 321-322, 844

in HDFN, 536

phenotypes and haplotypes, 319, 320, 844

position effect in, 319-320, 324

red cell abnormalities in, 326

RhAG (Rh-associated glycoprotein), 318

terminology, 315, 317, 318-319

in transplantation, 599, 628-629

Rh testing, 328-332

with autoagglutinins, 329, 331, 469-470

of autologous blood, 122

of blood components, 164, 165

of C, c, E, e, 320, 330-331

in children, 330, 415, 545, 563, 574

comparison with previous records, 413,

526

controls, 328-329, 330

of cord blood, 545

for D, 319-320, 328-330

of donors and recipients, 328-332, 410, 411,

413, 441

false-positive/false-negative results, 329,

330, 331-332

of Granulocytes, 144

in HDFN, 330, 539, 545

high-protein reagents in, 328-329, 330

low-protein reagents in, 329-330

microplate test, 328, 741

of Platelets Pheresis, 142

in prenatal evaluation, 538-539

slide test, 328, 739-740

in transfusion reaction evaluation, 654

for transplantation, 629

tube test, 740-741

of umbilical cord blood, 595

for weak D, 165, 323, 328, 411, 538-539,

741-744

RIBA (recombinant immunoblot assay), 170,

670-671, 672

Rigors, 633, 643

Risks of disease transmission

HIV infection, 677

with plasma derivatives, 699, 701

posttransfusion hepatitis, 673, 675

reducing, 618, 620, 699, 701-703

with tissue transplantation, 617-618,

619-620, 620

with transfusions, 700

RNA interference, 220

RNA (ribonucleic acid)

isolation of, 209-211

mRNA processing, 204-206

mRNA translation, 206-207

splicing, 206

transfer, 207

Rocky Mountain spotted fever, 696

Rodgers blood group. See Chido/Rodgers

Room temperature antibodies, 302-303

Room temperature storage, 185

Rosette test, 550, 793-794

Rouleaux

in ABO discrepancies, 298, 299, 303

in antibody detection, 412

in Rh typing, 329, 330

saline replacement technique, 303, 755-

756

RT-PCR. See Reverse transcriptase PCR

Run charts, 23

898 AABB Technical Manual

S/santigensandantibodies,336, 337, 340, 840

Safe work practices

for biosafety, 54-55

for electrical safety, 48-49

for fire prevention, 47-48

general guidelines, 44, 73-76

for radiation safety, 66

Safety goggles, 74

Safety program

accidents and injuries, 45-46

biosafety, 49-57, 77

chemical safety, 57-63, 78-88

disaster planning, 67-68

electrical safety, 48-49

emergency response plans, 44

employee monitoring programs, 45

engineering controls, 43

fire prevention, 47-48

first aid and follow-up, 45

general elements, 42-47

hazard identification and communication,

hepatitis prophylaxis, 45, 668, 672, 703

latex allergies, 46-47

management controls, 45

personal protective equipment, 43-44

policy manual, 45

radiation safety, 63-66

regulations and recommendations, 71-72

resources for information, 850

safe work practices, 44

safety officer, 42-43

shipping hazardous materials, 66

training, 43, 44

waste management, 67

in work environment, 27-28

Saline replacement technique, 303, 755-756

Saliva

ABH substances in, 290-291, 301, 736-739

Lewis substance in, 445, 736-739

Salvia lectins, 743

Samples. See Blood samples

SBO (standard blood orders), 92-93

Scianna system (ISBT 013), 226, 336, 349-350,

842

Sdaantigen, 357

agglutination pattern of antibody, 357, 412

neutralization of, 357, 445, 767-768

SDF-1 (stromal-derived growth factor-1), 594

Secretor gene (Se), 290, 291, 304-305, 737-739

Secretory component, 258

Segregation, 229, 230

Selectins, 246

Semen, 620, 621, 624

SEN-V virus, 668

Sensitization, 272-275

Separation techniques

in apheresis, 139-140

immunomagnetic, 596, 598

of multiple antibodies, 449

for transfused and autologous cells, 748-750

Sepsis

neonatal, 569-570

and positive DAT, 456

prevention, 693-694

transfusion-associated, 635, 643, 655,

691-692

Sequence-specific oligonucleotide probes

(SSOP), 214, 373, 395-396

Sequence-specific primers (SSPs), 214, 373, 395,

396

SERF (Cromer) antigen, 353, 354, 843

Serious Hazards of Transfusion (SHOT) initia-

tive, 641

Serologic centrifuges, calibration, 828-830

Serologic testing

additives in, 276-277, 443, 753-754

of autologous blood, 120, 122

of blood components, 165-166

in cold agglutinin syndrome, 460, 465

comparison with previous results, 413

of cord blood, 595

of Granulocytes, 144

in HDFN, 538-539, 544-546

for HLA antigens, 396-397

in IgM warm AIHA, 464

incubation temperatures, 443, 715

in mixed-type AIHA, 460, 466-467

in neonatal immune thrombocytopenia, 552

for organ and tissue transplantation, 620,

621-623, 629

in paroxysmal cold hemoglobinuria, 460,

467

for platelet antibodies, 375

of Platelets Pheresis, 142

with positive DAT, 456

in pretransfusion testing, 410-413, 562-563

Index 899

records of, 165

for syphilis, 165-166, 695

in transfusion reaction evaluations, 653-654

in warm autoimmune hemolytic anemia,

460, 461

in warm IgM AIHA, 464

Serotonin release assay, 377

Serum

dilution, 725-726

for pretransfusion testing, 409, 424

Serum hyperviscosity syndrome, 153-154

Serum proteins in typing discrepancies, 298,

299, 303, 331

Serum-to-cell ratio, 444

Services

critical, 6-7

nonconforming, 19, 20, 21

quality principles, 5

Severe acute respiratory syndrome (SARS), 590

Sex-linked traits, 233-234, 241

SH antigen, 378

Shelflifeofcomponents,178, 188, 189-190

Shipments

of autologous units, 120

of blood components, 66, 179, 195-196

cargo aircraft only, 719

of clinical specimens, 66, 717-718, 720-722

containers for, 195, 847

of dangerous goods, 66, 716-722

of hematopoietic components, 606-607

labeling, 718, 719

monitoring temperature during, 722

packaging with dry ice, 196, 719, 720-721

packaging with liquid nitrogen, 718, 719

Shock, 633, 640

Short supply agreement, 177

SHOT (Serious Hazards of Transfusion) initia-

tive, 641

Showers, emergency, 61

Sickle cell disease (SCD)

alloantibody production in, 576

autologous donation with, 119

in children, 574-576

delayed transfusion reactions in, 656-657

separation of transfused from autologous

cells, 749-750

transfusion in, 508-509, 574-576

treatment with apheresis, 155-156

Signal transduction, 246

Signs, safety, 47, 51, 60

Silent mutations, 207

Single-donor platelets. See Platelets Pheresis

Skin appearance

in donors, 102-103

in transfusion reactions, 639, 644-646

Skin banking, 619, 624, 625

Sl (Knops) antigens, 354, 843

Slide tests

for ABO group, 731-732

for Rh typing, 328, 739-740

Sodium, 187

Software, computer, 17

Solid organ transplantation. See Organ donation

and transplantation

Solid-phase red cell adherence assay (SPRCA)

in detecting antigen-antibody reactions,

283-284

in platelet antibody detection, 371, 372, 375

in platelet crossmatching, 364

Soluble antigens

ABH, 290-291, 298, 301, 445, 736-739

Chido/Rodgers, 445, 765-766

HLA, 391

Lewis, 445, 736-739

P1, 310, 445

Sda, 357, 445, 767-768

testing, 301, 445, 736-739

Solutions

additive, 176, 186, 564-565

colloid, 486, 507-508, 572

dilution, 726-727

hemoglobin, 512-513

IV, 530

phosphate buffer, 728

preparation, 723-725

Solvent/detergent-treated pooled plasma, 496,

702

Somatic cells, 223, 241

Source Plasma, 142

Southern blotting, 214, 216

SPA immunoadsorption, 158

Specification, defined, 31

Spills

blood, 55, 56

chemical, 61-62, 84-88

radioactive, 66

SPRCA. See Solid-phase red cell adherence assay

SRA (C-serotonin release assay), 377

900 AABB Technical Manual

SSOP (sequence-specific oligonucleotide

probes), 214, 373, 395-396

SSP (sequence-specific primers), 214, 373, 395,

396

Staffing, 8-9

Standard blood orders (SBO), 92-93

Standard Precautions, 49, 54

Standards

for autologous donations, 119

for hematopoietic transplantation, 609

ISO 9001, 1- 2, 3

performance improvement, 25

STAR (Scianna) antigen, 349, 350, 842

Statistical tables for binomial distribution,

33-35

Stem cells, pluripotent, 249, 251, 581

Sterile connection devices, 847

Sterility testing, 195

Storage

acceptable temperatures for, 184, 185, 715

antigen deterioration with, 431, 433

of autologous blood, 94-95, 123

biochemical changes with, 185, 186, 187

of biohazardous material, 57

of blood components, 93-94, 184-185, 186,

187-188

of blood samples, 184-185, 410

of directed donor units, 94-95

frozen, 184-185, 606

of Granulocytes, 144

of hazardous chemicals, 61

of hematopoietic progenitor cells, 604-606

liquid, 185, 186, 187-188

liquid nitrogen, 185, 606

of organs and tissue, 624

of Platelets, 185, 188, 694-695

of records, 18-19

of Red Blood Cells, 185, 186, 187-188

of Red Blood Cells, Deglycerolized, 192

of Red Blood Cells, Frozen, 184-185, 809

refrigerated, 184

room temperature, 185

of untested or infectious products, 606

vapor-phase, 606

Storage lesion

of platelets, 188

of red cells, 185, 186, 187-188

Stroma-free hemoglobin solution, 512-513

Stromal-derived growth factor-1 (SDF-1), 594

STS (serologic testing for syphilis), 165-166,

695

Subgroups of ABO system, 293-294

in ABO discrepancies, 302

confirmation of by adsorption/elution,

735-736

testing for, 296

Sulfhydryl reagents, 448-449

to alter blood group antigens, 342, 445, 446,

766-767

applications for, 448-449

in dispersing autoagglutination, 469-470,

744-745

to distinguish IgM from IgG, 764-765

effect on Kell antigens, 342

Supertransfusion programs, 575-576

Supplier relations, 9-10

Supplies, critical, 9, 10, 11

Suppressor genes, 235-236

Surgical blood order systems, 91-93, 414

Survey meters, 65

Survival studies of red cells, 441, 509, 655

Syncope, in donors, 107

Syngeneic HPC transplantation, 582, 587

Syphilis, 695

testing autologous blood, 120

testing blood components, 164, 165-166, 695

testing transplantation donors for, 590, 620,

621

T-cell receptor (TCR), 245, 254, 393, 394

T-cell reduction (depletion), 586, 598

T lymphocytes, 249, 253-255

cytotoxic T cells, 249, 253-254

depletion methods, 598

helper T cells, 249, 253-254

receptors (TCR), 245, 254, 393, 394

recognition by T cytotoxic cells, 254

stimulation of B cells, 254-255

subpopulations of, 249, 253

TA-GVHD. See Transfusion-associated

graft-vs-host disease

Tc (Cromer) antigens/antibodies, 353, 354, 843

TCR (T-cell receptor), 245, 254, 393, 394

Temperature

of donor, 101-102

effect on agglutination, 273-274, 336, 443

incubation, 443, 715

Index 901

monitoring systems, 197-198, 823-826

during shipments, 195-196, 722

storage, 184, 185, 715

Tendon transplants, 624

Test results, grading, 412, 728-729

Thalassemia, 508, 575-576

Thawed Plasma, 189, 191

Thawing

Cryoprecipitated AHF, 191, 815

devices for, 846

effects of, 181

Fresh Frozen Plasma, 191

hematopoietic components, 607

Red Blood Cells, Frozen, 191-192, 809

Therapeutic apheresis, 144-158

complications, 150-153

in HDFN, 145, 149

indications for, 146-148, 153-158, 500

photopheresis, 158

plasma volumes exchanged, 145, 149

removal of normal plasma constituents,

149-150

removal of pathologic substances, 145,

148-149

replacement fluids in, 150, 151

SPA immunoadsorption, 158

vascular access in, 145, 150

Therapeutic cells, 581. See also Hematopoietic

progenitor cell transplantation

Therapeutic plasma exchange (TPE), 145. See

also Therapeutic apheresis

Thermal amplitude studies, 441, 458, 465

Thermometers, 198

electronic oral, 822-823

liquid-in-glass, 821-822

standardization and calibration, 198,

821-823, 847

Thrombin, 502

Thrombin time, normal value, 837

Thrombocythemia, 154

Thrombocytopenia

in blood loss, 499

drug-induced, 374-377

and exchange transfusion, 567

heparin-induced, 375-377, 492

idiopathic thrombocytopenic purpura, 158,

373-374, 492, 553-554

neonatal alloimmune thrombocytopenia,

551-553, 568-569

posttransfusion purpura, 366, 370, 638,

659-660

secondary to maternal ITP, 553-554

thrombotic thrombocytopenic purpura,

154-155, 158, 492, 500

treatment, 158, 490-491

Thrombopoietin, recombinant human, 219

Thrombotic thrombocytopenic

purpura/hemolytic-uremic syndrome

(TTP/HUS), 154-155, 158, 492, 500

Tick-borne infections, 695-697

Timers/clocks, 847

Tissue antigens. See HLA system

Tissue oxygenation, 484-485, 511-512

Tissue transplantation

ABO compatibility in, 627

bone banking, 623, 625

consent for, 620-621

disease transmission in, 617, 619-620

donor eligibility, 620-621

heart valves, 625

preservation conditions and dating periods,

624

records, 626

recovery of tissue, 621

regulation, 626

risk reduction in, 618, 620

serologic testing for, 621-623

skills and experience appropriate for, 618

skin banking, 625

types of donors for, 622

Titration of antibodies

applications, 449

cold agglutinins, 458, 465, 778-779

maternal antibodies, 449, 539-540, 730-731,

796-798

methods for, 761-764, 796-798

TNF (tumor necrosis factor), 264, 640

Toxoplasmosis, 698

TPE (therapeutic plasma exchange), 145. See

also Therapeutic apheresis

Training

biosafety, 50

chemical safety, 58-59

for dry ice shipments, 721

electrical safety, 48

FDA cGMP, 8

fire safety, 47

general safety, 43, 44

902 AABB Technical Manual

new employee, 8

radiation safety, 65

Traits, 232-236

TRALI. See Transfusion-related acute lung injury

Tranexamic acid, 513-514

Trans effect, 319, 320

Transforming growth factor-β(TNF-β), 264

Transfusion-associated graft-vs-host disease

(TA-GVHD), 657-659

HLA system in, 399, 400

management, 637

manifestations, 658

in neonates, 560-561

pathophysiology, 637, 658

treatment and prevention, 658-659

Transfusion-associated sepsis, 635, 643, 655,

691-692

Transfusion Committee, 514

Transfusion reactions

acute

air embolism, 636, 651-652

allergic (urticarial), 522, 634, 644-647

anaphylactic, 635, 644-647

circulatory overload, 636, 648-649

citrate toxicity, 649-650

coagulopathy, 651

evaluation, 531, 652-656

febrile nonhemolytic, 379, 398-399, 576,

634, 643-644

hemolytic, 336, 399-400, 634, 639-642

hyper- and hypokalemia, 650

hypocalcemia, 561, 636, 649-650

hypotension associated with ACE inhibi-

tion, 636, 645

hypothermia, 511, 637, 650

nonimmune hemolytic, 636, 642-643

sepsis, 635, 643, 655, 691-692

signs and symptoms, 633, 639

transfusion-related acute lung injury,

379-380, 399, 635, 647-648

classification, 633, 634-638

delayed

alloimmunization, 637, 656-657

graft-vs-host disease, 399, 400, 560-561,

637, 657-659

hemolytic, 346, 575, 637, 656-657

immunomodulation, 638, 660

iron overload, 638, 660

Kidd antibodies in, 346, 656

posttransfusion purpura, 366, 370, 638,

659-660

management, 634-639

records, 660-661

reporting, 19, 661

Transfusion-related acute lung injury (TRALI),

647-648

evaluation, 656

and granulocyte transfusions, 648

HLA antibodies in, 399, 647

management, 635

neutrophil antibodies in, 379-380, 647

pathophysiology, 635, 647

prevention, 648

symptoms, 634, 647-648

treatment, 648

Transfusion-transmitted diseases

babesiosis, 695-696

bacterial sepsis, 635, 643, 655, 691-692

Chagas’ disease, 697-698

cytomegalovirus, 686-687

Epstein-Barr virus, 687

erlichiosis, 696

fatalities from, 702-703

hepatitis, 667-675

herpesviruses, 686-689

human immunodeficiency viruses, 675-682

human T-cell lymphotropic viruses, 682-683,

684

Lyme disease, 696-697

malaria, 697

management, 703

parasitic worms, 698-699

parvovirus, 688-689

reducing risks of, 699, 700, 701-702

reporting, 702

Rocky Mountain spotted fever, 696

syphilis, 695

tick-borne infections, 695-697

toxoplasmosis, 698

transmissible spongiform encephalopathies,

689-690

West Nile virus, 683, 685-686

Transfusion triggers

for autologous transfusions, 125

for platelet transfusions, 490-491, 569, 576

for red cell transfusions, 483, 486-488

Transfusions

administration procedures, 521-532, 565-566

Index 903

alternatives to, 512-514

of antiprotease concentrates, 505-506

assessments of, 23, 38, 514

in autoimmune hemolytic anemias,

462-464, 467, 468, 509-510

of autologous blood, 123

cause of ABO discrepancies, 298

cause of positive DAT, 455, 462

in children, 562-572, 574-577

of coagulation factors, 493-494, 495, 496,

497, 498-505

of colloid solutions, 486, 507-508, 572

consent for, 521-522

of Cryoprecipitated AHF, 500-502, 572

delays in starting, 525

equipment for, 523-524, 527-530

events following, 531-532

exchange, 546-547, 560, 566-568

fatalities from, 19, 641, 645, 661, 690-691,

702-703

of Fresh Frozen Plasma, 496, 498-500, 571

of Granulocytes, 144, 492, 569-570

and HLA system, 397-400

in HPC transplantation, 591-592, 599, 600,

601

identification of recipient and components,

524-526, 527

of Immune Globulin, Intravenous,

505, 506

as immunizing stimulus in HDFN,

537-538

of incompatible blood, 509, 641

indications for, 486-488, 563-564

infusion rates, 531, 566

infusion sets for, 527-529, 565-566

intrauterine, 541, 543-544

of irradiated components, 493

IV solutions for, 530

of leukocyte-reduced components, 492-493

massive, 419, 510-512, 628-629, 649-651

in neonates, 562-572

non-group-specific, 419-420, 510-511

in older infants and children, 574-577

patient care during, 530

perioperative, 486-487

of plasma derivatives and substitutes, 496,

503-508

of Platelets, 488-492, 553, 568-569

premedication in, 522

of protein C and protein S, 507

quality assurance, 514, 527, 532

of RBCs, 483-488, 558, 562-568

of recombinant proteins, 512

requests for, 407-408, 522-523

selection of components, 411, 486

in sickle cell disease, 508-509, 574-576

starting, 526-527

in thalassemia, 508, 575-576

in transplantation, 627, 629-630

in urgent situations, 419, 510-511,

522-523

of Whole Blood, 485, 486

Transmissible spongiform encephalopathies

(TSEs), 689-690

Transplantation. See Hematopoietic progenitor

cell transplantation; Organ donation and

transplantation; Tissue transplantation

Transplantation antigens. See HLA system

Transportation

of autologous units, 120

of blood components, 66, 179, 195-196

of blood to patient area, 524-525

cargo aircraft only, 719

of clinical specimens, 66, 717-718, 720-722

containers for, 195, 847

of dangerous goods, 66, 716-722

of hematopoietic components, 606-607

labeling, 718, 719

monitoring temperatures during, 722

packaging with dry ice, 196, 719, 720-721

packaging with liquid nitrogen, 718, 719

Trial to Reduce Alloimmunization to Platelets

(TRAP) Study Group, 365

Tropical spastic paraparesis (TSP), 682-683

Trypanosoma cruzi, 698, 700

TSEs (transmissible spongiform

encephalopathies), 689-690

TTP/HUS. See Thrombotic thrombocytopenic

purpura/hemolytic-uremic syndrome

TTV virus, 668

Tube tests

for ABO group, 732-733

for Rh type, 740-741

Tumor cell detection, 608

Tumor necrosis factor (TNF), 264, 640

Tumor purging, 597-598

Twitching, in donors, 108

Type and screen (T/S), 91-93, 414

904 AABB Technical Manual

U antigen/antibody (MNS system), 336, 337,

340, 840

Ulex europaeus (anti-H), 304, 743-744

Ultraviolet B (UVB) irradiation, 365

Umbilical cord blood. See Cord blood

UMC (Cromer) antigen, 353, 354, 843

Unasyn, 455

Uniforms, 73

Urgent release of blood, 419, 510-511, 522-523

Urine

examinations in transfusion reactions, 639,

653

neutralization of Sda, 357, 445, 767-768

Urticaria (hives), 634, 644-647

Utilization of blood. See Blood utilization

UVB (Ultraviolet B) irradiation, 365, 398

Vaccines, hepatitis, 45, 668, 672, 703

Valeri method for red cell cryopreservation,

810-812

Validation

of computer systems, 13-14, 415

defined, 31

of equipment, 13

of processes, 11-12

statistical tables for, 33-35

validation plans, 12-13

variances to, 11

Vapors, hazardous, 62, 63

Variant Creutzfeldt-Jakob disease (vCJD), 590,

689-690

Vascular access

in apheresis, 145, 150

in HPC collection, 593, 594

in neonates, 565-566, 568

for transfusions, 523-524

Vasovagal syndrome, 107, 141

vCJD (variant Creutzfeldt-Jakob disease), 590,

689-690

Vel collection, 355-356

Venipuncture, 105-106, 800-804

Venous access. See Vascular access

Venous transplants, 624

Verification, defined, 31

Vicia graminea (anti-N), 743

View boxes, 846

Vil antigen, 354

Viral marker testing. See Infectious disease test-

ing

Viruses

inactivation of, 699, 701-702

transmitted by transfusion

cytomegalovirus, 686-687

Epstein-Barr, 687

hepatitis, 667-675

HIV, 675-682

HTLV, 682-683, 684

human herpesviruses, 688

parvovirus, 688-689

transmissible spongiform

encephalopathies, 689-690

West Nile, 683, 685-686

Viscosity, relative, 835

Vital signs, 526-527, 531

Vitamin K, 498-499, 571

VLBW (very low birthweight) neonates, 557, 568.

AABB Technical Manual 15th Ed. 2005

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